CN118414082A - A novel GLV-phenolamide: biosynthesis and function in protecting plants from herbivores - Google Patents

Abstract

The present invention relates to compounds of general formula (I) that mediate resistance to leafhopper and planthopper pests or enantiomers, diastereomers, stereoisomers. The invention further relates to methods for preparing said compounds, methods for preparing said compounds enzymatically using at least a BBL2 polypeptide and PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H activity. Further contemplated are genetically modified organisms for preparing said compounds, expression cassettes for heterologous expression of the activity, the use of corresponding polypeptides and polynucleotides for preparing said compounds, compositions comprising said compounds, and the use of said compounds for plant protection.

Description

A novel GLV-phenolamide: biosynthesis and function in protecting plants from herbivores

Technical Field

The present invention relates to compounds of general formula (I) that mediate resistance to leafhopper and planthopper pests

or enantiomers, diastereomers, stereoisomers. The invention further relates to methods for preparing said compounds, methods for preparing said compounds enzymatically using at least a BBL2 polypeptide and PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H activity. Further contemplated are genetically modified organisms for preparing said compounds, expression cassettes for heterologous expression of the activity, the use of corresponding polypeptides and polynucleotides for preparing said compounds, compositions comprising said compounds, and the use of said compounds for plant protection.

Background technique

Plants are at the bottom of most terrestrial food chains and are constantly attacked by herbivores and pathogens (Kessler and Kalske, Annual Review of Ecology, Evolution, and Systematics 49, 115-138 (2018), Bednarek and Osbourn, Science 324, 746-748 (2009)). Plants provide a variety of resources for most herbivorous insect species, such as food, mating and oviposition sites, and shelter. Insect selection of host plants involves complex behavioral responses to various physical and chemical characteristics of host plants, which operate at different spatial scales and include long-range olfactory (e.g., plant-derived volatiles sensed by odorant receptors) and visual (e.g., plant shape, size, and color) cues as well as short-range chemotaxis and taste (e.g., surface metabolites sensed by chemoreceptors) cues (Thorsteinson, Ann. Rev. Entomol. 5, 193-218 (1960)). The physical and chemical characteristics of plants that insects use for host selection depend on the feeding guild and feeding behavior (eg, polyphagous or oligophagous) of the insect species (Prokopy and Owens, Entomol Exp Appl 24(3):609-62 (1978)).

Insects can also sense plant hormones. For example, Helicoverpa zea (Lepidoptera) larvae can sense jasmonic acid (JA) (Li et al., Nature 419:712-715 (2002)), which accumulates in food plants during attack and induces de novo synthesis of plant defense metabolites (Dicke and Baldwin, Trends Plant Sci, 15:167-175, (2010)). Therefore, insects may choose plants to eat based on their ability to produce certain plant hormones.

In some cases, insects can suppress the accumulation of plant defense metabolites as a mechanism of food plant selection (Howe and Jander, Annu Rev Plant Biol 59: 41-66 (2008)). These inhibitory mechanisms are often associated with changes in plant hormone biosynthesis or signaling pathways and may involve specific enzymes (e.g., glucose oxidase) produced by insects (Musser et al., Nature, 416: 599-600 (2002)) or vector microorganisms (Mayer et al. J Chem Ecol, 34: 1045-1049 (2008)).

For insects of the order Hemiptera, behavioral responses to host plant features involve a series of steps, including tapping with the lips and probing with their piercing mouthparts. These initial probing and feeding attempts also trigger the rapid accumulation of phytohormones (such as JA) and their mediated induced defense metabolites. For example, when Nicotiana attenuata plants are starved of JA by silencing the initial key steps of the JA biosynthetic pathway, they are severely attacked in nature by Hemiptera leafhoppers of the genus Empoasca.

Research on plant traits that provide resistance to biotic agents, such as Empoasca leafhoppers, has focused primarily on non-host resistance to pathogens (Fan et al., Science 331, 1185-1188 (2011); Peart et al., Proc Natl Acad Sci U S A 99, 10865-10869 (2002); Sohn et al., New Phytol 193, 58-66 (2012)) and host resistance to herbivores (Agrawal, Science 279, 1201-1202 (1998); Karban and Baldwin, University of Chicago Press, 104-166 (2007)). This difference in emphasis may reflect the greater physiological autonomy of herbivores, which selectively choose plants to attack, as well as the challenge of discovering host resistance traits that herbivores reject. In laboratory no-choice assays, plants that become "defenseless" due to abrogation of defense pathways can be attacked by non-host herbivores (Muller et al., J Chem Ecol 36, 905-913 (2010); Barth and Jander, Plant J 46, 549-562 (2006)).

However, these assays do not capture the selective programs by which insects choose their host plants in nature, limiting the inferences that can be drawn about non-host resistance from these laboratory studies. Due to a lack of field studies, the mechanisms and metabolic traits of non-host resistance to herbivores remain largely unknown.

While much is known about plant traits that contribute to non-host resistance against pathogens, less is known about non-host resistance against herbivores, despite its agricultural importance. For example, Empoasca leafhoppers recognize host plants by eavesdropping on an unknown output of jasmonic acid (JA)-mediated signaling.

There is therefore a need for means and methods which can improve the protection of plants against herbivores such as leafhoppers or plant hoppers.

Summary of the invention

The present invention solves this need and provides compounds of general formula (I)

in:

R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy;

R ⁵ , R ⁶ and R ⁷ are each independently H or (C ₁ -C ₆ )-alkyl;

X is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group,

Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , _NH- (CH2) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester;

Z is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group,

or an enantiomer, diastereomer, stereoisomer or salt thereof.

The inventors have surprisingly found that the compounds confer resistance to leafhoppers and potentially to all insects that feed by sucking or lay their eggs in leaves.

In a preferred embodiment of the compounds, two of R ¹ , R ² , R ³ and R ⁴ are OH; R ⁵ , R ⁶ and R ⁷ are each H; and X is straight-chain (C ₂ -C ₈ )-alkenyl.

In a preferred embodiment of said compounds, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, and X is -CH=CH-.

In a further preferred embodiment, the compound has the following formula (II):

wherein R ⁵ , R ⁶ , R ⁷ and Z are as defined above;

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In another preferred embodiment, the compound has formula (III)

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In a further aspect the invention relates to processes for the preparation of the compounds according to the invention.

In a preferred embodiment, the method is an enzymatic production method using at least a BBL2 (berberine bridge enzyme 2) polypeptide.

In a further preferred embodiment of the method, the BBL2 (berberine bridge enzyme 2) polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 1;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 1;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 1;

(d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 1;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 2;

(h) represented by a polypeptide fragment of SEQ ID NO: 2 having the function of BBL2 (berberine bridge enzyme 2);

(i) represented by the polypeptide domain of SEQ ID NO: 2 having the function of BBL2 (berberine bridge enzyme 2);

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2 and has the function of BBL2 (berberine bridge enzyme 2); or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In a further preferred embodiment of the method, the enzymatic preparation method additionally uses a PPO (polyphenol oxidase) activity or polypeptide, wherein preferably the PPO (polyphenol oxidase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 3 or 5;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 3 or 5;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 3 or 5;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 3 or 5;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 4 or 6;

(h) represented by a polypeptide fragment of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or 6 and having PPO (polyphenol oxidase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In a preferred embodiment, the enzymatic preparation method additionally uses a PPO (polyphenol oxidase) activity or polypeptide, wherein preferably the PPO (polyphenol oxidase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 3 or 5;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 3 or 5;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 3 or 5;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 3 or 5;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 4 or 6;

(h) represented by a polypeptide fragment of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 4 or 6 and having PPO (polyphenol oxidase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In a further preferred embodiment of the method, the enzymatic preparation method additionally uses AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide, wherein preferably the AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 7;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 7;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 7;

(d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 7;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 8;

(h) represented by a polypeptide fragment of SEQ ID NO: 8 having AT1 (polyamine hydroxycinnamoyltransferase 1) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 8 having AT1 (polyamine hydroxycinnamoyltransferase 1) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 and having AT1 (polyamine hydroxycinnamoyltransferase 1) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In a further preferred embodiment of the method, the enzymatic preparation method additionally uses an ODC (ornithine decarboxylase) activity or polypeptide and/or an HPL (hydroperoxide lyase) activity or polypeptide, wherein preferably

(A) The ODC (ornithine decarboxylase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 9 or 11;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 9 or 11;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 9 or 11;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 9 or 11;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 10 or 12;

(h) represented by a polypeptide fragment of SEQ ID NO: 10 or 12 having ODC (ornithine decarboxylase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 10 or 12 having ODC (ornithine decarboxylase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 10 or 12 and having ODC (ornithine decarboxylase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (A)(a) to (f); and

(B) The HPL (hydroperoxide lyase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 13, 15 or 17;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 13, 15 or 17;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 13, 15 or 17;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 13, 15 or 17;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (B)(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 14, 16 or 18;

(h) represented by a polypeptide fragment of SEQ ID NO: 14, 16 or 18 having HPL (hydroperoxide lyase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 14, 16 or 18 having HPL (hydroperoxide lyase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 14, 16 or 18 and has HPL (hydroperoxide lyase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in (B)(a) to (f).

In a further preferred embodiment of the method, the enzymatic preparation method additionally uses PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide and/or 4CL (4-coumarate: Coenzyme A ligase) activity or polypeptide and/or HCT (hydroxycinnamoyl-transferase) activity or polypeptide and/or C3H (coumarate 3-hydroxylase) activity or polypeptide, wherein preferably:

(A) The PAL (L-phenylalanine ammonia lyase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 19, 21, 23 or 25;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 19, 21, 23 or 25;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 19, 21, 23 or 25;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 19, 21, 23 or 25;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of A(a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in A(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 20, 22, 24 or 26;

(h) represented by a polypeptide fragment of SEQ ID NO: 20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 20, 22, 24 or 26 and having PAL (L-phenylalanine ammonia lyase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in A(a) to (f); and

(B) the C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 27;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 27;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 27;

(d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 27;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of B(a) to (d);

(f) encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in B(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 28;

(h) represented by a polypeptide fragment of SEQ ID NO: 28 having C4H (cinnamate 4-hydroxylase) activity;

(i) represented by the polypeptide domain of SEQ ID NO: 28 having C4H (cinnamate 4-hydroxylase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 28 and having C4H (cinnamate 4-hydroxylase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in B(a) to (f); and

(C) The 4CL (4-coumaric acid: Coenzyme A ligase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131;

(d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of C(a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in C(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132;

(h) represented by a polypeptide fragment of SEQ ID NO: 30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumaric acid:CoA ligase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumarate:CoA ligase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 and having 4CL (4-coumarate:CoA ligase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in C(a) to (f); and

(D) The HCT (hydroxycinnamoyl-transferase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43;

(d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of D(a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any of the polynucleotides specified in D(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 32, 34, 36, 38, 40, 42 or 44;

(h) represented by a polypeptide fragment of SEQ ID NO: 32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity;

(i) represented by a polypeptide domain of SEQ ID NO: 32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity;

(j) represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% similarity to the amino acid sequence of SEQ ID NO: 32, 34, 36, 38, 40, 42 or 44 and having HCT (hydroxycinnamoyl-transferase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in D(a) to (f); and

(E) The C3H (coumarate 3-hydroxylase) activity or polypeptide is:

(a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 45;

(b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 45;

(c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO:45;

(d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 45;

(e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of E(a) to (d);

(f) is encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in E(a) to (d);

(g) represented by a polypeptide having SEQ ID NO: 46;

(h) represented by a polypeptide fragment of SEQ ID NO: 46 having C3H (coumarate 3-hydroxylase) activity;

(i) represented by the polypeptide domain of SEQ ID NO:46 having C3H (coumarate 3-hydroxylase) activity;

(j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 46 and having C3H (coumarate 3-hydroxylase) activity; or

(k) represented by a polypeptide encoded by any one of the polynucleotides specified in E(a) to (f).

In a further preferred embodiment, the enzymatic preparation is performed by providing the activity or polypeptide in vitro. It is particularly preferred that the method is performed at a pH of 4.8 or lower, more preferably by solid phase extraction using an argon stream.

In a further preferred embodiment, the enzymatic preparation is performed by means of an activity or polypeptide provided in a living cell, tissue or organism.

In a further preferred embodiment, the enzymatic production is performed by and in genetically modified cells, tissues or organisms, wherein the genetic modification allows the heterologous expression of one or more activities or polypeptides as defined above.

In another aspect, the invention relates to an organism, tissue or cell for the preparation of a compound according to the invention, which is genetically modified, wherein the genetic modification allows the heterologous expression of one or more activities or polypeptides as defined herein.

In a preferred embodiment of the method, organism, tissue or cell, said genetic modification results in at least the expression of a BBL2 (berberine bridge enzyme 2) polypeptide as defined herein.

In a preferred embodiment of the method, organism, tissue or cell, the genetic modification results in at least the expression of a PPO (polyphenol oxidase) activity or a polypeptide as defined herein.

In a further preferred embodiment of the method, organism, tissue or cell, the genetic modification results in at least the expression of an AT1 (polyamine hydroxycinnamoyltransferase 1) activity or a polypeptide as defined herein.

In a further preferred embodiment of the method, organism, tissue or cell, the genetic modification results in at least the expression of an ODC (ornithine decarboxylase) activity or polypeptide and/or an HPL (hydroperoxide lyase) activity or polypeptide as defined herein.

In another preferred embodiment of the method, organism, tissue or cell, the genetic modification results in at least the expression of a PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or a C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide and/or a 4CL (4-coumarate:CoA ligase) activity or polypeptide or a HCT (hydroxycinnamoyl-transferase) activity or polypeptide and/or a C3H (coumarate 3-hydroxylase) activity or polypeptide as defined herein.

In another preferred embodiment of the method, organism, tissue or cell, the expression is conveyed by a native, regulated, tissue-specific or constitutive promoter.

It is particularly preferred that the promoter allows (i) polycistronic expression of an activity or polypeptide as defined herein, (ii) individual expression of an activity or polypeptide as defined herein; or (iii) group expression of at least two activities selected from the group of activities as defined herein.

It is further particularly preferred that the expression is overexpression.

In a further preferred embodiment, said overexpression is conveyed by a strongly regulated or strong constitutive promoter and/or by providing at least a second copy of the genetic element encoding said activity or polypeptide.

In another specific embodiment of the present invention, the enzyme activity or polypeptide is from an organism belonging to the genus Nicotiana. Particularly preferred, the enzyme activity or polypeptide is from the species Nicotiana attenuata.

It is further preferred that the polynucleotide is contained in one or more extrachromosomal vectors or plasmids and/or is integrated in the genome of the organism.

In a further preferred embodiment, the genetically modified organism, tissue or cell is eukaryotic. Particularly preferred is that the genetically modified organism is a plant, or wherein the tissue is a plant tissue, or wherein the cell is a plant cell.

In a further preferred embodiment, the genetically modified organism belongs to, or the tissue or cell is derived from, a higher plant attacked by insect herbivores, preferably a higher plant attacked by insects that feed by tearing and scouring and/or sucking, more preferably a higher plant of the genera Nicotiana, Solanum, Oryza, Zea mays, Phaseolus vulgaris or Camellia.

In another aspect, the present invention relates to an expression cassette for heterologous expression in a eukaryotic host cell, wherein the expression cassette comprises a polynucleotide as defined herein. Preferably, the host cell is a plant cell.

In another aspect, the present invention relates to a vector or an insertion construct comprising a polynucleotide as defined herein or an expression cassette as defined herein.

In yet another aspect, the invention relates to the use of a polypeptide or polynucleotide as defined herein, an expression cassette as defined herein or a vector or insertion construct as defined herein for the preparation of a compound of the invention.

In another aspect, the invention relates to the use of an organism, tissue or cell as defined herein, or an organism, tissue or cell comprising an expression cassette or a vector or an insertion construct as defined herein, for the preparation of a compound of the invention.

In another aspect, the present invention relates to a composition comprising a compound according to the present invention or a compound prepared by a method according to the present invention or a compound prepared by an organism, tissue or cell as defined herein. Optionally, the composition further comprises an acceptable carrier, stabilizer and/or spreading agent.

In yet another aspect, the invention relates to the use of a compound of the invention or a compound prepared by a process according to the invention or prepared from an organism, tissue or cell as defined herein for protecting plants from attack by herbivores, or a composition as defined herein for protecting plants from attack by anti-herbivores.

In a preferred embodiment, the herbivore is an insect.

In another preferred embodiment, the insects are insects that feed by tearing and scouring and/or piercing and sucking.

In a further preferred embodiment, the insect is a leafhopper or a planthopper. Particularly preferably, the insect belongs to Aphrodinae, Bathymatopohorinae, Cicadellinae, Coelidiinae, Deltocephalinae, Erromeninae, Euacanthelinae, Eurymelinae, Evacantinae, Hylicinae, Iassinae, Jascopinae, Ledrinae, Megophthalminae, Mileewinae, Nastlopinae, Neobaliane, Neocoelidinae, Nioniinae, Phereurhinina, Portaninae, Signoretiinae, Tartesinee, Typhlory bina or Ulopinae family, more preferably Empoasca, Circulifer, Nilaparvata, Sogatella, Nepotettix or Cicadulina.

In another aspect, the invention relates to the use of a compound of the invention or a compound prepared by a method of the invention or a compound prepared by an organism, tissue or cell as defined herein or a composition as defined herein as an insecticide. Preferably, the insecticide is directed against insects as defined above.

In another preferred embodiment, the present invention relates to a method for plant protection which comprises contacting a plant or a part of a plant with a compound or composition of the invention as defined above.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a natural insect-guided multi-omics profile of a forward genetic line of a field-grown N. attenuata MAGIC population, highlighting deviations from typical jasmone signaling for non-host resistance to Empoasca leafhoppers. The figure shows a field planting of a MAGIC RIL population of natural N. attenuata plants in their natural growing environment at the WCCER field station in Arizona, USA. The figure shows the natural opportunistic Empoasca leafhopper community and the damage to leaves caused by their feeding. The leafhoppers attack these plants in a jasmonic acid (JA)-dependent manner and preferentially choose plants lacking JA as hosts (Image: R. Halitschke, D. Kessler, A. Kessler).

Fig. 2 is a schematic diagram of high-throughput phenotyping of 674 MAGIC RILs and parental lines in the field for phytohormones (1816 samples), transcriptomes (350 samples) and metabolomes (1706 samples) as well as Empoasca phenotypes (1907 samples). In order to eliminate the confounding factors caused by the stochasticity of insect attacks in nature, we simulated herbivory feeding as follows: oral secretions (OS) of freshly collected tobacco hornworm (Manduca sexta) larvae were immediately applied to standardized puncture wounds (W) of leaves in standardized leaf positions. This procedure is called W+OS treatment, which standardizes the initiation by precisely timing the onset of the response triggered by herbivory. Plant hormone and transcriptome data sets were collected 1 hour after W+OS treatment, and metabolome data sets were collected 72 hours later. PCC, Pearson correlation coefficient.

Fig. 3 Co-association network constructed from associations among metabolome, transcriptome, phytohormone and SNPs (PCC cutoff P < 0.05), and the top 5 most significant SNPs for each gene (small grey circles), metabolite or phytohormone from eQTL or mQTL filling were retained. Phytohormones and metabolites of different compound classes and JA-related genes were labeled differently. JA-Ile, JA signaling genes (NaAOC, NaMYC2a, NaMYC2b, NaJAZi), NaMYB8 and phenolamides (N-coumarylputrescine, N-caffeoylputrescine, N-feruloylputrescine) formed a central cluster and were labeled.

FIG4 Heatmap of co-expression of metabolites, JA-related genes and phytohormones with Empoasca phenotypes (number and lesion area) calculated by pairwise PCC (only significant correlations with P < 0.05 are shown and marked with differences).

FIG. 5 Reverse genetics combined with unbiased metabolomics based on information theory reveals a JA-JAZi module triggered by Empoasca spp., which regulates an unknown putrescine-containing phenolic amide induced by Empoasca spp. The phenotypes of leaves damaged by Empoasca spp. of transgenic Nicotiana acuminate lines (n=10) were randomly placed in an open selection greenhouse environment containing Empoasca spp. leafhoppers ( FIG. 26 ). Representative leaves of each genotype are shown, with insets highlighting damage to ovJAZi and irMYB8 lines. Different letters indicate significant differences [one-way analysis of variance (ANOVA), followed by Tukey's post hoc multiple comparisons].

Figure 6 Tissue-specific expression profiles of NaJAZh and NaJAZi in N. attenuata WT (left); heatmap differential markers depict Z-score scaled TPMs. Kinetics of relative transcript accumulation of NaJAZh and NaJAZi in leaves of N. attenuata (n=3) in response to continuous E. decipiens and M. sexta feeding, samples harvested 0 to 24 hours after the start of feeding. FLB, flower bud; STI, stigma; COE, early corolla; PED, pedicel; SED, seed; OFL, flowering; SNP, style; LEC, leaf control; ROT, root of oral secretion treated plants; OVA, ovary; NEC, nectary; ANT, anther; STT, treated stem; LET, treated leaf.

Figure 7 is a scatter plot of the diversity (Hj) of the specialized metabolome of leaves (n=4) relative to the index of specialization (δj) for 4 transgenic lines selected from a collection of 16 transgenic lines after 72 hours of feeding by E. decipiens and M. sexta (see Figure 32 for the complete data set). An increase in metabolome specialization (δj) indicates that, on average, more herbivory/genotype-specific metabolites are produced, while an increase in metabolome diversity (Hj) indicates that more metabolites are produced qualitatively, or that the overall metabolic frequency spectrum is more evenly distributed quantitatively. Differential markers represent different insects; symbols represent different treatments: triangles, insect feeding; circles, untreated control standards.

FIG8 Ranked metabolite specific (SI) index distribution plots for each metabolite calculated based on the metabolome specifically triggered by E. decipiens in the four transgenic lines shown in FIG7 (left), and further associated with PCC co-expression heatmaps between metabolites and E. decipiens numbers and injury phenotypes (right), where data from all reverse genetic lines shown in FIG5 were used to enhance the statistical power of PCC calculations. Points are labeled based on compound class annotations. Only significant correlations with P < 0.05 are shown in color.

FIG. 9 An in vivo Empoasca selection assay (n=8) (top) by infiltrating irMYC2 leaves with synthetic N-coumaroylputrescine (CoP), N-caffeoylputrescine (CP), or N-feruloylputrescine (FP) diluted in 0.1% DMSO solution, and an in vitro Empoasca non-selection assay (n=3, 25 Empoasca leafhoppers per replicate) (bottom) by feeding Empoasca with synthetic CoP, CP, or FP diluted in 10% glucose solution, show that these phenolamides do not affect leafhopper behavior or performance.

Figure 10 Elucidation of herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathways by combining MS/MS structural metabolomics with forward and reverse genetics. The figure shows the biclustering of 518 idMS/MS spectra constructed from 15 RILs of the field-grown MAGIC population based on shared fragments (based on similarity of NDP) and shared neutral losses (based on similarity of NL), revealing 7 distinct modules (M) in the molecular network (left); close-up of module 5, where putrescine- or caffeoyl-derived phenolic amides are enriched (right), containing an unknown metabolite m/z 347.19, which is directly related to both isomers of CP (circled in green). CS, N-caffeoylspermidine; CoCS, N’,N”-coumaryl, caffeoylspermidine; CFS, N’,N”-caffeoyl, feruloylspermidine; DCS, N’,N”-dicaffeoylspermidine; CPD, caffeoylputrescine dimer; CGA, chlorogenic acid; Unk., unknown.

Figure 11 Elucidation of herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathway by combining MS/MS structural metabolomics with forward and reverse genetics: The figure shows the accumulation of m/z 347.19 in EV and irMYC2 lines triggered by Empoasca (top) and in MeJA-induced EV, irMYC2, irMYB8, and ovJAZi lines (bottom).

Figure 12 Elucidation of herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathways by combining MS/MS structural metabolomics with forward and reverse genetics: The figure shows the Manhattan plot of herbivory-induced unknown m/z 347.19 from mQTL analysis of W+OS-induced leaves of MAGIC RIL population grown in greenhouse and extracted using a procedure that minimizes the loss of phenolamide moieties (left). The core JA signaling gene NaMYC2a, the phenolamide regulator NaMYB8, the CP biosynthesis gene NaAT1 and two unknown biosynthesis candidate genes NaPPO1 and NaPPO2 (P value cutoff = ^10-3 ), as well as the uncharacterized candidate gene NaBBL2 (P = 0.0013) were entered in the mQTL analysis. The gene co-expression network was constructed using a previously published microarray dataset of irMYB8 plants harvested 1 hour and 5 hours after W+OS induction. The phenolamide biosynthesis genes NaAT1 and NaDH29 were used as baits (diamonds), the dots in the center depict genes co-expressed with both baits, while the lower dots (NaAT1) and upper dots (NaDH29) depict genes co-expressed with a single bait (right).

Figure 13 Elucidation of herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathway by combining MS/MS structural metabolomics with forward and reverse genetics: The figure shows virus-induced gene silencing (VIGS) of biosynthetic gene candidates involved in the preparation of m/z 347.19. Silencing of NaPPO1, NaPPO2, NaAT1, and NaBBL2 expression abolished W+OS treatment-induced m/z 347.19 observed in EV control plants (C, untreated control standard).

Figure 14 Elucidation of herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathway by combining MS/MS structural metabolomics with forward and reverse genetics: The figure shows the proposed trifurcated biosynthetic pathway of the m/z 347.19 preparation induced by Empoasca spp., which requires LOX2-HPL-dependent C6GLV metabolism, LOX3-dependent and JA-regulated phenylpropanoid metabolism, and polyamine metabolism, whose products are presumably conjugated with NaBBL2- and NaPPO1/2-dependent reactions.

Figure 15 Elucidating herbivory-induced GLV-caffeoylputrescine metabolites and their trifurcated biosynthetic pathway by combining MS/MS structural metabolomics with forward and reverse genetics: The figure shows a scatter plot of the metabolite abundance of m/z 347.19 relative to (Z)-3-hexen-1-ol in the MAGIC accession from the greenhouse (top), and the accumulation of m/z 347.19 in leaves of stably transformed EV, asHPL, irLOX2 and irLOX2/3 lines after W+OS treatment (bottom).

Figure 16 Structure elucidation, biosynthesis, function and engineering of m/z 347.19 in vitro and in planta: The figure shows that the in vitro enzyme assay for the preparation of m/z 347 shows that NaPPO1 or NaPPO2 can catalyze the condensation of CP with (Z)-3-hexenal (Z3H) to form m/z 347. The extracted ion chromatograms (EICs) of the doubly charged CP dimer (m/z 250.13) and m/z 347.19 of NaPPO1 and NaPPO2 expressed in E. coli and CP and Z3H or (E)-2-hexenal. Compounds 1, 2, 3, and 4 represent isomers of CP dimer; compounds 5 and 6 represent isomers of m/z 347.19.

Figure 17 Structural elucidation, biosynthesis, function and engineering of m/z 347.19 in vitro and in plants: The figure shows the proposed enzymatic reaction for the synthesis of m/z 347.19 mediated by NaPPO1 or NaPPO2: NaPPO1/2 oxidizes CP to caffeoylquinoneputrescine and activates Z3H for Michael addition reaction, followed by aromatization to produce m/z 347.19.

Fig. 18 Mortality of Empoasca in vitro non-selection assay (n=4, 25 Empoasca leafhoppers per replicate) where Empoasca were fed for 6 h with 1 μM m/z 347.19 synthesized using NaPPO1/NaPPO2 and CP and Z3H and diluted in 10% glucose solution, or with 10% glucose as control (top). Empoasca in planta selection assay (n=8) using VIGS plants of EV, PPO1, PPO2 and AT1 (bottom).

Figure 19 Genes involved in the biosynthesis of m/z 347.19.

FIG20 Engineering of biosynthetic pathways to produce m/z 347.19 in plants. Neither Vicia faba nor Solanum chilense accumulate m/z 347.19 after priming; S. chilense accumulates CP after MeJA priming, while V. faba does not (inserted heat map). S. chilense can be engineered to produce m/z 347.19 by expressing NaPPO1, NaPPO2, and NaBBL2 in MeJA-primed S. chilense leaves infiltrated with Z3H. m/z 347.19 accumulates only in S. chilense expressing NaPPO1/NaPPO2 and NaBBL2 together, but not in S. chilense expressing NaPPO1/NaPPO2 alone. S. chilense can be engineered to produce m/z 347.19 by expressing NaPPO1, NaPPO2, and NaBBL2 in leaves infiltrated with Z3H and CP. Mortality of an in vivo Empoasca feeding assay (n=4, 25 Empoasca leafhoppers per replicate) in which Empoasca fed for 10 hours on reconstituted Chilean tomato and broad bean leaves, respectively. EIC, extracted ion chromatogram.

Figure 21 A 2019 field MAGIC colony grown at the WCCER field station in Prescott, Arizona, USA. The design of the MAGIC colony shows the distances between the main lines (8 in an east-west direction), branches (4 on each main line), and emitters (18 on each branch).

Fig. 22 Growth stages of plants in the MAGIC colony during herbivore screening. Four RILs were planted around each emitter (sprinkler) at a spacing of 50 cm.

Fig. 23 Plant treatments and herbivore screening of MAGIC populations. W+OS treatments and herbivore screening.

Figure 24 MAGIC group photo of field team members in 2019.

Figure 25 Multi-omics co-association network annotation. Detailed annotation of the outer layer of the multi-omics co-association network that is not closely related to JA signaling (additional annotation of the network provided in Figure 3 is provided). Different compound classes, plant hormones, JA-related genes and SNPs are shown in different grayscale tones.

Fig. 26 Setup of Empoasca selection assay in Isserstedt greenhouse. Different Nicotiana acuminate transgenic lines (n=10 plants per genotype) were randomly placed on greenhouse benches at least 40 cm apart to achieve an open choice experimental setup for Empoasca selection. Leafhoppers were released from colonies reared on broad bean plants enclosed in mesh tents located on both sides of the greenhouse benches.

Figure 27 Transgenic Gradient Leaf Tobacco Transcript Abundance of Target Genes in Lines.Relative mRNA Abundance of Genes in Rosette Plants of Different Transgenic Lines.For lines transformed with inverted repeat (ir) constructs designed to silence gene expression, transcripts of mature +2 leaves older than source-sink transition leaves from each line and size-matched EV plants and untreated control plants (C) of each genotype were quantified 1 hour after W+OS initiation.For plants transformed with overexpression (ov) constructs, only uninduced control plants were used.Asterix indicates significant differences between W+OS treatment and untreated control (C) [Student t-test for paired differences, *P<0.05, **P<0.01, and ***P<0.001; n=6 plants for each treatment/genotype]. N.S., not significant.

Figure 28 Tissue-specific expression profiles of 13 JAZs in N. attenuata WT. Heat map markers depict Z-score scaled TPMs. FLB, flower bud; STI, stigma; COE, early anther; PED, pedicel; SED, seed; OFL, open flower; SNP, style; LEC, leaf control; ROT, root from plants with OS-treated leaves; OVA, ovary; NEC, nectary; ANT, anther; STT, treated stem; LET, treated leaf.

FIG29 Interaction between NaMYC2a/b, NaMYB8 and NaJAZi proteins in Y2H assay. GAL4 DNA-BD-NaMYC2a/b/NaMYB8 and AD-JAZ were co-transformed into yeast. Transformants were grown on QDO (SD-Ade/-His/-Leu/-Trp, supplemented with 2 mM 3-AT, at 30° C.) at the indicated dilutions.

Figure 30 Setup for Empoasca and Manduca bioassay. Setup for Empoasca (right) and Manduca (left) feeding experiment in a growth chamber. Note: Clip cages were used to restrict Empoasca attack to specific leaves.

FIG. 31 Empoasca selective (top) and non-selective (bottom) assays in a growth chamber.

Figure 32 Metabolome diversity and specialization affected by insect herbivory in different Nicotiana acuminate transgenic lines. Scatter plot of the diversity (Hj index) of the specialized metabolome relative to specialization (δj index) in the presence and absence of Empoasca peach leafhopper (E.Decipiens) (n=3) and tobacco hornworm (M.Sexta) (n=4) for 72 hours of attack on 16 different Nicotiana acuminate transgenic lines. This figure provides the complete data set described in Figure 7.

Figure 33 Accumulation of phenolic derivatives and phenolamides in leaves of Nicotiana acuminate in response to continuous feeding by two herbivore species in 16 different transgenic lines. Simplified pathways reveal associations between major phenylpropanoid-quinic acid and -polyamine conjugates in response to feeding by Empoasca or Sextus manduca insects. CGA, chlorogenic acid; CoP, N-coumaroylputrescine; CP, N-caffeoylputrescine; FP, N-feruloylputrescine; CS, N-caffeoylspermidine; FS, N-feruloylspermidine; DCS, N',N"-dicaffeoylspermidine; CFS, N',N"-caffeoyl,feruloylspermidine. Asterisks indicate significant differences between insect treatments and controls (Student's t-test, *P<0.05, **P<0.01, ***P<0.001; n=3 to 4 plants per treatment/genotype). Figure 33 A) Overview, B) CGA, C) Phenylalanine, D) CoP, E) CS, F) CP, G) FS, H) FP, I) DCS and J) CFS.

Figure 34 Standard curves for CoP, CP and FP. Standard curves for CoP, CP and FP were constructed by preparing synthetic CoP, CP and FP, and used to quantify phenolamide levels in leaves and insect diets. LC-MS conditions are provided in the Examples.

Figure 35 Annotated MS/MS spectrum and structure of unknown metabolite m/z 347.19. The IdMS/MS of m/z 347.19 is annotated by highlighting the neutral losses of _{putrescine and C6H8O and} the corresponding structure of m/z 347.19 as shown on the right.

Figure 36 Association between herbivory-induced accumulation of m/z 347.19 and accumulation of jasmonic acid and Empoasca genus phenotypes in MAGIC RIL populations grown in the 2019 field season. Paired Pearson correlation coefficient (PCC) analysis was performed using the amount of m/z 347.19 quantified 72 hours after W+OS treatment of leaves (1706 samples) from MAGICRIL populations grown at the Arizona field station and the levels of different jasmonic acids quantified 1 hour after W+OS treatment of leaves (1816 samples) and Empoasca genus phenotypes (1907 samples). The replicate samples were averaged. A significant correlation of P value <0.05 was shown.

Figure 37 Transcript accumulation of m/z 347.19 biosynthetic gene in response to OS treatment in N. attenuata leaves of different transgenic lines. Transcript accumulation of m/z 347.19 biosynthetic gene in response to OS treatment of leaves in WT and irMYB8 lines after 1 hour and 5 hours, respectively. *P<0.05, **P<0.01, ***P<0.001.

Figure 38 Detailed kinetics of transcript accumulation of the same biosynthetic genes in WT plants in response to OS treatment of leaves. *P<0.05, **P<0.01, ***P<0.001.

FIG39 Transcript abundance of m/z 347.19 biosynthetic genes in EV and irMYC2 lines 7 hours after OS treatment, quantified by RNAseq and expressed as fragments per kilobase of transcription per million sequencing reads (FPKM). Asterisks indicate significant differences compared to untreated control plants (two-way ANOVA followed by post hoc multiple comparisons, *P<0.05, **P<0.01, ***P<0.001; n=3 plants per treatment/genotype). N.S., not significant.

Figure 40 Silencing efficiency of NaAT1, NaPPO1, NaPPO2 and NaBBL2 in VIGS plants. Relative mRNA abundance of NaAT1, NaPPO1, NaPPO2 and NaBBL2 in the rosette stage of Nicotiana attenuata VIGS plants, respectively. Asterisks indicate significant differences between W+OS treatment and untreated controls (C) (Student t-test for paired differences, *P<0.05, **P<0.01, and ***P<0.001; n=8 to 11 plants per treatment/genotype). N.S., not significant.

Figure 41 Accumulation of phenolamides in leaves of N. attenuata in response to OS treatment in different virus-induced gene silenced (VIGS) plants. Silencing NaPPO1 and NaPPO2 did not significantly alter herbivory-induced phenolamide accumulation compared to EV. CoP, N-coumaroylputrescine; FP, N-feruloylputrescine; CS, N-caffeoylspermidine; FS, N-feruloylspermidine; CoCS, N',N"-coumaroyl, caffeoylspermidine; CFS, N',N"-caffeoyl, feruloylspermidine; DCS, N',N"-dicaffeoylspermidine; DFS, N',N"-dicaffeoylspermidine. Different letters indicate significant differences (two-way ANOVA followed by Tukey's post hoc multiple comparisons; n = 4 to 8 plants per treatment/genotype).

Figure 42 Association between herbivory-induced m/z 347.19 and volatile organic compounds (VOCs) in a MAGIC RIL population. The Pearson correlation coefficient (PCC) between the accumulation of 72hm/z 347.19 after W+OS treatment of leaves and the release of VOCs during 24 hours after W+OS treatment was calculated for 650 MAGIC RILs grown in a greenhouse. The dotted line indicates a significant PCC correlation threshold with a P value <0.05.

FIG. 43 SDS-PAGE (sodium eicosate-polyacrylamide gel electrophoresis) analysis of recombinant NaPPO1 protein during the purification process. SDS-PAGE separation of NaPPO1. Lanes: 1, uninduced control sample (1 mL); 2, induced overnight fraction sample (1 mL); 3, insoluble fraction sample (5 mL); 4, soluble fraction sample; 5-8, samples collected after gradient washing with 40 mM (10 mL), 80 mM (1 mL), 250 mM (1 mL), and 250 mM (1 mL) imidazole buffer. Arrows indicate purified NaPPO1 protein.

FIG. 44 SDS-PAGE analysis of recombinant NaPPO2 protein during the purification process. SDS-PAGE separation of NaPPO2. Lanes: 1, uninduced control sample (1 mL); 2, induced overnight fraction sample (1 mL); 3, insoluble fraction sample (5 mL); 4, soluble fraction sample; 5-8, samples collected after gradient washing with 40mM (10 mL), 80mM (1 mL), 250mM (1 mL), and 250mM (1 mL) imidazole buffer. Arrows indicate purified NaPPO2 protein.

Figure 45 SDS-PAGE analysis of recombinant NaBBL2. Lanes: 1, uninduced control sample (1 mL); 2, induced overnight fraction sample (1 mL); 3, insoluble fraction sample (5 mL); 4, soluble fraction sample; 5-8, samples collected after gradient washing with 40mM (10 mL), 80mM (1 mL), 250mM (1 mL) and 250mM (1 mL) imidazole buffer. Arrows indicate purified NaBBL2 protein.

FIG46 Comparison of the MS/MS spectra of m/z 347.19 prepared in vitro and m/z 347.19 triggered by Empoasca in leaves. The MS/MS spectrum of m/z 347.19 prepared in vitro is the top spectrum (black) and is compared with the MS/MS spectrum of m/z 347.19 triggered by OS in N. acuminate leaves (red: bottom spectrum). The MS/MS spectra were acquired by MS/MS data acquisition targeting m/z 347.19 using an 8Da mass isolation window and a collision energy of 25eV. The corresponding extracted ion chromatogram (EIC) of m/z 347.19 (25 min LC method) is on the left.

Figure 47 Comparison of the MS/MS spectra of the unknown m/z 250.132 prepared in vitro and the N-caffeoylputrescine standard. The MS/MS spectrum of the unknown m/z 250.132 prepared in vitro is shown (top spectrum). Compared with the MS/MS spectrum of the N-caffeoylputrescine standard (bottom spectrum), the unknown doubly charged m/z 250.132 (C ₂₆ H ₃₆ N ₄ O ₆ ²⁺ ) is annotated as an N-caffeoylputrescine dimer.

Figure 48 In vitro production of m/z 347.19 does not require NaBBL2. EIC of m/z 347.19 in E. coli expressing NaPPO1 and NaPPO2 and NaBBL2 in the presence of CP and (Z)-3-hexenal in the culture medium. Arrows indicate the two isomers of m/z 347.19 produced in the in vitro enzyme assay.

FIG. 49 Michaelis-Menten kinetics of PPO1 and BBL2 at different concentrations of caffeoylputrescine and (Z)-3-hexenal. Steady-state kinetics of the conversion of caffeoylputrescine or (Z)-3-hexenal to CPH catalyzed by PPO1 purified from E. coli Bl21 expressing PPO1. Error bars represent the standard error of the mean (SEM) of three independent measurements of the reaction rate. Kinetic constants, apparent Km and Vmax (Table 2) and their standard errors were inferred using nonlinear Michaelis-Menten curve fitting in GraphPad Prism (v.9.0d). The substrate concentration of (Z)-3-hexenal was 1 mM.

Figure 50 Michaelis-Menten kinetics of PPO2 and BBL2 at different concentrations of caffeoyl putrescine and (Z)-3-hexenal. Steady-state kinetics of the conversion of caffeoyl putrescine or (Z)-3-hexenal to CPH catalyzed by PPO2 purified from E. coli Bl21 expressing PPO2. Error bars represent the standard error of the mean (SEM) of three independent measurements of the reaction rate. Kinetic constants, apparent Km and Vmax (Table 2) and their standard errors were inferred using nonlinear Michaelis-Menten curve fitting in GraphPad Prism (v.9.0d). The substrate concentration of (Z)-3-hexenal was 1 mM.

Figure 51 Michaelis-Menten kinetics of PPO1 and BBL2 at different concentrations of caffeoylputrescine and (Z)-3-hexenal. Steady-state kinetics of the conversion of caffeoylputrescine or (Z)-3-hexenal to CPH catalyzed by PPO1 purified from E. coli Bl21 expressing PPO1. Error bars represent the standard error of the mean (SEM) of three independent measurements of the reaction rate. Kinetic constants, apparent Km and Vmax (Table 2) and their standard errors were inferred using nonlinear Michaelis-Menten curve fitting in GraphPad Prism (v.9.0d). The substrate concentration of caffeoylputrescine was 80 μM.

Figure 52 shows the Michaelis-Menten kinetics of PPO2 and BBL2 at different concentrations of caffeoyl putrescine and (Z)-3-hexenal. Steady-state kinetics of the conversion of caffeoyl putrescine or (Z)-3-hexenal to CPH catalyzed by PPO2 purified from E. coli Bl21 expressing PPO2. Error bars represent the standard error of the mean (SEM) of three independent measurements of the reaction rate. Kinetic constants, apparent Km and Vmax (Table 2) and their standard errors were inferred using nonlinear Michaelis-Menten curve fitting in GraphPad Prism (v.9.0d). The substrate concentration of (Z)-3-hexenal was 1 mM. The substrate concentration of caffeoyl putrescine was 80 μM.

FIG53 Substrate specificity of NaPPO1 and NaPPO2 enzymes. Overlay of total ion chromatograms (TIC) of enzyme assays of authentic chlorogenic acid (CGA standard), N-coumaroyl putrescine (CoP standard), (Z)-3-hexenal or (E)-2-hexenal with NaPPO1/NaPPO2 purified from E. coli. Compounds 1 and 2 represent CGA and CoP; 3 and 4 represent contaminating peaks from (Z)-3-hexenal and CoP standards, respectively.

Figure 54 ¹ H NMR spectrum (nuclear magnetic resonance hydrogen spectrum) and chemical shift of m/z 347.19. ¹ H-NMR spectrum of the enzyme product at m/z 347.19 in 700 μL MeOH-d ₃ , doped with 0.1% formic acid, pre-saturated with water, 32 scans (left), ¹ H and ¹³ C chemical shifts of m/z 347.19 (right).

Figure 55 Half-life estimation of m/z 347.19. NMR spectra of purified m/z 347.19 acquired at different times are shown. The half-life of m/z 347.19 was estimated from the signal at position 2 at 6 ppm to be about 22 hours at room temperature in acidified methanol- _d3 (0.1% formic acid) in the dark.

FIG. 56 Accumulation of m/z 347.19, CP, and biosynthetic enzymes in different plant species. MeJA-induced accumulation of m/z 347.19 in closely related tobacco species. Asterisks indicate significant differences between lanolin wounding (W) or MeJA treatment and lanolin control (C) (Student t-test for paired differences, *P<0.05, **P<0.01, and ***P<0.001; n=6 plants per treatment/genotype). At, N. attenuata; Ac, N. acuminata; Li, N. linearis; Mi, N. miersii; Ob, N. obtusifolia; Pa, N. pauciflora; Sp, N. spegazzinii;

Figure 57 Phylogenetic tree of sequenced plant genomes created using the taxonomic universal tree from NCBI. Heatmap markers scaled by percentage of amino acid identity of top BLAST (blastp) hits querying the respective plant proteomes for NaAT1, NaPPO1, NaPPO2, and NaBBL2 homologs (left), and the heatmap on the right represents the presence and absence (intensity threshold>500) of MeJA-induced caffeoylputrescine and m/z 347.19 in different species.

Figure 58 Localization of NaPPO1 and NaPPO2 in Nicotiana attenuata. (A) The 'thylakoid transfer domain' of the plastidial PPO was identified in the amino acid sequences of NaPPO1 and NaPPO2 (Joy et al., Plant Physiol 107, 1083-1089 (1995)) by N-terminal amino acid sequence alignment (the thylakoid transfer domain is marked with a red rectangle). The N-terminal amino acid sequences of NaPPO1 and NaPPO2 were aligned with those of plastid polyphenol oxidase (PPO) precursors (antirrhinol synthase (AmAS1; AB044884.1) from Antirrhinum majus, PPO (BAA08234.1) from Vitis vinifer, PPO (AAA85122.1) from Solanum tuberosum, and PPO (PAP1; BAA08234.1) from Phytolacca americana.

Figure 59 Subcellular localization of 35S::NaPPO1-GFP or 35S:μ:NaPPO2-GFP, GFP fused to the C-terminus of the open reading frames of NaPPO1 and NaPPO2, transiently expressed in leaves of Nicotiana attenuata. Control: uninfected plants, scale bar: 20 μm.

Figure 60 proposes a mechanism for non-host resistance against Empoa spp. leafhoppers in Nicotiana attenuata. A model for the in vivo enzyme reaction that produces m/z 347.19 is proposed. NaPPO1 and NaPPO2 are located in the plastid and are therefore physically separated from caffeoyl putrescine (CP) that may be produced in the cytoplasm. After Empoa spp. damage, the Empoa spp.-induced jasmonic acid signaling pathway is activated, in which JA-Ile or other JA derivatives recruit NaJAZi and release their interaction with NaMYC2 and NaMYB8 to activate CP production. In the reaction shown in Figure 17, NaPPO1 or NaPPO2 condenses CP with (Z)-3-hexenal transported from the plastid with the help of NaBBL2, and m/z 347.19 accumulates in the central vacuole because it is relatively stable under weak acetic acid conditions that usually characterize the vacuole. Alternatively, chloroplasts and the GLVs and PPOs they carry may be engulfed in response to Empoasca attack and transported to the central vacuole to produce m/z 347.19, where the CP resides.

FIG61 Amino acid sequence alignment of NaBBL2 with other characterized berberine bridge enzymes. The red boxes highlight the residues of the berberine bridge enzyme involved in the divalent attachment of FAD (His and Cys). The Cys residue was found to be mutated in the NaBBL2 enzyme (C ₁₇₃ to G ₁₇₃ mutation). The alignment was performed using the MUSCLE algorithm. Accession numbers: Nicotiana acuminate BBL2 (XM_019396928), E. californica BBE (CAK49173), P. somniferum BBE (XP_001394039), C. sativa THCA synthase (AMQ48632), C. sativa CBDA synthase (A6P6V9), C. japonica BBL (BAJ40864).

Detailed ways

Although the invention will be described with respect to specific embodiments, this description should not be construed in a limiting sense.

Before describing exemplary embodiments of the present invention in detail, definitions important for understanding the present invention are given.

As used in this specification and in the appended claims, the singular forms "a", "an" and "an" include the corresponding plural forms as well, unless the context clearly indicates otherwise.

In the context of the present invention, the terms "about" and "approximately" represent an interval of accuracy that a person skilled in the art will understand still ensures the technical effect of the feature in question. The term generally indicates a deviation of ±20% from the indicated numerical value, preferably ±15%, more preferably ±10%, and even more preferably ±5%.

It should be understood that the term "comprising" is not limiting. For the purposes of the present invention, the term "consisting of" or "consisting essentially of" are considered preferred embodiments of the term "comprising..." If a group is defined hereinafter as comprising at least a certain number of embodiments, this is also intended to cover a group that preferably consists only of these embodiments.

In addition, the terms "(i)", "(ii)", "(iii)" or "(a)", "(b)", "(c)", "(d)" or "first", "second", "third", etc., in the specification or in the claims are used to distinguish between similar elements and not necessarily to describe a sequential or chronological order. It should be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in an order different from that described or illustrated herein. If the terms refer to steps of a method, procedure or use, there is no time or time interval continuity between the steps, that is, the steps can be performed simultaneously or there may be time intervals of seconds, minutes, hours, days, weeks, etc. between the steps, unless otherwise indicated.

It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present invention, which will only be limited by the appended claims. Unless otherwise defined, all technical and scientific terms used in this article have the same meanings as commonly understood by those of ordinary skill in the art.

As mentioned above, the present invention relates in one aspect to compounds of formula (I)

in:

R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy;

R ⁵ , R ⁶ and R ⁷ are each independently H or (C ₁ -C ₆ )-alkyl;

X is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group;

Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , _NH- (CH2) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester;

Z is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group,

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In a further preferred embodiment, the present invention relates to compounds of general formula (I), wherein:

Two of R ¹ , R ² , R ³ and R ⁴ are OH; and the remaining groups of R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy;

R ⁵ , R ⁶ and R ⁷ are each H;

X is a straight chain (C ₂ -C ₈ )-alkenyl group;

Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , _NH- (CH2) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester;

Z is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group,

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In a further preferred embodiment, the present invention relates to compounds of general formula (I), wherein:

^R1 and ^R4 are H;

^R2 and ^R3 are OH,

R ⁵ , R ⁶ and R ⁷ are each H;

X is -CH=CH-;

Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , _NH- (CH2) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester;

Z is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group,

or its enantiomers, diastereomers, stereoisomers or salts

In one embodiment, X is straight-chain (C ₂ -C ₈ )-alkenyl.

In one embodiment, X is straight-chain (C ₂ -C ₆ )-alkenyl.

In one embodiment, X is straight-chain (C ₂ -C ₄ )-alkenyl.

In one embodiment, X is -CH=CH-.

In one embodiment, Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 3.

In one embodiment, Y is selected from NH-( _CH2 ) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester.

In one embodiment, Y is selected from -( _CH2 ) _m - _NH2 , -( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , -( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each 1.

In one embodiment, Y is selected from NH-( _CH2 ) _m - _NH2 , NH-( _CH2 ) _n -NH-( _CH2 ) _o - _NH2 , and NH-( _CH2 ) _p -NH-( _CH2 ) _q -NH-( _CH2 ) _r - _NH2 , wherein m, n, o, p, q and r are each 1, or tyramide.

In one embodiment, compound (I) has the general formula (Ia)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ , R ⁷ , X and Z are as defined above for formula (I),

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In one embodiment, at least one of R ¹ , R ² , R ³ and R ⁴ in formula (Ia) is OH; and the remaining groups in R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy. Preferably, in formula (Ia), R ¹ and R ⁴ are H, and R ² and R ³ are OH.

In one embodiment, R ⁵ and R ⁶ in formula (Ia) are both H.

In one embodiment, R ⁵ , R ⁶ and R ⁷ in formula (Ia) are each H.

In one embodiment, X in formula (Ia) is straight-chain (C ₂ -C ₆ )-alkenyl.

In one embodiment, X in formula (Ia) is -CH=CH-.

In one embodiment, Z in formula (Ia) is linear or branched (C ₁ -C ₈ )-alkyl.

In one embodiment, compound (I) has the general formula (Ib)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , X and Z are as defined above for formula (I),

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In one embodiment, at least one of R ¹ , R ² , R ³ and R ⁴ in formula (Ib) is OH; and the remaining groups in R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy. Preferably, in formula (Ib), R ¹ and R ⁴ are H, and R ² and R ³ are OH.

In one embodiment, R ⁵ and R ⁶ in formula (Ib) are both H.

In one embodiment, R ⁵ , R ⁶ and R ⁷ in formula (Ib) are each H.

In one embodiment, X in formula (Ib) is straight-chain (C ₂ -C ₆ )-alkenyl.

In one embodiment, X in formula (Ib) is -CH=CH-.

In one embodiment, Z in formula (Ib) is straight-chain or branched (C ₁ -C ₈ )-alkyl.

In one embodiment, compound (I) has the general formula (Ic)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ , X and Z are as defined above for formula (I),

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In one embodiment, at least one of R ¹ , R ² , R ³ and R ⁴ in formula (Ic) is OH; and the remaining groups in R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy. Preferably, in formula (Ic), R ¹ and R ⁴ are H and R ² and R ³ are OH.

In one embodiment, R ⁵ and R ⁶ in formula (Ic) are both H.

In one embodiment, R ⁵ , R ⁶ and R ⁷ in formula (Ic) are each H.

In one embodiment, X in formula (Ic) is straight-chain (C ₂ -C ₆ )-alkenyl.

In one embodiment, X in formula (Ic) is -CH=CH-.

In one embodiment, Z in formula (Ic) is linear or branched (C ₁ -C ₈ )-alkyl.

In one embodiment, compound (I) has the general formula (Id)

wherein R ¹ , R ² , R ³ , R ⁴ , R ⁵ , R ⁶ and R ⁷ and Z are as defined above for formula (I),

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In one embodiment, at least one of R ¹ , R ² , R ³ and R ⁴ in formula (Id) is OH; and the remaining groups in R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy. Preferably, in formula (Id), R ¹ and R ⁴ are H, and R ² and R ³ are OH.

In one embodiment, R ⁵ and R ⁶ in formula (Id) are both H.

In one embodiment, R ⁵ , R ⁶ and R ⁷ in formula (Id) are each H.

In one embodiment, X in formula (Id) is straight-chain (C ₂ -C ₆ )-alkenyl.

In one embodiment, X in Formula (Id) is -CH=CH-.

In one embodiment, Z in formula (Id) is linear or branched (C ₁ -C ₈ )-alkyl.

In one embodiment, at least one of R ¹ , R ² , R ³ and R ⁴ in formula (I), (Ia), (Ib), (Ic) or (Id) is OH; and the remaining groups in R ¹ , R ² , R ³ and R ⁴ are each independently H, OH or (C ₁ -C ₆ )-alkoxy.

In one embodiment, at least two of R ¹ , R ² , R ³ and R ⁴ in formula (I), (Ia), (Ib), (Ic) or (Id) are OH; and the remaining groups of R ¹ , R ² , R ³ and R ⁴ are each independently H, OH, (C ₁ -C ₆ )-alkyl or (C ₁ -C ₆ )-alkoxy.

In one embodiment, at least two of R ¹ , R ² , R ³ and R ⁴ in formula (I), (Ia), (Ib), (Ic) or (Id) are OH; and the remaining groups of R ¹ , R ² , R ³ and R ⁴ are each independently H, OH or (C ₁ -C ₆ )-alkoxy.

In one embodiment, two of R ¹ , R ² , R ³ and R ⁴ in formula (I), (Ia), (Ib), (Ic) or (Id) are OH; and the remaining two of R ¹ , R ² , R ³ and R ⁴ are each H. Preferably, R ¹ and R ⁴ are H, and R ² and R ³ are OH.

In a further particularly preferred embodiment, the invention relates to compounds of formula (II)

in:

R ⁵ , R ⁶ and R ⁷ are each independently H or (C ₁ -C ₆ )-alkyl;

and Z is a linear or branched (C ₁ -C ₈ )-alkyl group or a linear or branched (C ₂ -C ₈ )-alkenyl group.

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In one embodiment, X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H. In one embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , R3 and ^R4 are H. In one embodiment, X is -CH=CH-, ^R3 is OH, R1, ^R2 and ^R4 are H. In one embodiment, X is -CH=CH-, R3 is OH, ^R1 , ^R2 and R4 are H. In one embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H.

In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 and ^R3 is OH.

In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, and ^R2 and ^R3 are OH.

In one embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 .

In one embodiment, Y is -(CH ₂ ) ₄ NH ₂ . In one embodiment, Y is -(CH ₂ ) ₃ -NH-(CH ₂ ) ₄ -NH ₂ . In one embodiment, Y is -(CH ₂ ) ₃ -NH-(CH ₂ ) ₄ -NH ₂ . In _one embodiment, Y is -(CH ₂ ) ₃ -NH-( _{CH 2 ) 4} -NH ₂ .

In one embodiment, R ⁵ and R ⁶ are H and Z is (C ₁ -C ₆ )-alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is C ₂ -alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is C ₃ -alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is C ₄ -alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is C ₅ -alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is C ₆ -alkyl. In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH(CH ₃ ). In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH ₂ CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH(CH ₃ )CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH(CH ₃ ). In one embodiment, R ⁵ and R ⁶ are H and Z is CH(CH ₂ CH ₃ ). In one embodiment, R ⁵ and R ⁶ are H and Z is C(CH ₃ ) ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH ₂ CH ₂ CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH(CH ₃ )CH ₂ CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH(CH ₃ )CH 2 . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH(CH ₃ )CH _{2 .} In one embodiment, R ⁵ and R ⁶ are H and Z is C(CH ₃ ) ₂ CH ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ C(CH ₃ ) ₂ . In one embodiment, R ⁵ and R ⁶ are H and Z is CH ₂ CH(CH ₂ CH ₃ ). In one embodiment, R ⁵ and R ⁶ are H and Z is CH(CH ₂ CH ₃ )CH ₂ . Preferably, in the above embodiment, wherein R ⁵ and R ⁶ are H, R ⁷ is also H.

In another embodiment, X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, and Y is -( _CH2 ) _4NH2 . In one embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, and Y is -( _CH2 ) _{4NH2 .} In one embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, and Y is -( _CH2 ) _4NH2 . In one embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, and _{Y is} -( _CH2 ) _{4NH2 .} In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, and Y is -( _CH2 ) _4NH2 . In one embodiment, X is -CH=CH-, R1 and ^R4 are H, ^R2 and ^R3 are OH, and Y is -( _CH2 ) _4NH2 . In one embodiment, X is -CH= ^CH- , ^R1 and ^R4 _are H, ^{R2 and} _R3 are OH _, and Y is -( _CH2 ) _{4NH2 .}

In another embodiment, X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 . In one embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, and Y is -( _CH2 ) _{3 -} NH-( _CH2 ) ₄ - _NH2 .

In another embodiment, X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 . In one embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 . In one embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R3 and ^R4 are H, and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 . In one embodiment, X is -CH=CH-, R ⁴ is OH, R ¹ , R ² and R ³ are H, and Y is -(CH ₂ ) ₃ -NH-(CH ₂ ) ₄ -NH-(CH ₂ ) ₃ -NH _2. In one embodiment, X is -CH=CH-, R ¹ and R ⁴ are H, R ² is -OCH ₃ , R ³ is OH, and Y is -(CH ₂ ) ₃ -NH-(CH ₂ ) ₄ -NH-(CH ₂ ) ₃ -NH _2. In one embodiment, X is -CH=CH-, R ¹ and R ⁴ are H, R ² and R ³ are OH, and Y is -(CH ₂ ) ₃ -NH-(CH ₂ ) ₄ -NH-(CH ₂ ) ₃ -NH ₂ . In one embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 , and Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 .

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is _-CH =CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and _Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is _-CH =CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and _Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is _-CH =CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and _Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 _is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl.

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, ^X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl.

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R2 is OH, R1, ^R3 and ^R4 are H, ^Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl. In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is ( _C1 - _C6 )-alkyl.

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH ( CH3} ), CH( _{CH2CH3 )} and C( _{CH3 ) 2} . In another particularly preferred embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH( _CH2CH3 ) and _C ( _{CH3 ) 2} . In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH( _CH2CH3 ) and _C ( _{CH3 ) 2} . In another particularly preferred embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH( _CH2CH3 ) and _C ( _{CH3 ) 2} . In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are _H , and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH ( CH3} ), CH( _CH2CH3 ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) _{4NH2 ,} ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH( _CH2CH3 ) and _C ( _{CH3 ) 2} . In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 _is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) _4NH2 , ^R5 , ^R6 and ^R7 are _H , and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH ( CH3} ), CH( _CH2CH3 ) and C( _CH3 ) _{2 .}

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ) _, CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), _{CH (CH2CH3 )} and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), _{CH (CH2CH3 )} and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), _{CH (CH2CH3 )} and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 , ^R3 is OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _{CH2 , CH2CH} ( _CH3 ), CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , R5, ^R6 and ^R7 are H, and ^Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), _{CH (CH2CH3 )} and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _{CH2 , CH2CH} ( _CH3 ), CH _{( CH2CH3} ) and C( _CH3 ) _{2 .}

In another particularly preferred embodiment, the compound has the general formula (I) or an enantiomer, diastereomer, stereoisomer or salt thereof, wherein X is -CH=CH-, ^R1 is OH, ^R2 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _{CH2CH2CH2 ,} CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R2 is OH, ^R1 , ^R3 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R3 is OH, ^R1 , ^R2 and ^R4 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R4 is OH, ^R1 , ^R2 and ^R3 are H, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 is _-OCH3 and ^R3 is OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), CH( _{CH2CH3 )} and C( _{CH3 ) 2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 and ^R4 are H, ^R2 and ^R3 are OH, Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _{CH2CH (CH3 )} , CH _{( CH2CH3} ) and C( _CH3 ) _{2 .} In another particularly preferred embodiment, X is -CH=CH-, ^R1 is H, ^R3 is OH, ^R2 and ^R4 are _-OCH3 , Y is -( _CH2 ) ₃ -NH-( _CH2 ) ₄ -NH-( _CH2 ) ₃ - _NH2 , ^R5 , ^R6 and ^R7 are H, and Z is _C3 -alkyl, wherein the _C3 -alkyl is selected from _CH2CH2CH2 , CH( _CH3 ) _CH2 , _CH2CH ( _CH3 ), CH( _{CH2CH3 )} and C( _{CH3 ) 2 .}

In the most preferred embodiment, the present invention relates to compounds having the following formula (III):

or an enantiomer, diastereomer, stereoisomer or salt thereof.

In certain specific embodiments of the present invention, in the compounds as defined above, the alkyl and alkenyl chains, i.e., the straight or branched (C ₁ -C ₈ )-alkyl of X, the straight or branched (C ₂ -C ₈ )-alkenyl of X, the straight or branched (C ₁ -C ₈ )-alkyl of Z, and the straight or branched (C ₂ -C ₈ )-alkenyl of Z, may be optionally substituted with suitable substituents. Suitable substituents include, but are not limited to, OH, F, Cl, Br or CN.

In another aspect, the present invention relates to a method for preparing a compound of the present invention as defined above, for example a compound of formula (I), preferably a compound of formula (II) and more preferably a compound of formula (III). As used herein, the term "preparing a compound of the present invention" means generating a compound of the present invention in any suitable amount and with any suitable purity by any suitable procedure. For example, such a procedure may be based on components present in an in vitro environment, or based on components present in an in vivo environment.

The preparation is preferably an enzymatic preparation method. The term "enzymatic preparation method" used herein refers to one or more reactants or precursors of the compound according to the present invention being modified, converted, combined or otherwise changed into one or more products, and finally with the help of one or more enzymes and optional other factors (such as energy carriers, cofactors, reducing equivalents, ions, etc.) and under specific conditions (such as specific pH values, specific ion concentrations) in a specific environment (such as aqueous environment, etc.) to produce the compound according to the present invention. The enzymatic preparation method may involve 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more enzymatic conversions, conversions or modification steps. The method may further include non-enzymatic steps, such as allowing the correct positioning of the compound, allowing the interaction of two or more polypeptides, or allowing polypeptides or activities to be recruited to complexes or reaction sites. The order of these steps may vary. Generally, the order of the conversion, conversion or modification steps follows a specific chemical logic, starting with a simple reactant and ending with a complex product. In certain embodiments, the method may utilize a precursor or reactant, which may be the product of another enzymatic preparation method or enzymatic activity, or may be the product of non-enzymatic synthesis or provided from scratch. The presence, identity, and amount of such a precursor may have an impact on the total number of steps of the enzymatic preparation method. For example, a precursor or reactant that only requires enzymatic steps 4, 5, 6, etc. to produce a compound according to the present invention may be provided, because the precursor or reactant is the same or similar to the product of enzymatic steps 1 to 3; or a precursor that requires enzymatic steps 8, 9, and 10 to produce a compound according to the present invention may be provided, because the precursor or reactant is the same or similar to the product of enzymatic steps 1 to 7. In addition, different precursors or reactants for different enzymatic steps may be provided simultaneously or sequentially during the preparation method, for example, 2, 3, 4, 5 or more different precursors or reactants may be provided to produce a compound according to the present invention. In certain embodiments, the presence of enzyme activity may depend on the presence of precursors or reactants in the environment in which the preparation method is performed.

In a specific embodiment, a precursor, reactant or substrate may be provided from an external site to a reaction site (e.g. a living cell or organism, preferably a plant as defined herein). For example, such a precursor, reactant or substrate may be provided in a preform or proform that requires one or more metabolic steps to produce a form that can be used in the method of the invention. These steps may preferably be performed by standard transformation procedures of living cells, particularly plant cells or plants.

In another specific embodiment, the precursor, reactant or substrate can be provided to a living cell, for example a plant cell as defined herein, in the form of a lipophilic ester (e.g., as a methyl ester). Such an ester can be removed by a suitable enzyme, such as an esterase. Esterases are typically present in living cells, for example, in higher plant cells.

For example, precursors, reactants or substrates can be provided to living cells, such as plants as defined herein, by spraying techniques (more information can be obtained from, for example, Daware and Lokhande, International Journal of Innovations in Engineering and Science, 4, 8 (2019). In other embodiments, they can be mixed with surfactants or spreading agents, such as those defined below.

In a preferred embodiment, the enzymatic preparation method uses at least BBL2 (berberine bridge enzyme 2) polypeptide. The term "polypeptide" used herein refers to a continuous and unbranched peptide chain of a specific length. In contrast, "peptide" refers to any type of amino acid sequence or its functional derivative containing more than 2 amino acids. In addition, peptides can be combined with other chemical groups or functional groups. For example, a polypeptide can have a length of more than 20 to 50 amino acids. The term "protein" used in this article refers to the arrangement of one or more polypeptides. Therefore, a protein can contain one polypeptide or consist of one polypeptide, and is therefore synonymous with a polypeptide. In other embodiments, a protein may contain two or more polypeptides, which may be organized in units or subunits of a higher-order structure in the form of a protein. The term "activity" used in this article refers to a polypeptide, preferably an enzyme, that realizes a certain biological function, preferably an enzyme function. In certain embodiments, the activity may be the function of an enzyme that converts a reactant into a product.

"BBL2 (berberine bridge enzyme 2) polypeptide" relates to a BBL2 polypeptide or realizes a biological function of a BBL2 polypeptide. Berberine bridge enzymes are described as FAD-linked oxidases that have a special C-terminal structural element adjacent to the substrate binding region. Generally, the FAD binding module is formed by the N-terminal and C-terminal parts of the protein. There is a substrate binding module that cooperates with the isoalloxazine ring of FAD to provide an environment for efficient substrate binding and oxidation.

Without wishing to be bound by theory, it is assumed that BBL2 plays a role as a non-catalytic protein in the enzymatic preparation method described herein in the context of the present invention. This function may be similar to the non-catalytic chalcone isomerase (CHIL) in flavonoid metabolism. It is further assumed that BBL2 interacts with the reactive PPO-activated N-caffeoyl putrescine (CP) or its derivatives as a reaction intermediate to stabilize it in the cellular environment and avoid conversion into byproducts, which cannot react with (Z)-3-hexenal to form compounds according to the present invention, such as CPH (caffeoyl putrescine-3-hexenal compounds). Therefore, it is believed that BBL2 can conduct substrates more effectively between active enzymes, such as in the form of metabolites. Metabolites generally promote the conduction of unstable and toxic intermediates, and increase local substrate concentrations and prevent undesirable metabolic crosstalk. Such dynamic assembly and disassembly allow rapid reorganization of metabolic properties in response to environmental challenges, and are considered to involve scaffold proteins. Thus, the scaffolding function for BBL2 may allow for the dynamic assembly and disassembly of metabolites conducting reaction intermediates and maximize the catalytic efficiency toward the preparation of compounds according to the invention (eg, CPH).

Preferably, BBL2 is (a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 1; (b) encoded by a polynucleotide that is a variant of SEQ ID NO: 1; (c) encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 1; (d) encoded by a polynucleotide that is a species homolog of SEQ ID NO: 1; (e) encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% similarity to a polynucleotide as defined in any one of (a) to (d); (f) encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) represented by a polypeptide having SEQ ID NO: 2; (h) represented by a polypeptide fragment of SEQ ID NO: 2 having the function of BBL2 (berberine bridge enzyme 2); (i) represented by a polypeptide domain of SEQ ID NO: 2 having the function of BBL2 (berberine bridge enzyme 2); (j) represented by a polypeptide domain comprising the same polypeptide as SEQ ID NO: 2; NO:2 has an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% similarity and a polypeptide representative having the function of BBL2 (berberine bridge enzyme 2) or (k) a polypeptide represented by any one of the polynucleotides specified in (a) to (f).

The term "polynucleotide" as used herein refers to a nucleic acid or nucleic acid molecule known to those skilled in the art, such as DNA, RNA, single-stranded DNA, cDNA or derivatives thereof. The nucleic acid may further be linear or circular. Preferably, the term refers to a DNA molecule.

As used herein, the term "allelic variant" refers to variant polynucleotides that exist in two or more different allelic forms at a particular locus.

As used herein, a "fragment", "variant" or "homologue" of a polynucleotide refers to a polynucleotide comprising or consisting of a nucleotide sequence having at least 75%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or nucleotide sequence identity to the nucleotide sequence of a reference polynucleotide (e.g., a reference polynucleotide of SEQ ID NO: 1). In certain embodiments, fragments, variants and homologues may encode a polypeptide capable of performing one, multiple or all of the functions performed by a corresponding reference polypeptide (e.g., a reference polypeptide of SEQ ID NO: 2).

Similarly, a "fragment", "variant" or "homologue" of a polypeptide refers to a polypeptide comprising or consisting of an amino acid sequence having at least 75%, preferably 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more amino acid sequence identity to the amino acid sequence of a reference polypeptide (e.g., a reference polypeptide of SEQ ID NO: 2). In certain embodiments, fragments, variants and homologues of a reference polypeptide may be capable of performing one, multiple or all of the functions performed by a reference polypeptide (e.g., a reference polypeptide of SEQ ID NO: 2).

The term "species homolog" refers to homologous (i.e., highly similar) polynucleotide or amino acid sequences from species different from a reference sequence (e.g., SEQ ID NO: 1 or 2). Such a high degree of similarity is generally a strong evidence that the two sequences are related by evolutionary changes from a common ancestral sequence. Species homologs are generally functional homologs, i.e., not only are the nucleotide or amino acid sequences of the homologs similar to the reference sequence, but the function (e.g., enzymatic activity) of the corresponding polypeptide is also similar or identical to that of the reference polypeptide.

The term "stringent conditions" or "stringent hybridization conditions" as used herein refers to incubation overnight at 42° C. in a solution comprising 50% formamide, 5×SSC (750 mM sodium chloride, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 g/m) denatured sheared salmon sperm DNA, followed by washing the filter with 0.1×SSC at about 65° C. Stringency changes in hybridization and signal detection are primarily achieved by manipulating formamide concentration (lower formamide percentages result in lower stringency), salt conditions, or temperature. For example, lower stringency conditions include overnight incubation at 37°C in a solution containing 6x SSPE (20x SSPE = 3M NaCl; 0.2M NaH ₂ PO ₄ ; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA; followed by washing with 1x SSPE, 0.1% SDS at 50°C. In addition, to achieve even lower stringency, washing after stringent hybridization can be performed at higher salt concentrations (e.g., with 5x SSC). Further variations of the above conditions can be achieved by including and/or replacing alternative blocking reagents for suppressing hybridization experimental background. Typical blocking reagents to be used in the context of the present invention include Denhardt's reagent, BLOTTO, heparin or denatured salmon sperm DNA. The inclusion of a specific blocking reagent may require modification of the above hybridization conditions due to compatibility issues.

A nucleic acid comprising a nucleotide sequence having, for example, at least 95% "identity" with a reference nucleotide sequence of the present invention means that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the nucleotide sequence may include up to 5 point mutations in every 100 nucleotides of the reference nucleotide sequence encoding a polypeptide. In other words, in order to obtain a polynucleotide comprising a nucleotide sequence having at least 95% identity with a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or replaced with another nucleotide, or a number of nucleotides accounting for up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence. The query sequence may be a complete sequence or any fragment as described herein. Whether any particular nucleic acid molecule has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with a nucleotide sequence of the present invention can be routinely determined using known computer programs. Using the FASTDB computer program based on Brutlag et al., 1990, Comp.App.Biosci.6:237-245 algorithm, it is possible to determine the preferred method for determining the best overall match (also referred to as overall sequence alignment) between the query sequence (sequence of the present invention) and the subject sequence. In the nucleotide sequence alignment, the query sequence and the subject sequence are both DNA sequences. RNA sequences can be compared by converting U to T. The result of the overall sequence alignment is represented by percent identity. In the FASTDB alignment of DNA sequences, the preferred parameters used when calculating percent identity are: matrix=unit, k-tuple=4, mismatch penalty=1, connection penalty=30, randomization group length=0, cutoff score=1, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject nucleotide sequence (taking the shorter one). If the subject sequence is shorter than the query sequence due to 5' or 3' deletions (rather than due to internal deletions), the result must be manually corrected. This is because the FASTDB program does not consider the 5' and 3' truncation of the subject sequence when calculating the identity percentage.For the subject sequence truncated at 5' or 3', relative to the query sequence, by calculating the mismatch/misalignment of the subject sequence at 5' and 3' ends in the query sequence, the percentage of the total bases of the query sequence is corrected.Whether Nucleotide matches/aligns is determined by the result of the FASTDB sequence comparison.Then the percentage can be deducted from the identity percentage calculated using the specified parameters of the above-mentioned FASTDB program to obtain the final identity percentage score.The score of this correction is just the score for the purpose of the present invention.For the purpose of manually adjusting the identity percentage score, only calculate the bases that do not match/misalign with the query sequence beyond the 5' and 3' bases of the subject sequence shown by the FASTDB comparison.

A polypeptide comprising an amino acid sequence that is, for example, at least 95% "identical" to a query amino acid sequence of the invention means that the amino acid sequence of the subject polypeptide is identical to the query sequence except that the subject polypeptide sequence may include up to 5 amino acid variations per 100 amino acids in the query amino acid sequence. In other words, up to 5% of the amino acid residues in the subject sequence may be inserted, deleted (indels), or substituted with another amino acid in order to obtain a polypeptide comprising an amino acid sequence that is at least 95% identical to the query amino acid sequence. These variations of the reference sequence may occur at the amino or carboxyl terminal positions of the reference amino acid sequence, or anywhere between those terminal positions, interspersed individually between residues in the reference sequence, or interspersed in one or more contiguous groups within the reference sequence. Whether any particular polypeptide has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity with, for example, the amino acid sequence of the present invention can be routinely determined by using known computer programs. Using the FASTDB computer program based on the algorithm of Brutlag et al., 1990, Comp.App.Biosci.6:237-245, it is possible to determine the preferred method for determining the best overall match (also referred to as overall sequence alignment) between the query sequence (sequence of the present invention) and the subject sequence. In the amino acid sequence alignment, the query sequence and the subject sequence are both amino acid sequences. The result of the overall sequence alignment is given in percent identity. The preferred parameters used in the FASTDB amino acid alignment are: matrix=PAM 0, k-tuple=2, mismatch penalty=1, connection penalty=20, randomization group length=0, cutoff score=1, window size=sequence length, gap penalty=5, gap size penalty=0.05, window size=500 or the length of the subject amino acid sequence (whichever is shorter). If the subject sequence is shorter than the query sequence due to N-terminal or C-terminal deletions (rather than due to internal deletions), the result must be manually corrected. This is because the FASTDB program does not consider the N-terminal and C-terminal truncation of the subject sequence when calculating the overall identity percentage. For the subject sequence truncated at the N-terminal and C-terminal ends, relative to the query sequence, the percentage of the total bases of the query sequence is corrected by calculating the percentage of the residues that are not matched/not aligned with the corresponding subject residues in the query sequence N-terminal and C-terminal. Whether the residues match/align can be determined by the result of the FASTDB sequence alignment. This percentage is then deducted from the identity percentage calculated using the specified parameters of the above-mentioned FASTDB program to derive the final identity percentage score. This final identity percentage score is just the score for the purpose of the present invention. For the purpose of manually adjusting the identity percentage score, only the residues outside the N-terminal and C-terminal of the subject sequence that does not match/align with the query sequence are considered. That is, the query residues that are only located outside the farthest N-terminal and C-terminal residues of the subject sequence.

According to specific embodiments, for the purposes of sequence alignment and comparison, alternative local sequence identity algorithms may be used. For example, the algorithm of Smith and Waterman, 1981, Adv.Appl.Math.2:482, the sequence identity alignment algorithm of Needleman and Wunsch, 1970, J.Mol.Biol.48:443, the search similarity method of Pearson and Lipman, 1988, Proc.Nat.Acad.Sci.U.S.A.85:2444, or computerized implementations of these algorithms, such as the multiple sequence alignment tool Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/), T-coffee/M-coffee (http://tcoffee.crg.cat/), BLAST (https://blast.ncbi.nlm.nih.gov), FASTA (https://www.ebi.ac.uk/Tools/sss/fasta/), etc., preferably using default settings, can be used. Another example of a useful algorithm is PILEUP. PILEUP uses progressive alignments to create multiple sequence alignments from a group of related sequences. It can also draw a clustering tree for creating alignments. PILEUP uses a simplified version of the progressive alignment method of Feng and Doolittle, 1987, J.Mol.Evol.35:351-360. Another example of a useful algorithm is the BLAST algorithm described in Altschul et al., 1990, J.Mol.Biol.215:403-410, or the WU-BLAST-2 program. WU-BLAST-2 uses several search parameters, most of which are set to default values. Another useful algorithm is the gap BLAST using BLOSUM-62 to replace the scoring matrix.

In another embodiment, the enzymatic production method additionally uses a PPO (polyphenol oxidase) activity or polypeptide. Preferably, the enzymatic production method uses a BBL polypeptide in combination with a PPO activity or polypeptide.

"PPO (polyphenol oxidase) activity or polypeptide" relates to a PPO polypeptide or to an enzymatic function or biological function that realizes a PPO polypeptide. The polyphenol oxidase enzymes generally accept monophenols and/or ortho-diphenols as substrates. The enzymes are described as catalyzing the ortho-hydroxylation of monophenol molecules, wherein the benzene ring contains a single hydroxyl substituent to form ortho-diphenols. They further catalyze the oxidation of ortho-diphenols to form ortho-quinones. In addition, PPO was found to catalyze the polymerization of ortho-quinones to form polyphenols.

In a particularly preferred embodiment, the PPO (polyphenol oxidase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 3 or 5; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 3 or 5; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 3 or 5; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 3 or 5; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of (a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) is represented by a polypeptide having SEQ ID NO: 4 or 6; (h) is represented by a polypeptide fragment of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity; (i) is represented by a polypeptide fragment of SEQ ID NO: 4 or 6 having PPO (polyphenol oxidase) activity. NO:4 or 6 polypeptide domain representative; (j) comprises and has the amino acid sequence of SEQ ID NO:4 or 6 and has PPO (polyphenol oxidase) activity polypeptide representative; or (k) is represented by any one of the polypeptides encoded by the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic production method additionally uses AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide. Preferably, the enzymatic production method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide.

"AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide" relates to an AT1 polypeptide or to an enzyme function or biological function that realizes an AT1 polypeptide. The polyamine hydroxycinnamoyltransferase has been described as an enzyme that catalyzes the transfer of acyl groups from, for example, p-coumaroyl-CoA to polyamines such as agmatine or putrescine. It has been shown that it can use feruloyl-CoA, caffeoyl-CoA and sinapoyl-CoA as acyl donors.

In a particularly preferred embodiment, the AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 7; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 7; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 7; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 7; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of (a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) is represented by a polypeptide having SEQ ID NO: 8; (h) is represented by a polypeptide fragment of SEQ ID NO: 8 having AT1 (polyamine hydroxycinnamoyltransferase 1) activity; (i) is represented by a polypeptide fragment of SEQ ID NO: 8 having AT1 (polyamine hydroxycinnamoyltransferase 1) activity. (j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 8 and has AT1 (polyamine hydroxycinnamoyltransferase 1) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic production method additionally uses an ODC (ornithine decarboxylase) activity or polypeptide.Preferably, the enzymatic production method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide.

"ODC (ornithine decarboxylase) activity or polypeptide" relates to an ODC polypeptide or realizes an enzyme function or biological function of an ODC polypeptide. The ornithine decarboxylase has been described as an enzyme that catalyzes the decarboxylation of ornithine to form putrescine. The decarboxylation reaction catalyzed by ornithine decarboxylase is the first and key step in the synthesis of polyamines such as putrescine, spermidine and spermine.

In a particularly preferred embodiment, the ODC (ornithine decarboxylase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 9 or 11; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 9 or 11; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 9 or 11; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 9 or 11; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of (a) to (d); (f) is encoded by a polynucleotide that can hybridize with any one of the polynucleotides specified in (a) to (d) under stringent conditions; (g) is represented by a polypeptide having SEQ ID NO: 10 or 12; (h) is represented by a polypeptide having SEQ ID NO: 10 or 12; NO:10 or 12; (i) represented by a polypeptide domain of SEQ ID NO:10 or 12 having ODC (ornithine decarboxylase) activity; (j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:10 or 12 and having ODC (ornithine decarboxylase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic preparation method additionally uses an HPL (hydroperoxide lyase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide. In other embodiments, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO and ODC and HPL activity or polypeptide; or a BBL polypeptide in combination with a PPO and AT1 and HPL activity; or a BBL polypeptide in combination with a PPO and AT1 and ODC and HPL activity.

"HPL (hydroperoxide lyase) activity or polypeptide" relates to an HPL polypeptide or realizes an enzymatic function or biological function of an HPL polypeptide. The hydroperoxide lyase has been described as catalyzing the cleavage of the C-C bond in the hydroperoxides of fatty acids.

In a particularly preferred embodiment, the HPL (hydroperoxide lyase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 13, 15 or 17; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 13, 15 or 17; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 13, 15 or 17; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 13, 15 or 17; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of (a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any of the polynucleotides specified in (a) to (d); (g) is represented by a polypeptide having SEQ ID NO: 14, 16 or 18; (h) is represented by a polypeptide having SEQ ID NO: 14, 16 or 18; NO:14, 16 or 18; (i) represented by a polypeptide domain of SEQ ID NO:14, 16 or 18 having HPL (hydroperoxide lyase) activity; (j) represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:14, 16 or 18 and having HPL (hydroperoxide lyase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic preparation method additionally uses a PAL (L-phenylalanine ammonia lyase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide and/or a PAL activity or polypeptide. In other embodiments, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO and ODC and PAL activity or polypeptide, or a BBL polypeptide in combination with a PPO and AT1 and ODC and PAL activity or polypeptide; or a BBL polypeptide in combination with a PPO and ODC and HPL and PAL activity; or a BBL polypeptide in combination with a PPO and AT1 and ODC and HPL and PAL activity.

"PAL (L-phenylalanine ammonia lyase) activity or polypeptide" relates to a PAL polypeptide or to an enzyme function or biological function that realizes a PAL polypeptide. L-phenylalanine ammonia lyase is found to catalyze the reaction that converts L-phenylalanine into ammonia and trans-cinnamic acid. Phenylalanine ammonia lyase (PAL) is the first and key step in the phenylpropanoid pathway and is involved in the biosynthesis of polyphenolic compounds (such as flavonoids, phenylpropanoids and lignin) in plants.

In particularly preferred embodiments, the PAL (L-phenylalanine ammonia lyase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 19, 21, 23 or 25; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 19, 21, 23 or 25; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 19, 21, 23 or 25; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 19, 21, 23 or 25; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of A(a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) is encoded by a polynucleotide having SEQ ID NO: 19, 21, 23 or 25. NO:20, 22, 24 or 26; (g) represented by a polypeptide having SEQ ID NO:20, 22, 24 or 26; (h) represented by a polypeptide fragment of SEQ ID NO:20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity; (i) represented by a polypeptide domain of SEQ ID NO:20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity; (j) represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with the amino acid sequence of SEQ ID NO:20, 22, 24 or 26 and having PAL (L-phenylalanine ammonia lyase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic preparation method additionally uses C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide and/or a PAL activity or polypeptide and/or a C4H activity or polypeptide. In other embodiments, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO and an ODC and an HPL and a C4H activity or polypeptide, or a BBL polypeptide in combination with a PPO and an AT1 and an ODC and an HPL and a PAL and a C4H activity or polypeptide; or a BBL polypeptide in combination with a PPO and an ODC and an HPL and a PAL and a C4H activity; or a BBL polypeptide in combination with a PPO and an AT1 and an ODC and an HPL and a PAL and a C4H activity or polypeptide.

"C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide" relates to a C4H polypeptide or to an enzyme function or biological function that realizes a C4H polypeptide. Trans-cinnamate 4-hydroxylase is described as a reaction that catalyzes the conversion of trans-cinnamic acid (CA) to p-coumaric acid (COA) in the phenylpropanoid/lignin biosynthetic pathway of plants.

In a particularly preferred embodiment, the C4H (L-phenylalanine ammonia lyase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 27; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 27; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 27; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 27; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of B (a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) is represented by a polypeptide having SEQ ID NO: 28; (h) is represented by a polypeptide having SEQ ID NO: 29; NO:28; (i) represented by a polypeptide domain of SEQ ID NO:28 having C4H (cinnamate 4-hydroxylase) activity; (j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:28 and having C4H (cinnamate 4-hydroxylase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic preparation method additionally uses 4CL (4-coumaric acid: CoA ligase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide and/or a PAL activity or polypeptide and/or a C4H activity or polypeptide and/or a 4CL activity or polypeptide. In other embodiments, the enzymatic production method uses a BBL polypeptide in combination with PPO and ODC and HPL and PAL and C4H and 4CL activities or polypeptides, or a BBL polypeptide in combination with PPO and AT1 and ODC and HPL and PAL and C4H and 4CL activities or polypeptides; or a BBL polypeptide in combination with PPO and ODC and HPL and PAL and C4H and 4CL activities; or a BBL polypeptide in combination with PPO and AT1 and ODC and HPL and PAL and C4H and 4CL activities; or a BBL polypeptide in combination with PPO and HPL and PAL and 4CL activities.

"4CL (4-coumarate:CoA ligase) activity or polypeptide" relates to a 4CL polypeptide or to an enzyme function or biological function that realizes a C4H polypeptide.

4-Coumarate:CoA ligase is a ligase that specifically forms carbon-sulfur bonds as an acid-thiol ligase. It catalyzes the formation of hydroxycinnamate-CoA esters and plays a crucial role at the bifurcation point of the major branching pathway from general phenylpropanoid metabolism to coumarin.

In a particularly preferred embodiment, the 4CL (4-coumaric acid: CoA ligase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131; (c) is encoded by a polynucleotide that is a variant of SEQ ID NO: 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131. NO:29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131; (d) encoded by a polynucleotide that is a species homolog of SEQ ID NO:29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131; (e) encoded by a polynucleotide that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to a polynucleotide as defined in any one of (a) to (d); (f) encoded by a polynucleotide that is capable of hybridizing under stringent conditions to any of the polynucleotides specified in C(a) to (d); (g) encoded by a polynucleotide having SEQ ID NO:29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131. NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132; (h) represented by a polypeptide fragment of SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumaric acid: CoA ligase) activity; (i) represented by a polypeptide fragment of SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumaric acid: CoA ligase) activity. NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132; (j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 and has 4CL (4-coumaric acid: Coenzyme A ligase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

In another embodiment, the enzymatic preparation method additionally uses HCT (hydroxycinnamoyl-transferase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide and/or a PAL activity or polypeptide and/or a C4H activity or polypeptide and/or a 4CL activity or polypeptide and/or an HCT activity or polypeptide. In other embodiments, the enzymatic production method uses a BBL polypeptide in combination with PPO and ODC and HPL and PAL and C4H and 4CL activities or polypeptides, or a BBL polypeptide in combination with PPO and AT1 and ODC and HPL and PAL and C4H and 4CL and HCT activities or polypeptides; or a BBL polypeptide in combination with PPO and ODC and HPL and PAL and C4H and 4CL and HCT activities; or a BBL polypeptide in combination with PPO and AT1 and ODC and HPL and PAL and C4H and HCT activities; or a BBL polypeptide in combination with PPO and HPL and PAL and C4H and 4CL and HCT activities.

"HCT (hydroxycinnamoyl-transferase) activity or polypeptide" relates to an HCT polypeptide or realizes an enzyme function or biological function of an HCT polypeptide. Hydroxycinnamoyl-transferase, also known as shikimate hydroxycinnamoyltransferase, is involved in phenylpropanoid biosynthesis. It uses 4-coumaroyl-CoA and shikimate as substrates.

In a particularly preferred embodiment, the HCT (hydroxycinnamoyl-transferase) activity or polypeptide is: (a) encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43; (b) encoded by a polynucleotide that is a variant of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43; (c) encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43; (d) encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 31, 33, 35, 37, 39, 41 or 43. NO:31, 33, 35, 37, 39, 41 or 43; (e) encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of (a) to (d); (f) encoded by a polynucleotide capable of hybridizing to any of the polynucleotides specified in (a) to (d) under stringent conditions; (g) represented by a polypeptide having SEQ ID NO:32, 34, 36, 38, 40, 42 or 44; (h) represented by a polypeptide fragment of SEQ ID NO:32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity; (i) represented by a polypeptide fragment of SEQ ID NO:32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity. NO:32, 34, 36, 38, 40, 42 or 44 polypeptide domain representative; (j) comprises and SEQ ID NO:32, 34, 36, 38, 40, 42 or 44 amino acid sequence has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity polypeptide and has HCT (hydroxycinnamoyl -transferase) activity representative; Or (k) by any one of the polypeptides encoded by the polynucleotides specified in (a) to (f) representative.

In another embodiment, the enzymatic preparation method additionally uses C3H (coumarate 3-hydroxylase) activity or polypeptide. Preferably, the enzymatic preparation method uses a BBL polypeptide in combination with a PPO activity or polypeptide and an AT1 activity or polypeptide and/or an ODC activity or polypeptide and/or an HPL activity or polypeptide and/or a PAL activity or polypeptide and/or a C4H activity or polypeptide and/or a 4CL activity or polypeptide and/or an HCT activity or polypeptide and/or a C3H activity or polypeptide. In other embodiments, the enzymatic production method uses a BBL polypeptide combined with PPO and ODC and HPL and PAL and C4H and 4CL activities or polypeptides, or a BBL polypeptide combined with PPO and AT1 and ODC and HPL and PAL and C4H and 4CL and HCT and C3H activities or polypeptides; or a BBL polypeptide combined with PPO and ODC and HPL and PAL and C4H and 4CL and HCT and C3H activities or polypeptide activities; or a BBL polypeptide combined with PPO and AT1 and HPL and PAL and C4H and 4CL and HCT and C3H activities or polypeptide activities; or a BBL polypeptide combined with PPO and HPL and PAL and 4CL and HCT and C3H activities or polypeptide activities.

"C3H (coumarate 3-hydroxylase) activity or polypeptide" relates to a C3H polypeptide or realizes an enzyme function or biological function of a C3H polypeptide. Coumarate 3-hydroxylases are found to catalyze the direct 3-hydroxylation of 4-coumaric acid to caffeic acid in lignin biosynthesis.

In a particularly preferred embodiment, the C3H (coumarate 3-hydroxylase) activity or polypeptide: (a) is encoded by a polynucleotide having a nucleotide sequence of SEQ ID NO: 45; (b) is encoded by a polynucleotide that is a variant of SEQ ID NO: 45; (c) is encoded by a polynucleotide that is an allelic variant of SEQ ID NO: 45; (d) is encoded by a polynucleotide that is a species homolog of SEQ ID NO: 45; (e) is encoded by a polynucleotide that has at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with a polynucleotide as defined in any one of E (a) to (d); (f) is encoded by a polynucleotide that can hybridize under stringent conditions with any one of the polynucleotides specified in (a) to (d); (g) is represented by a polypeptide having SEQ ID NO: 46; (h) is represented by a polypeptide having SEQ ID NO: 47 having C3H (coumarate 3-hydroxylase) activity. NO:46; (i) represented by a polypeptide domain of SEQ ID NO:46 having C3H (coumarate 3-hydroxylase) activity; (j) represented by a polypeptide comprising an amino acid sequence that is at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO:46 and having C3H (coumarate 3-hydroxylase) activity; or (k) represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

According to the present invention, the enzymatic preparation can be carried out in any environment or background suitable for producing a compound as defined herein. The environment can be an in vitro environment or an in vivo environment. The term "in vitro" as used herein refers to the preparation using biomolecules (e.g., activities or polypeptides as defined herein) and optionally using additional factors (such as energy carriers, cofactors, reducing equivalents, ions, etc.) outside their normal biological background (e.g., in a tube or reactor or reaction vessel, etc.). Cells or tissues are generally not present in an in vitro environment.

The environment may alternatively be an "in vivo" environment. In such an environment, biomolecules may be provided in a cellular environment, preferably in a living cell, tissue or organism. This may include providing the activity or polypeptide produced by a cell or a group of cells, or providing additional factors, such as energy carriers, cofactors, reducing equivalents, ions, etc., by a cell or cell environment. In certain embodiments, one or more of these components may be provided from the outside of a cell, tissue or organism, such as via a culture medium, injection, etc. The in vivo environment may be a homologous environment or a natural environment, i.e., an environment in which biomolecules, etc. are provided in their natural context. Alternatively, the in vivo environment is a heterologous environment. The term "heterologous" as used herein refers to at least one activity or polypeptide required for enzymatic preparation or a sequence encoding such an activity or polypeptide or any other additional factors (such as energy carriers, cofactors) that are not usually or naturally present in the cells, tissues or organisms used for preparation, but are introduced into the cells, tissues or organisms or modified in the production cells, tissues or organisms. The heterologous environment can be, for example, a higher plant species, into which one or more activities or polypeptides required for enzymatic production, or sequences encoding one or more activities or polypeptides, have been introduced (e.g., by genetic engineering). In other embodiments, the heterologous environment can be a microbial cell, such as a bacterial or fungal cell, comprising one or more activities or polypeptides required for enzymatic production, or sequences encoding one or more activities or polypeptides that have been introduced into the cell, for example, by genetic engineering or transformation.

In a specific preferred embodiment, the enzymatic preparation according to the present invention is performed under any suitable conditions using an activity or polypeptide provided in an in vitro environment. The activity or polypeptide required for the preparation may include an activity or polypeptide defined herein, such as a BBL2 polypeptide and a PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H polypeptide or activity. One or more of these activities or polypeptides may be provided in any suitable purity, concentration or amount. For example, one or more activities or polypeptides may be obtained, extracted and/or purified according to a suitable scheme known to those skilled in the art. For example, one or more activities or polypeptides required for the preparation of the compound according to the present invention may be expressed from a vector or plasmid in a suitable microorganism (i.e., a bacterial expression system), preferably by using a suitable overexpression promoter. The expression may be carried out under suitable temperature conditions, such as at 37° C. or 16° C. Subsequently, the bacterial cells may be collected, lysed and homogenized. The supernatant may be incubated with a suitable resin or incubated on a suitable column to purify and separate the polypeptide fraction. In a particularly preferred embodiment, the preparation is carried out in the manner described in the examples, particularly in Example 16.

According to certain embodiments, in vitro enzymatic preparation can be carried out by different groups or combinations or selections of activity or polypeptide. Grouping can usually be defined according to the steps or progress in the overall approach to prepare the compound of the present invention, such as as shown in Figure 19. According to the selection of activity or polypeptide, specific starting metabolites or compounds may be required. For example, if a group of activities or polypeptides comprising PPO and BBL2 is selected, starting metabolites or starting compounds such as N-caffeoyl putrescine may be required, and therefore provided with suitable amount, purity or concentration in an in vitro environment. In addition, another reactant, such as (Z)-3-hexenal (Z3H) can be provided.

For example, if a group of activities or polypeptides comprising AT1, ODC, HPL, PPO and BBL2 is selected, a starting metabolite or starting compound such as putrescine, spermine or spermidine may be required and thus provided in an appropriate amount, purity or concentration in an in vitro environment. In addition, another reactant such as caffeoyl-CoA may be provided.

For example, if a group of activities or polypeptides including PAL, C4H, 4CL, HCT, C3H AT1, ODC, HPL, PPO and BBL2 is selected, a starting metabolite or starting compound such as phenylalanine may be required and thus provided in an appropriate amount, purity or concentration in an in vitro environment. In addition, another reactant may be provided.

In addition to the above examples, the present invention further contemplates any other selection or grouping of polypeptides or activities defined herein for compounds of the present invention, as well as any other suitable reaction, starting compound or addition of metabolites. It is also contemplated that a subgroup of an activity or polypeptide defined herein is used to produce a certain intermediate, and that the intermediate is transferred to a different environment comprising a different or other subgroup of an activity or polypeptide defined herein. In other embodiments, the activity or polypeptide may be provided in a combined form, for example, all groupings or subgroups may be introduced into an in vitro environment in a combined manner, or they may be provided continuously, for example, providing a second, third, fourth, etc. activity, etc., after a specific time period has passed in the previous provision.

In other embodiments, the preparation method and/or its efficiency can be controlled by sampling after a specific time period. Such samples can be tested subsequently to determine certain compounds and/or to measure the amount of such compounds, such as intermediates or compounds according to the present invention as defined herein. For such tests, mass spectrometry means and methods can be used, for example.

The preparation method can be carried out with any suitable buffer (e.g., acetate buffer). Particularly preferably, the in vitro preparation method is carried out under the conditions of about 4.8 or lower pH values, such as pH 4.7, 4.6, 4.5, 4.4., 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, etc., or any value between the above values or any lower value, particularly preferably pH 4.8. Such pH values can be obtained in> 80mM acetate buffer. In other embodiments, the method is carried out at a temperature of about 5 to 15°C, preferably at a temperature of about 6 to 12°C, more preferably at a temperature of about 7 to 10°C, and most preferably at a temperature of about 8°C. In other embodiments, the method is carried out for any suitable time period, preferably 24 to 72 hours, more preferably about 48 hours. Other details can be drawn from the examples, for example from Example 16 or 17. Alternatively, the activity or polypeptide may be purchased from any suitable supplier.

In a further preferred embodiment, the preparation method is performed by using or involving the use of a solid phase extraction (SPE) protocol. It is particularly preferred that the SPE uses an argon flow.

The term "preparation" used in the context of in vivo production refers to the preparation or synthesis of the compounds of the invention by suitable living cells or tissues or organisms. The synthesized compounds may further be accumulated by the cells, tissues or organisms. The terms "accumulation" or "accumulation" refer to the storage of the synthesized compounds within the cells and/or excretion into the surrounding environment, in both cases resulting in an overall increase in the concentration of the compound compared to a natural or wild-type cell or organism, which, for example, does not contain or express an activity or polypeptide as defined herein, such as BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H activity.

In order to prepare the compound of the present invention in vivo, the present invention considers in certain embodiments in suitable microbial cells or organisms to carry out heterologous preparation.The example of suitable microbial cells or organisms includes prokaryotic or eukaryotic expression host.For example, microbial cells or organisms can be bacterium, such as Klebsiella, Clostridium, Bacillus, Arthobacter, Streptomyces, Corynebacterium, Erwinia, Xanthomonas, Lactobacillus, Caldicellulosiruptor, Pseudomonas, Alcanivorax, Brevibacterium, Bifidobacterium, Escherichia or Staphylococcus. Preferably, the bacterium may belong to the genus Escherichia, more preferably, the bacterium is Escherichia coli. It is also contemplated to use fungi, such as Aspergillus, Candida, Saccharomyces, Ustilago, Cryptococcus, Fusarium, Rhizopus, Magnaporthe, Komagataella, Trichderma, Penicillium, Acremonium, Mucor, Alternaria, Botrytis, Entomophilus, and the like. The present invention also provides a kind of fungus of the genera of the genus ...

In a specific embodiment, the method contemplates cultivating the microbial cells or organisms in a culture medium. As used herein, the term "cultivating in a culture medium" refers to the use of any suitable means and methods known to those skilled in the art that allow the growth of cells or organisms as defined herein and that are suitable for the synthesis and/or accumulation of the compounds of the present invention in the cells or organisms. For example, the culture medium can be adapted to the growth pattern of the organism, for example, including a carbon source, or without a carbon source in the case of an autotrophic organism.

In the specific embodiment of E. coli and related organisms, culture media such as Terrific Broth (TB), Luria-Bertani medium (LB) or M9 minimal medium can be used. Those skilled in the art will further appreciate that other culture media suitable for E. coli are also considered herein and their preparation, for example from suitable literature sources or databases. Typically, TB culture media can include 12g Bacto tryptone, 24g Bacto yeast extract, 4mL glycerol in 1 liter unit, add distilled water to 900ml, autoclave, and then add 100mL sterile 0.17MKH ₂ PO ₄ and 0.72MK ₂ HPO ₄ to complete. Typically, LB culture media can include 10g Bacto tryptone, 5g yeast extract, 10g NaCl in 1 liter unit, add distilled water to 1000ml, and then autoclave. Typically, M9 minimal medium may comprise, in 1 liter unit, 880 ml sterile water, 100 ml M9 salt stock solution, 1 ml autoclaved 1 M MgSO ₄ , 0.1 ml autoclaved 1 M CaCl ₂ and 20 ml 20% glucose (sterile), wherein the M9 salt stock solution (10x) comprises 60 g Na ₂ HPO ₄ x 7H ₂ O, 30 g KH ₂ PO ₄ , 5 g NaCl, 10 g NH ₄ Cl, to which water is added to 1000 ml and then autoclaved.

The culture may be carried out in a batch process or a continuous fermentation process. Preferably, the cells or organisms are cultured in the presence of precursors such as phenylalanine or other suitable amino acids. Methods for carrying out batch or continuous fermentation processes are well known to those skilled in the art and are described in the literature, for example in Li et al., Microb Cell Fact, 2015, 14 (83). The culture may be carried out under specific temperature conditions, for example, between 15°C and 37°C, preferably between 20°C and 30°C or between 15°C and 30°C, more preferably between 20°C and 30°C, and most preferably at about 24°C. In another embodiment, the culture may be carried out in a pH range of pH 3 to 4.8. The fermentation time may vary depending on the degree of fermentation, the culture medium used, the organism used, etc. In certain embodiments of the present invention, a fermentation time of about 6 to 72 h may be used, preferably a fermentation time of about 10 to 24 h, more preferably a fermentation time of 12, 14, 16, 18 or 20 h, or any value between the above values. Fermentation can be started from a newly inoculated culture, for example from a solid medium or a frozen standby. Preferably, fermentation is started from a pre-culture, for example an overnight culture. Therefore, cells can be transferred to the main culture at a specific OD ₆₀₀ value, for example at an OD ₆₀₀ of 0.5 to 0.8, preferably at 0.6. If batch fermentation is performed, cells can be cultured at high density in a defined minimal medium restricted by glucose, and formation can be stopped at a specific OD ₆₀₀ value of about 1 to 100, preferably about 100. Other details can be obtained from suitable literature sources, such as Li et al., Microb Cell Fact, 2015, 14 (83).

In further specific embodiments, the culture medium may include other substances. An example of such other substances is an antibiotic, such as tetracycline, ampicillin, kanamycin. Such antibiotics may be used as a selection tool for the extrachromosomal elements comprising the corresponding resistance box, or as an inducer for the corresponding regulated promoter, such as defined below. They may be used at any suitable concentration, such as for ampicillin, a suitable concentration range of 50 to 400 μg/ml, such as 50,100,150 μg/ml, or for kanamycin, a suitable range of 25 to 50 μg/ml, such as 25 or 50 μg/ml. Other details are known to those skilled in the art, or may be obtained from suitable literature sources.

For the in vivo preparation of the compounds of the invention, the present invention alternatively contemplates in certain embodiments a heterologous preparation in a suitable eukaryotic organism, preferably a higher eukaryotic cell or organism, or in a tissue of said organism, preferably an organism, cell or tissue as defined below.

In an embodiment, the compounds of the invention may be prepared in vivo in higher eukaryotic cells or organisms (such as plant cells, plant tissues or plant organisms) in order to accumulate the compounds in the plant cells or tissues for the purpose of subsequently extracting them, for example by means of a suitable plant material extraction method, preferably as described in the examples. In an alternative embodiment, the compounds of the invention may be prepared in vivo in higher eukaryotic cells or organisms (such as plant cells, plant tissues or plant organisms) in order to increase the resistance of the prepared cells, tissues or organisms to insect herbivores, such as herbivores that typically attack the plants prepared as defined herein. Thus, the corresponding prepared cells, tissues or organisms may be protected from such attacks, thereby increasing harvests and yields.

In order to carry out heterologous preparation in suitable cells, tissues or organisms, the cells, tissues or organisms may be genetically modified. Such genetic modification allows expression or heterologous expression of one or more activities or polypeptides as defined herein. For example, the genetic modification allows expression of BBL2 polypeptide and PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H activities or polypeptides. The genetic modification may also additionally allow expression or heterologous expression of any additional factors, elements, polypeptides or activities that are necessary according to the preparation method of the present invention, or that facilitate auxiliary steps such as transport, accumulation, input, output, excretion, stabilization, precursor modification, energy supply, reactant supply, etc.

The term "genetically modified" or "genetically modified" as used herein refers to changing cells, tissues or organisms by any suitable gene means and methods known to those skilled in the art to prepare the compounds of the present invention. Similarly, the term "genetically modified cells, tissues or organisms" as used herein refers to cells, tissues or organisms that have been modified or changed by any suitable gene means and methods known to those skilled in the art so that it synthesizes the compounds according to the present invention. The cells, tissues or organisms can also further accumulate and/or excrete or export the compounds. The term also includes modifications to genetically modified cells or organisms, such as starting cells or organisms. In the present invention, cells, tissues or organisms are genetically modified to express one or more activities or polypeptides defined herein. These activities or polypeptides are generally associated with phenylpropanoid pathways, polyamine pathways, green leaf volatiles (GLV) pathways and/or caffeoyl putrescine-hexenal (CPH) pathways, such as shown in Figure 19. The present invention contemplates further activities such as transport activity, export or import activity, secretion or excretion activity, activity leading to transport of precursors or intermediates, accumulation activity, precursor or pre-precursor modification activity, activity involved in energy supply or reactant supply, etc.

Methods for genetically modifying organisms are known to those skilled in the art and are described in the literature. They include conventional methods for introducing genetic elements or materials into cells, tissues or organisms for inclusion in cells, integration into chromosomes or extrachromosomal (see, e.g., Pfeifer et al., Science 291, 1790-1792 (2001); Wang et al., Appl Microbiol Biotechnol 77 (2007)), or conventional methods for removing or destroying or modifying genetic elements or sequences present in genomes or organisms (see, e.g., Peiru et al., Microbial Biotechnology (2008) 1 (6), 476-486; Zhang et al., Biotechnol Prog. 28 (1), 52-59 (2012)).

The term "genetic element" used herein refers to any molecular unit capable of transmitting genetic information. Therefore, it relates to a gene, preferably a chimeric gene, an exogenous gene, a transgenic or a codon optimized gene. The term "gene" refers to a nucleic acid molecule or fragment expressing a specific protein, preferably it refers to a nucleic acid molecule comprising a regulatory sequence before (5' non-coding sequence) and after (3' non-coding sequence) a coding sequence. The term "chimeric gene" refers to any gene as a non-natural gene, which includes a regulatory sequence and a coding sequence that are not found together in nature. Therefore, a chimeric gene may include regulatory sequences and coding sequences from different sources, or regulatory sequences and coding sequences arranged in a different manner from the same source and found in nature. According to the present invention, "foreign gene" refers to a gene that is not usually found in an organism or cell, but is introduced into the organism or cell by gene transfer, or has been modified in an organism corresponding to the foreign gene. Foreign genes may include natural genes inserted into non-natural organisms, or chimeric genes. The term "transgenic" refers to a gene that has been introduced into a genome by a transformation process.

The term "coding sequence" refers to a DNA sequence that encodes a specific amino acid sequence. The term "regulatory sequence" refers to a nucleotide sequence that is located upstream (5' non-coding sequence), internal or downstream (3' non-coding sequence) of a coding sequence, and that affects the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, enhancers, translation leader sequences, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures.

The term "promoter" refers to a DNA sequence that can control the expression of a coding sequence or functional RNA. Typically, the coding sequence is located at the 3' of the promoter sequence. The promoter can be from an organism in which expression occurs, can be from a natural gene or an exogenous gene, or be composed of different elements from different promoters found in nature, such as in different taxa or categories, or even include synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters can guide the expression of genes at different developmental stages or in response to different environments or physiological conditions. The promoter that causes gene expression in most genetic backgrounds and/or most of the time is generally referred to as a "constitutive promoter". Typically, due to the fact that the exact boundaries of regulatory sequences are not yet fully defined, DNA fragments of different lengths may have the same promoter activity. On the other hand, the promoter that causes gene expression only under a specific environment (e.g., based on the presence of a specific factor, a growth stage, a temperature, a pH, or the presence of a specific metabolite, etc.) is understood to be a "regulatable promoter". The promoter is generally operably connected to the coding sequence to be expressed as defined herein. The term "natural promoter" used herein relates to a promoter that is operably connected to a coding sequence to be expressed in the wild-type case.

The term "3' non-coding sequence" refers to a DNA sequence located downstream of a coding sequence. This may include sequences encoding regulatory signals that can affect mRNA processing or gene expression. The 3' region can affect transcription, i.e., the presence of an RNA transcript, RNA processing or stability, or translation of the associated coding sequence. The term "RNA transcript" refers to the product produced by transcription of a DNA sequence catalyzed by RNA polymerase. When the RNA transcript is a complete complementary copy of a DNA sequence, it is referred to as a primary transcript, or it may be an RNA sequence derived from post-transcriptional processing of the primary transcript and is referred to as a mature RNA. The term "mRNA" refers to a messenger RNA that can be translated into protein by the cell.

The term "operably linked" refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. In the context of a promoter, the term means that a coding sequence is able to affect the expression of the coding sequence, i.e., the coding sequence is under the transcriptional control of the promoter. Such control can affect one gene or open reading frame (monocistron), or it can affect a group of genes or open reading frames.

In a particularly preferred embodiment, the genetic modification results in at least the expression of a BBL2 (berberine bridge enzyme 2) polypeptide, preferably as defined above. For example, the following may be introduced as genetic elements into the cells, tissues or organisms used for the preparation: a polynucleotide having the nucleotide sequence of SEQ ID NO: 1, or a variant thereof, or a species homolog thereof, or a polynucleotide having 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with the polynucleotide of SEQ ID NO: 1. The introduction may be carried out using a suitable promoter as defined herein to allow overexpression of BBL2.

In a particularly preferred embodiment, the genetic modification additionally results in the expression of a PPO (polyphenol oxidase) activity or polypeptide, preferably as defined above. For example, the following may be introduced as genetic elements into the cells, tissues or organisms used for the preparation: a polynucleotide having the nucleotide sequence of SEQ ID NO: 3 or 5, or a variant thereof, or a species homologue thereof, or a polynucleotide having 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with the polynucleotide of SEQ ID NO: 3 or 5. The introduction may be carried out using a suitable promoter as defined herein to allow overexpression of PPO.

In another particularly preferred embodiment, the genetic modification additionally results in the expression of AT1 (polyamine hydroxycinnamoyl transferase 1) activity or polypeptide, preferably as defined above. For example, the following may be introduced as genetic elements into the cells, tissues or organisms used for the preparation: a polynucleotide having the nucleotide sequence of SEQ ID NO: 7, or a variant thereof, or a species homologue thereof, or a polynucleotide having 75%, 80%, 90%, 95%, 97%, 98% or 99% identity with the polynucleotide of SEQ ID NO: 7. The introduction may be carried out using a suitable promoter as defined herein to allow overexpression of AT1.

In another particularly preferred embodiment, the genetic modification additionally results in the expression of ODC (ornithine decarboxylase) activity or polypeptide and/or HPL (hydroperoxide lyase) activity or polypeptide, preferably as defined above. For example, the following substances can be introduced as genetic elements into the cells, tissues or organisms used for preparation: a polynucleotide with a nucleotide sequence of SEQ ID NO:9 or 11 and/or 13, 15 or 17, or a variant thereof, or a species homologue thereof, or a polynucleotide with 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide of SEQ ID NO:9 or 11 and/or 13, 15 or 17. Suitable promoters defined herein can be used to introduce to allow the overexpression of ODC and/or HPL.

In another particularly preferred embodiment, the genetic modification additionally results in the expression of a PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or a C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide and/or a 4CL (4-coumarate:CoA ligase) activity or polypeptide and/or a HCT (hydroxycinnamoyl-transferase) activity and/or a C3H (coumarate 3-hydroxylase) activity or polypeptide, preferably as defined above. For example, the following can be introduced as genetic elements into production cells, tissues or organisms: polynucleotides having the nucleotide sequences of SEQ ID NO: 19, 21, 23 or 25, and/or 27 and/or 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131 and/or 31, 33, 35, 37, 39, 41 or 43, and/or 45, or variants thereof, or species homologues thereof, or ... NO: 19, 21, 23 or 25, and/or 27 and/or 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131 and/or 31, 33, 35, 37, 39, 41 or 43, and/or 45 have 75%, 80%, 90%, 95%, 97%, 98% or 99% identity. Suitable promoters as defined herein may be used for introduction to allow overexpression of PAL, C4H, 4CL and/or HCT.

The introduction can be controlled using suitable methods (e.g. sequencing or PCR amplification). In a further embodiment, the expression of BBL2, optionally the expression of PPO, further optionally the expression of AT1, further optionally the expression of ODC and/or HPL, further optionally the expression of PAL, C4H, 4CL, HCT and/or C3H can be tested using extraction and functional assays known to those skilled in the art or described in the examples.

The expression is usually controlled or transmitted by a natural, regulated, tissue-specific or constitutive promoter as defined herein. For example, in the case where the genetically modified organism is taxonomically related to the source organism of the heterologous gene or genetic element, or if it is known that these organisms have the use of similar promoters or transcription initiation structures, a natural promoter can be adopted. In addition, if it is known that the genetically modified organism and the source organism of the heterologous gene or genetic element have similar genomic GC content, it is considered to use a natural promoter. Alternatively, all or some of the relevant natural promoter structures or regions of any gene or genetic element with the above-mentioned characteristics that allows heterologous expression of the activity or polypeptide according to the present invention can be replaced or partially replaced or modified to cause a regulated or constitutive promoter function. Examples or regulated promoters that can be used in the context of the present invention include inducible promoters. Therefore, regulation may be made according to extracellular factors, such as the presence of temperature, certain metabolites or small molecules. Suitable examples include tetracycline inducible promoter PTet. Examples of suitable constitutive promoters include gltA, yfgF or glyA. The E. coli promoters lac, trp, phoA, araBAD, rha and tac are also contemplated. Further contemplated are tissue-specific promoters, i.e. promoters that are active only in certain tissues or after certain developmental steps. Such promoters can, for example, be used for tissue-specific expression of an activity or polypeptide as described above, ultimately leading to tissue-specific accumulation of the compounds of the invention only in a subset of tissues of the organism, for example only in tissues exposed to herbivore attack (such as leaves or flowers).

In a preferred embodiment, the promoter allows polycistronic expression of an activity or polypeptide as defined above. The term "polycistronic" as used herein refers to a sequence encoding a variety of different polypeptides or activities. In the case of polycistronic transcription, control can be provided by the organizational concept of an "operon", which is understood to be a functional unit of genomic DNA representing a gene cluster controlled by a single promoter. The genes of an operon are usually transcribed together into one mRNA chain and translated together, or trans-spliced to create a single translatable monocistronic mRNA, for example, several mRNA chains each encoding a single gene product. In such a case, the genes contained in the operon are usually expressed together, or they are not expressed. Therefore, multiple genes are usually transcribed together in an operon. In general, the expression of a prokaryotic operon results in the production of polycistronic mRNA. Typically, an operon includes three genetic components: (i) a promoter, i.e., a sequence that enables a gene to be transcribed, for example, as defined herein; (ii) an operator, i.e., a fragment that can be bound by a repressor, which can hinder the transcription of a gene; and (iii) a co-expressed structural gene. In addition, there may be a regulatory gene encoding a repressor protein that can bind to the operator gene sequence. Polycistronic arrangements may also exist in eukaryotic organisms (e.g., higher eukaryotic organisms). If polycistronic transcription occurs, there may be intercistronic or intergenic regions between open reading frames. Such intercistronic regions may include ribosome binding sites, for example, Shine-Dalgarno sequences or other functional elements that have an impact on transcription and/or translation.

In certain embodiments, the operon can comprise all coding sequences of BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT, and C3H, or any subset thereof (e.g., BBL2 and PPO, or BBL2 and PPO and AT1, or ODC and HPL, or ODC and HPL and PAL, or C4H and 4CL, HCT and C3H, etc.).

In a further preferred embodiment, the promoter allows the individual expression of an activity or polypeptide as defined above. For example, each of BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and C3H can be equipped with its own promoter. In certain embodiments, the promoter may be regulatable. It is also possible to consider using different regulatable promoters for different activities, for example, resulting in differential expression patterns of the activities, for example according to the order of preparation or necessity.

In another preferred embodiment, it is contemplated to express in groups a group of at least two activities selected from the activities defined above. For example, a group of BBL2 and PPO, AT1 and ODC, HPL and PAL, C4H and 4CL, HCT and C3H, or any other combination of 2, 3, 4, 5, 6, 7, 8 or more activities may be expressed. Grouped expression may be achieved using an operon structure as defined above or a differentially regulated promoter as defined above.

In a further preferred embodiment, the expression as mentioned above is overexpression. The term "overexpression" refers to more transcript accumulation, particularly more polypeptide and activity accumulation, than when the natural copy of the genetic element that produces the polypeptide or activity in the case of the source organism is expressed. In a further alternative embodiment, the term can also refer to more transcript accumulation, particularly more polypeptide or activity accumulation, than when a typical, moderately expressed housekeeping gene (such as actin, GAPDH or ubiquitin) is expressed.

In preferred embodiments, such overexpression may result in an increase in gene transcription rate of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more, or any value in between, compared to the corresponding wild-type or native transcript (without modification or overexpression) in the source organism. In a preferred embodiment, such an increase in gene transcription rate can provide at least one or more than one (e.g., 2, 3, 4, 5, 6, 7, 8 or all) of the following: a gene or genetic element encoding BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H; or a gene or genetic element having SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, or a homologous sequence thereof as defined herein.

In another preferred embodiment, the overexpression may result in an increase in the amount of the polypeptide encoded by the overexpressed gene by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more than 1000% or any value in between these values compared to the corresponding wild-type or original polypeptide amount in the source organism (without modification or overexpression). In a preferred embodiment, such an increase in the amount of a polypeptide encoded by an overexpressed gene may provide at least one or more than one (e.g., 2, 3, 4, 5, 6, 7, 8 or all) of the following: a polypeptide of BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H; or a polypeptide having SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, or a homologous sequence thereof as defined herein.

In a particularly preferred embodiment, at least the genes encoding BBL2 and PPO activities or polypeptides are overexpressed. Such overexpression can result in an increase in the amount of transcripts and/or polypeptides encoded by the overexpressed genes by about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, 500%, 600%, 700%, 800%, 900%, 1000% or more than 1000% or any value in between these values compared to the corresponding wild-type or original polypeptide amount in the source organism (without modification or overexpression).

In one embodiment, overexpression as defined above can be communicated by using a promoter as defined above. The promoter that can be used for overexpression of a gene as described herein, which the present invention contemplates, can be a constitutive promoter or a regulated promoter. In a preferred embodiment, the promoter is a heterologous promoter or a synthetic promoter, such as a strong heterologous promoter or a regulated heterologous promoter. In a particularly preferred embodiment, a strongly regulated or strongly constitutive promoter is used. Examples of such strong constitutive or strongly regulated promoters include T7 promoters (constitutive E. coli), rhaP (inducible E. coli) or, for example, the S35 promoter of a plant. Other suitable plant promoters considered herein may be from Plant Prom, a plant promoter sequence database (accessed on December 21, 2021, at http://www.softberry.com/berry.phtml?topic=plantprom&group=data&subgroup=plantprom), or PlantPromoterdb, another plant promoter database (accessed on December 21, 2021, at https://ppdb.agr.gifu-u.ac.jp/ppdb/cgi-bin/index.cgi).

Alternatively, in other embodiments, the overexpression defined above can be communicated at least by a second copy of the genetic elements encoding the activity or polypeptide. For example, a gene or genetic element may have 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, 200, 300, 400, 500 or more than 500 copies in a single cell, or any value between the above values. These copies may be present in the genome of an organism, or on a non-genomic plasmid or vector. In the case of a plasmid or vector, the number and compatibility of gene copies can be defined and regulated by the form and properties of a plasmid or vector source or ori. The example of a suitable bacterial source is from a plasmid, such as pBR322, pUC, pSC101 or p15A. Therefore, the plasmid or vector considered by the present invention can be from these plasmids, or its modified version.

In another embodiment, expression or overexpression as defined above may be conveyed by optimization of codon usage, for example by adapting the codon usage of the gene or genetic element as defined above to the codon usage of the most frequently transcribed or expressed genes in the target organism or cell, or to the codon usage of the highest expressed genes (compared to housekeeping genes, e.g. as defined above). Examples of such codon usage of highly expressed genes may include the codon usage of a group of 5, 10, 15, 20, 25 or 30 or more of the highest expressed genes in the organism in which expression occurs.

In another particularly preferred embodiment, the one or more enzyme activities or polypeptides used for the preparation of the compounds according to the invention, for example one or more of BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and C3H as defined herein, are from an organism belonging to the genus Nicotiana. The genus usually includes at least the following species: Nicotiana acaulis, Nicotiana acuminata, Nicotiana africana, Nicotiana alata, Nicotiana ameghinoi, Nicotiana amplexicaulis, Nicotiana arentsii, Nicotiana attenuata, Nicotiana azambujae, Nicotiana benavidesii, Nicotiana benthamiana, Nicotiana bonariensis, Nicotiana burbidgeae, Nicotiana cavicola, Nicotiana aclevelandii, Nicotiana cordifolia, Nicotiana corymbosa), Nicotiana cutleri, Nicotiana debneyi, Nicotiana excelsior, Nicotiana exigua, Nicotiana forgetiana, Nicotiana fragrans, Nicotiana glauca Graham, Nicotiana glutinosa, Nicotiana goodspeedii, Nicotiana gossei, Nicotiana hesperis, Nicotiana heteroantha, Nicotiana ingulba, Nicotiana kawakamii, Nicotiana knightiana, Nicotiana langsdorffii, Nicotiana linearis, Nicotiana longibracteata, Nicotiana longiflora, Nicotiana amaritima, Nicotiana megalosiphon, Nicotiana miersii, Nicotiana mutabilis, Nicotiana nesophila, Nicotiana noctiflora, Nicotiana nudicaulis, Nicotiana aocidentalis, Nicotiana obtusifolia, Nicotiana otophora, Nicotiana paa, Nicotiana palmeri, Nicotiana apaniculata, Nicotiana pauciflora, Nicotiana petuniodes, Nicotiana plumbaginifolia, Nicotiana quadrivalvis, Nicotiana raimondii, Nicotiana spathulata ... repanda), Nicotiana arosulata, Nicotiana rotundifolia, Nicotiana rustica, Nicotiana setchellii, Nicotiana simulans, Nicotiana solanifolia, Nicotiana spegazzinii, Nicotiana astenocarpa, Nicotiana stocktonii, Nicotiana suaveolens, Nicotiana sylvestris, Nicotiana tabacum, Nicotiana thrysiflora, Nicotiana tomentosa, Nicotiana atomosiformis, Nicotiana trigonophylla, Nicotiana truncata, Nicotiana umbratica, Nicotiana wavy undulata), velvet tobacco (Nicotiana velutina), Weigandi tobacco (Nicotiana wigandioides), Utkeni tobacco (Nicotiana wuttkei). Particularly preferred, the one or more enzyme activities or polypeptides used to prepare the compounds according to the invention (for example one or more of BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and C3H as defined herein) are from an organism belonging to the species Nicotiana attenuata.

In another embodiment, a polynucleotide as defined herein (e.g. encoding BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT or C3H) is contained in one or more extrachromosomal vectors or plasmids and/or integrated in the genome of the organism, or is contained in an expression cassette, preferably for heterologous expression in a eukaryotic host cell, more preferably for expression in a plant cell.

Therefore, in a further aspect, the present invention also relates to a vector or an insertion construct or an expression cassette comprising said polynucleotide as defined herein.

Suitable carriers can be, for example, plasmids, BACs or phage vectors, preferably plasmids.Term " plasmid " refers to any plasmid suitable for transforming bacteria, fungi or higher eukaryotes (such as plants) or any other suitable host organisms according to the present invention known to those skilled in the art, and refers in particular to any plasmid suitable for expressing proteins in higher eukaryotes such as bacteria (such as Escherichia coli), fungi or plants, for example, plasmids capable of autonomous replication.Polynucleotides according to the present invention (such as polynucleotides as defined above) can be connected to a carrier containing a selectable marker to breed in a host.Usually, plasmid vectors are introduced into a precipitate (such as a calcium phosphate precipitate), or in a complex with a charged lipid.If the carrier is a phage, it can be packaged in vitro using a suitable packaging cell line, and then transduced into a host cell.Preferably, the carrier will include at least one selectable marker.Such markers include, for example, tetracycline, kanamycin or ampicillin resistance genes for cultivating in Escherichia coli and other bacteria. Preferred vectors for use in bacteria include, but are not limited to, pQE70, pQE60, and pQE9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; pKK223-3, pKK233-3, pDR540, pRIT5 (available from Pharmacia Biotech, Inc.), and pET vectors (available from Novagen). Preferred vectors for use in plants include, but are not limited to, pBI121, pCAMBIA, pEarleyGate 201, pPZP100, pBI221, pCAMBIA1381Xb, pEarleyGate 202, pPZP101pBINPLUS, pCAMBIA1381Xc, pEarleyGate 203, pPZP102, pBin19, pCAMBIA1381Z, pEarleyGate 204, pPZP111, pCAMBIA0105.1R, pCAMBIA1390, pEarleyGate 205, pPZP112, pCAMBIA0305.1, pKANNIBAL, pHANNIBAL, pGreenll, and pGreen. Other suitable vectors are known to those skilled in the art or can be obtained from suitable literature sources, such as Chen Biotechnol Adv. 30(5), 1102-1107 (2012).

According to the present invention, the introduction of the vector defined above into cells, tissues or organisms can be achieved by any suitable technique, such as calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, chemical transformation, transduction, Agrobacterium tumefaciens-based transformation or gene gun-based transformation.

For genomic integration, one, two or more copies of genetic elements as described above can be introduced into cells or organisms by "insertion constructs", and thus placed in chromosomes. The integration site can be located near the original copy (if present), or is preferably located at any suitable position, for example, at different positions. Advantageously, insertion can be pre-selected by selecting the necessary homologous flanks for integration. Therefore, the insertion site can be determined according to the known characteristics of the genome, such as the transcriptional activity of the chromosome region, the possible distance from the first copy (original gene), the direction of the first copy (original gene), the presence of other genes inserted, etc. In certain embodiments, the insertion construct can include a copy of genetic elements, such as the polynucleotides encoding BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H, or other copies can be provided in tandem repeat form. For example, the insertion construct may include BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT or C3H alone, or include BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and C3H as a group, or include any subgroup thereof. In other embodiments, the present invention contemplates the use of non-tandem repeats. In certain embodiments, the copies of the genetic elements may be kept as different and/or distant as possible. Such differences may be based on the use of different promoters, modification of the genomic side of the gene, or in a specific embodiment, modification of the nucleotide sequence of the second copy or other copies of the gene relative to the first copy (original version), or modification of the third copy of the gene relative to the second copy and/or first copy (original version), etc.

"Expression cassette" used herein relates to a polynucleotide construct comprising a gene or genetic element and a regulatory sequence to be expressed by a transfected cell. The expression cassette generally directs the mechanism of the cell to produce RNA and protein. In certain embodiments, the expression cassette can be designed for modular cloning of protein coding sequences. In other embodiments, the expression cassette can be composed of more than one gene or genetic element and the sequence controlling their expression, the gene or genetic element being, for example, a polynucleotide encoding BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and C3H in a group or any subgroup thereof. For example, the expression cassette can include a promoter sequence, an open reading frame and a 3' non-translated region, optionally including (for example in eukaryotic organisms) a polyadenylation site.

In another important aspect, the present invention relates to an organism, tissue or cell for preparing a compound according to the invention. Said organism, tissue or cell may be genetically modified, preferably as defined above, for example by introduction of a vector or plasmid, an insertion construct or an expression cassette according to the invention.

Another preferably considered possibility for the genetic modification of cells, organisms or tissues (cell populations) is the molecular modification of the genome, preferably using genome editing systems.

"Genome editing systems" advantageously allow providing genome modifications without the need to insert an antibiotic resistance cassette or any additional selection marker. For example, such genome editing approaches can be based on the use of CRISPR/Cas systems, TALEN-systems or zinc finger nucleases (ZFN)-systems.

Particularly preferred is the use of CRISPR (clustered regularly spaced short palindromic repeats)/Cas system. CRISPR/Cas can be used to reduce the expression of a specific gene (or group or similar gene) or edit a genomic sequence. This is usually achieved by the expression of single-stranded RNA and CRISPR genes or nucleases. The technology usually relies on the expression of CRISPR genes (such as Cas9) or other similar genes and RNA guide sequences (see, for example, Cong et al., Science, 339 (6121), 819-823 (2013)). Therefore, the expression of appropriate flanking RNA guide sequences can be used to target double-stranded cleavage to a specific sequence, and the flanking RNA guide sequence can be provided as a component of a multi-component system, for example, provided together with Cas9 or similar functions. In a preferred embodiment, RNA guide sequences and CRISPR gene expression (such as Cas9) can be included as part of an expression construct.

The term "TALEN-system" relates to the use of TALEN, i.e., transcription activator-like effector nuclease, which is an artificial restriction enzyme produced by fusing a TAL effector DNA binding domain with a DNA cleavage domain. TAL effectors are proteins typically secreted by Xanthomonas bacteria or related species, or proteins derived therefrom and modified. The DNA binding domain of a TAL effector may comprise a highly conserved sequence, such as a sequence of about 33-34 amino acids, except for the 12th and 13th amino acids, which are highly variable (repeated variable di-residues or RVDs) and generally show a strong correlation with specific nucleotide recognition. The TALEN DNA cleavage domain may be derived from a suitable nuclease. For example, a DNA cleavage domain from a FokI endonuclease or a FokI endonuclease variant may be used to construct a hybrid nuclease. Due to the properties of the FokI domain (which acts as a dimer), TALEN may preferably be provided as a separate entity. TALEN or TALEN components may preferably be engineered or modified to target any desired DNA sequence. Such engineering can be performed according to a suitable method, for example Zhang et al., Nature Biotechnology, 1-6 (2011), or Reyon et al., Nature Biotechnology, 30, 460-465 (2012).

The term "zinc finger nuclease (ZFN)-system" as used herein refers to a system of artificial restriction enzymes, which are generally produced by fusing a zinc finger DNA-binding domain to a DNA-cleavage domain. The zinc finger domain may preferably be engineered or modified so as to target any desired DNA sequence. Such engineering methods are known to those skilled in the art or may be obtained from suitable literature sources, such as Bae et al., Nat Biotechnol, 21, 275-80 (2003); Wright et al., Nature Protocols, 1, 1637-1652 (2006)). Typically, a non-specific cleavage domain from a type II restriction endonuclease (e.g., from FokI) may be used as a cleavage domain in a ZFN. Since the cleavage domain dimerizes to cleave DNA, a pair of ZFNs is generally required to target a non-palindromic DNA site. The ZFN contemplated by the present invention may further comprise a fusion of non-specific cleavage to the C-terminus of each zinc finger domain. For example, to allow two cleavage domains to dimerize and cleave DNA, two separate ZFNs are typically required to bind to opposite strands of DNA with C-termini provided at a specific distance. It should be understood that the linker sequence between the zinc finger domain and the cleavage domain may require that the 5' end of each binding site be separated by about 5 to 7 bp. The present invention contemplates any suitable ZNF format or variant, such as a classic FokI fusion or an optimized version of FokI, as well as enzymes with modified dimerization interfaces, improved binding functions or variants that are capable of providing heterodimeric species.

According to a preferred embodiment of the invention, the genetic modification allows or expresses one or more activities or polypeptides as defined herein, such as BBL2, PPO, AT1, ODC, HPL, PAL, C4H, 4CL, HCT and/or C3H. The expression may be heterologous expression as defined herein, preferably overexpression as defined herein. The genetic modification of a cell, organism or tissue is a genetic modification as defined in detail above. In particular, the genetic modification may result in at least the expression of a BBL2 (berberine bridge enzyme 2) polypeptide as defined above, additionally the expression of a PPO (polyphenol oxidase) activity or polypeptide as defined above, additionally the expression of an AT1 (polyamine hydroxycinnamoyltransferase 1) activity or polypeptide as defined above, additionally the expression of an ODC (ornithine decarboxylase) activity or polypeptide and/or an HPL (hydroperoxide lyase) activity or polypeptide as defined above, and additionally the expression of a PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or a C4H (trans-cinnamate 4-hydroxylase) activity or polypeptide and/or a 4CL (4-coumarate:CoA ligase) activity or polypeptide and/or an HCT (hydroxycinnamoyl-transferase) activity or polypeptide and/or a C3H (coumarate 3-hydroxylase) activity or polypeptide as defined above.

In a preferred embodiment, the genetically modified organism, tissue or cell is prokaryotic, such as bacteria or fungi, preferably Klebsiella, Clostridium, Bacillus, Arthobacter, Streptomyces, Corynebacterium, Erwinia, Xanthomonas, Lactobacillus, Caldicellu The invention also provides a bacterium of the genus losiruptor, Pseudomonas, Alcanivorax, Brevibacterium, Bifidobacterium, Escherichia or Staphylococcus, more preferably Escherichia coli; or a bacterium of the genus Aspergillus, Candida, Saccharomyces, Ustilago, Cryptococcus, etc. cus), Fusarium, Rhizopus, Magnaporthe, Komagataella, Trichderma, Penicillium, Acremonium, Mucor, Alternaria, Botrytis, Endothia, Rhizoctonia, Sclerotinia, Kluyveromyces lyveromyces, Torulopsis, Sporotrichum, Geotrichum, Verticillium, Botryosphaeria, Trichothecium, Hansenula, Schizosaccharomyces, Brettanomyces or Neurospora, preferably Candida or Saccharomyces.

It is further preferred that the genetically modified organism, tissue or cell is eukaryotic. It is more preferred that the genetically modified organism is a plant, or the tissue is a plant tissue, or the cell is a plant cell. In another particularly preferred embodiment, the genetically modified organism is a higher plant attacked by an insect herbivore, or the genetically modified tissue belongs to a higher plant attacked by an insect herbivore, or the genetically modified cell is a higher plant cell, wherein the plant is attacked by an insect herbivore.

The term "insect herbivore" as used herein relates to insects that are anatomically and physiologically adapted to eat plant material, such as leaves as the main component of their diet. Due to their plant feeding, herbivorous insects generally have mouthparts adapted for rasping or grinding. It is particularly preferred that the insect herbivore is an insect that feeds by tearing and scouring and/or by piercing and sucking.

In a specific embodiment, the plant is a higher plant attacked by insects that feed in a tearing and scouring and/or piercing and sucking manner. The feeding mechanism of "tearing and scouring" insects generally involves repeated insertion and withdrawal of a stinger from plant tissue. "Piercing and sucking" refers to piercing plant cells with a stinger, liquefying them, and then using saliva to suck the ruptured cell contents with a suction mouth. More information can be obtained from suitable textbooks such as Strong et al., Insects on Plants, Harvard University Press (1984).

In other embodiments, the plant is a plant attacked by an insect that lays its eggs in the leaves of the plant. Non-limiting examples of such insects include bagworms, aphids, mealybugs, cicadas, earworms, beetles, lace bugs, leafhoppers, and scales.

In a further preferred embodiment, the herbivore insect is a leafhopper or a planthopper. Even more preferably, the insect belongs to Aphrodinae, Bathymatopohorinae, Cicadellinae, Coelidiinae, Deltocephalinae, Erromeninae, Euacanthelinae, Eurymelinae, Evacantinae, Hylicinae, Iassinae, Jascopinae, Ledrinae, Megophthalminae, Mileewinae, Nastlopinae, Neobaliane, Neocoelidinae, Nioniinae, Phereurhinina, Portaninae, Signoretiinae, Tartesinee, Typhlory Even more preferably, the insect belongs to the genus Empoasca, Circulifer, Nilaparvata, Sogatella, Nepotettix or Cicadulina. In a specific embodiment, the plant is therefore a higher plant attacked by leafhoppers or planthoppers, more preferably by plants belonging to the subfamily Aphrodinae, Bathymatopohorinae, Cicadellinae, Coelidiinae, Deltocephalinae, Erromeninae, Euacanthelinae, Eurymelinae, Evacantinae, Hylicinae, The present invention is preferably attacked by insects belonging to the family Iassinae, Jascopinae, Ledrinae, Megophthalminae, Mileewinae, Nastlopinae, Neobaliane, Neocoelidinae, Nioniinae, Phereurhinina, Portaninae, Signoretiinae, Tartesinee, Typhlory bina or Ulopinae, or even more preferably by insects belonging to the genera Empoasca, Circulifer, Nilaparvata, Sogatella, Nepotettix or Cicadulina.

In a more preferred embodiment, the higher plants are crop plants attacked by insects that feed by tearing and scouring and/or piercing and sucking.

In a most preferred embodiment, the higher plant belongs to the genus Nicotiana, Solanum, Oryza, Zea, Phaeseolus or Camellia.

In another aspect, the invention relates to the use of a polypeptide or a polynucleotide as defined above, an expression cassette as defined above or a vector or an insertion construct as defined above for the preparation of a compound according to the invention (eg as defined above).

In another aspect, the present invention relates to a composition comprising a compound according to the present invention as defined above, or a compound prepared by a method as defined above, or a compound prepared by an organism, tissue or cell (preferably a genetically modified organism, tissue or cell) as defined above. The composition may preferably be an agricultural composition. The term "agricultural composition" used herein refers to a composition suitable for agriculture or gardening, such as a composition comprising an effective concentration and amount of a compound of the present invention such as defined herein. In a specific embodiment, the composition may further be used for use on or in a plant in a non-agricultural environment.

In one embodiment, the composition may be designed for use in or on a plant or at the locus of a plant. Typically, it may be used in the foliage or flowering region of a plant, alternatively in the root region. The term "plant locus" is to be understood as any type of environment, soil, area or material in which a plant grows or is expected to grow. Preferably, the term relates to the soil on which the plant grows.

An "effective amount" or "effective concentration" of a compound as defined herein can be determined according to suitable in vitro and in vivo tests known to those skilled in the art. These amounts and concentrations may be adjusted according to the location, plant species or variety, soil, climatic conditions or any other suitable parameter that may have an effect on herbivore attack.

The composition may include a suitable carrier, preferably an agricultural chemical carrier. The term "agricultural chemical carrier" used herein is a material or composition that helps agricultural chemicals (including compounds as defined herein) to be delivered and/or released in the field of use, particularly on or in plants. Examples of suitable agricultural chemical carriers include solid carriers, such as mineral soils, such as silicates, silica gel, talc, kaolin, limestone, lime, chalk, red soil, loess, clay, dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate, magnesium oxide, ground synthetic materials; fertilizers, such as, for example, ammonium sulfate, ammonium phosphate, ammonium nitrate, urea, and products of plant origin, such as cereal powder, bark powder, wood powder and nut shell powder, cellulose powder, and other solid carriers. Other suitable examples of carriers include fumed silica or precipitated silica, which can be used, for example, as flow aids, anti-caking aids, grinding aids, and as carriers of liquid active ingredients in solid formulations. Another example of a suitable carrier is microparticles, such as microparticles that adhere to plant leaves and release their contents within a specific time period. In a specific embodiment, such an agricultural chemical carrier may be a composite gel microparticle, which can be used to deliver plant protection active ingredients, for example as described in US 6,180,141; or a composition comprising at least one plant active compound and an encapsulated adjuvant, wherein the adjuvant comprises fungal cells or fragments thereof, for example as described in WO 2005/102045; or a carrier particle, which is coated with a lipophilic tackifier on the surface, wherein the carrier particle adheres to the surface of plants, grasses and weeds, for example as disclosed in US 2007/0280981. In a further specific embodiment, such a carrier may include specific, strong binding molecules, which ensure that the carrier adheres to the plant until its contents are completely delivered. For example, the carrier may be or comprise a cellulose binding domain (CBD), which has been described as a useful agent for attaching molecular species to cellulose (see US 6,124,117); or a direct fusion between a CBD and an enzyme; or a multifunctional fusion protein that can be used to deliver an encapsulated agent, wherein the multifunctional fusion protein may consist of a first binding domain (which is a carbohydrate binding domain) and a second binding domain, wherein either the first binding domain or the second binding domain can bind to microparticles (see also WO 03/031477). Other suitable examples of carriers include bifunctional fusion proteins consisting of a CBD bound to microparticles and an anti-RR6 antibody fragment. In another specific embodiment, the carrier may be an active ingredient carrier particle adhered to a plant surface using a water-active coating, such as gum arabic, guar gum, karaya gum, tragacanth gum, and locust bean gum. When the particles are applied to plant surfaces, water from precipitation, irrigation, dew, water co-applied with the particles in special application equipment, or water from the plants themselves can provide sufficient moisture for the particles to adhere to the plant surface (see also US 2007/0280981).

In other embodiments, the composition may comprise or may additionally comprise a stabilizer. The term "stabilizer" as used herein refers to any suitable compound or composition which allows reducing the reactivity of the compound according to the invention and/or which prevents or slows down the reactive moieties of the composition according to the invention from interacting with each other and/or autocatalytically polymerizing. In a preferred embodiment, the stabilizer is a compound or composition capable of reducing the pH and/or maintaining a low pH, more preferably maintaining a pH of 4.8 or below. In other embodiments, the stabilizer may be a detergent, such as a zwitterionic detergent, etc.

In other embodiments, the composition may comprise or may additionally comprise a spreading agent. The term "spreading agent" as used herein refers to a substance whose addition to a liquid or semisolid composition results in an enlargement of the surface covered by the composition. Spreading agents typically act by, for example, reducing the surface tension of the composition or by increasing the wettability of the treated surface. Thus, it facilitates the uniform distribution of the composition and its ingredients, and also facilitates the formation of a uniform film on the treated surface (e.g., plant leaves). The spreading agent typically inhibits the tendency of compositions with high surface tension (e.g., aqueous solutions) to form droplets on the treated surface, which would result in a spotty concentration of substances present in the composition. Suitable examples of spreading agents to be used in the context of the present invention include oils or fats, such as short-chain fatty acids (C6 to C10), for example coconut oil and babassu seed oil, vegetable fats, squalene; medium-chain fatty acids (C12 to C16) or with a high lecithin or squalene content, for example avocado oil, sesame oil, grape seed oil or amaranth oil; or long-chain fatty acids (C18 to C24), for example evening primrose oil, borage oil, hemp oil and wild rose oil. Another class of suitable spreading agents is the hydrophobins, i.e. small, cysteine-rich proteins with a length of about 100-150 amino acids, which occur in nature only in filamentous fungi.

In other embodiments, the composition according to the present invention may include an adjuvant. The "adjuvant" used refers to a compound that assists or changes the effect of the main component of the composition (e.g., the compound according to the present invention). Adjuvants can serve as wetting agents, adhesives, accelerators (perpetrators) or activators. In specific embodiments, they can be used for spraying.

An example of an adjuvant contemplated by the present invention is a surfactant. A "surfactant" is a surface active agent that is generally amphipathic and comprises a hydrophobic group and a hydrophilic group. They can be anionic, cationic, nonionic or amphoteric. Examples include LAS, LES, CTAC, DODAC, APEO, FAEO and AEO.

The composition according to the present invention may further include additional active ingredients. For example, the composition may include, for example, at least one insecticidal compound. For example, the composition may additionally include at least one herbicidal compound and/or at least one fungicidal compound and/or at least one insecticidal compound. Suitable examples of herbicidal compounds include, but are not limited to, 2,4-dichlorophenoxyacetic acid, aminopyralid, atrazine, clopyralid, dicamba, glufosinate, fluazifop-butyl, clofopyralid, glyphosate, metazapine, imazamox, imazamox, linuron, 2-methyl-4-chlorophenoxyacetic acid, isopropyl metolachlor, paraquat, pendimethalin, pyralid, pyralid, fluazifop-butyl or metsulfuron. Suitable examples of fungicidal compounds include, but are not limited to, carbendazim, thiophanate-methyl, thiabendazole, flusilazole, azoxystrobin, oxadiazole, kasugamycin or rice blast. Suitable examples of insecticidal compounds include, but are not limited to, contact insecticides such as cephate, carbaryl, fipronil, pyrethrins, pyrethroids such as bifenthrin, cyfluthrin, cypermethrin, deltamethrin, lambda-cyhalothrin, permethrin, es-cypermethrin, tefluthrin or tralomethrin, and fipronil or spinosad; or surfactant insecticides, including, for example, neonicotinoid-based compounds and silicone-based surfactants. P450 inhibitors are also contemplated, such as piperniyl butoxide, which is a general P450 inhibitor. These inhibitors can be used in combination with the contact insecticides or surfactant insecticides mentioned above.

In a particularly preferred embodiment, the composition is designed or prepared for plant protection, in particular for plant protection against herbivores, e.g. as defined herein. For example, the composition can be formulated in any suitable manner to allow application to plants, e.g. higher plants, preferably crops as defined herein. For example, the composition can be formulated to include an adjuvant, a spreading agent and/or a surfactant, which allows effective application, e.g. by spraying or any other suitable method.

In another aspect, the invention relates to the use of a compound according to the invention as defined above, or a compound prepared by a method as defined above, or a compound prepared by an organism, tissue or cell (preferably a genetically modified organism, tissue or cell) as defined above, or a composition as defined above, for plant protection. In a preferred embodiment, the invention relates to the use of a compound according to the invention as defined above, or a compound prepared by a method as defined above, or a compound prepared by an organism, tissue or cell (preferably a genetically modified organism, tissue or cell) as defined above, or a composition as defined above for plant protection, for plant protection against herbivores.

Preferably, the herbivore is an insect herbivore as defined above, preferably a leafhopper or a planthopper, more preferably belonging to the family Aphrodinae, Bathymatopohorinae, Cicadellinae, Coelidiinae, Deltocephalinae, Errhomeninae, Euacanthelinae, Eurymelinae, Evacantinae, Hylicinae, The insect belongs to the family of Iassinae, Jascopinae, Ledrinae, Megophthalminae, Mileewinae, Nastlopinae, Neobaliane, Neocoelidinae, Nioniinae, Phereurhinina, Portaninae, Signoretiinae, Tartesinee, Typhlory bina or Ulopinae. Even more preferably, the insect belongs to the genus Empoasca, Circulifer, Nilaparvata, Sogatella, Nepotettix or Cicadulina.

Therefore, the compound can be provided by the organism, tissue or cell. It can be provided in any suitable amount, concentration or form known to those skilled in the art. The compound can, for example, accumulate in the organism, tissue or cell, and thereby help protect the organism, tissue or cell from herbivore attacks. Alternatively, the compound can be excreted or exported or otherwise delivered to the outside of the organism, cell or tissue, and then used for different purposes, for example as a component in a plant protection composition.

In another aspect, the invention relates to the use of a compound according to the invention as defined above, or of a compound prepared by a method as defined above, or of a compound prepared by an organism, tissue or cell (preferably a genetically modified organism, tissue or cell) as defined above, or of a composition as defined above as an insecticide. The insecticide may be employed in any suitable situation or environment, or on or in any plant as deemed suitable by the skilled person. Preferably, the insecticide is a specific insecticide against insect herbivores, more preferably against insect herbivores that feed by tearing and scouring and/or by sucking, even more preferably against leafhoppers or planthoppers, for example, belonging to the family Aphrodinae, Bathymatopohorinae, Cicadellinae, Coelidiinae, Deltocephalinae, Errhomeninae, Euacanthelinae, Eurymelinae, Evacantinae ), Hylicinae, Iassinae, Jascopinae, Ledrinae, Megophthalminae, Mileewinae, Nastlopinae, Neobaliane, Neocoelidinae, Nioniinae, Phereurhinina, Portaninae, Signoretiinae, Tartesinee, Typhlory bina or Ulopinae. Even more preferably, the insect belongs to the genus Empoasca, Circulifer, Nilaparvata, Sogatella, Nepotettix or Cicadulina.

In another aspect, the present invention relates to organisms as defined herein, tissues or cells, or organisms, tissues or cells comprising expression cassettes as defined herein, or vectors as defined herein or insertion constructs for the preparation of the purposes of compounds according to the present invention. Therefore, the compound can be provided by the organism, tissue or cell. It can be provided in any suitable amount, concentration or form known to those skilled in the art. The compound can, for example, accumulate in the organism, tissue or cell, and thereby contribute to protecting the organism, tissue or cell from herbivorous attacks. Alternatively, the compound can be excreted or exported or otherwise delivered to the outside of the organism, cell or tissue, and then used for different purposes, for example as a component in a plant protection composition.

In another embodiment, the present invention relates to the use of an organism as defined herein (e.g. an organism comprising an expression cassette as defined herein, or an organism genetically modified to express a compound of the present invention) for agricultural, horticultural or ornamental plant production. Preferably, the organism is a higher plant, such as a crop, which is used to produce a food product, or it may be a plant used for horticultural purposes or as an ornamental plant. By producing a compound of the present invention, the organism (e.g. a plant) is protected from herbivorous animals and can therefore produce a food product, or be sold as a horticultural or ornamental plant.

Purpose according to the present invention can be continuous use or cycle use.For example, cycle use can for example mean use in a specific time period, for example 1,2,3,4,5,6,7 days or more days, 1,2,3,4,5,6,7,8,9,10 weeks or more weeks, 1,2,3,4,5,6,7,8,9,10,11,12 months or more months, or 1,2,3,4,5,6,7,8,9,10 years or more years, or any time period between the above values.Described purposes can be based on any suitable amount, concentration or form of using compound or composition according to the present invention.

In a final aspect, the present invention relates to a method for plant protection, which comprises contacting a plant or a part of a plant with a compound according to the invention or a composition as defined herein. The method may comprise the steps of preparing the compound or composition according to the invention for the application form contemplated, for example by formulating as a spray, etc.; optionally packaging into suitable containers, for example for transport or storage, or directly for use in an application device; spreading the compound or composition to the target, for example higher plants in a field susceptible to herbivorous attack, by a suitable application technique. Alternatively, the method may be based on the use of pre-prepared materials, for example propagation materials that have been contacted with the compound or composition at a location different from the location of the plant.

The method may be performed once or more than once, for example 2, 3, 4, 5, 6, 7, 8 times, within a specific time period, such as a week, a month or a year.

The application step may include, for example, contacting the compound according to the present invention or its composition with a plant or plant propagation material (e.g., seed). This may include spraying, applying, coating, granulating, dusting or soaking application methods. Alternatively, the compound according to the present invention or composition may be applied by a ground machine or an aircraft or an unmanned aircraft (UAV) such as a drone.

The following examples and figures are provided for illustrative purposes. It should therefore be understood that the examples and figures should not be interpreted as limiting. It will be apparent to those skilled in the art that further modifications to the principles described herein can be considered.

Example

Example 1

General Strategy

In order to uncover the non-host resistance trait of JA-induced in Nicotiana attenuata, we adopted a forward genetic strategy.The repeated population of 650 recombinant inbred lines (RIL) from 26 parent multi-parent advanced generation intercross (MAGIC) colonies was planted in the natural growth environment of Arizona (Fig. 1 and Figure 21 and 22).Under this environment, the leafhopper of Empoasca genus is a large amount, and destroys its natural host cucumber (stinky melon (Cucurbita foetidissima)).The leafhopper attack (Kallenbach et al., Proc Natl Acad Sci U S A 109, E1548-1557 (2012)) of the Nicotiana attenuata system lacking JA, and its attack rate is different (Fig. 1) in the MAGIC colony.Based on the high-throughput analysis of plant hormones, transcriptome and metabolome, the leafhopper attack level in 1907 individual plants of 674 RILs and parental lines of the MAGIC colony grown in field was quantified, and a multi-omics data set was constructed. This multi-omics dataset was generated from leaves induced by simulated herbivory treatments. Herbivore attack was simulated by treating standardized puncture wounds (W+OS) with oral secretions of tobacco hornworm larvae to eliminate confounding factors caused by the stochastic nature of insect attack in nature and to capture transiently expressed genes and metabolites in these samples from field-grown plants (Figures 2 and 23 and 24).

In order to analyze the association between genetic and metabolic responses of this multi-omics data set, JA signaling-related genes were focused on, and the knowledge of leaf chemistry of Nicotiana acuminate obtained previously (Li et al., Proc Natl Acad Sci USA 112, E4147-4155 (2015), Li, S. et al., Proc Natl Acad Sci USA 113, E7610-E7618 (2016)) was used to construct a co-association network of JA-dependent modules. This network not only takes into account the correlation between metabolites, plant hormones and gene expression, but also takes into account the shared SNPs (Figure 3, Figure 25) inferred by the analysis of metabolic quantitative trait loci (mQTL) or expression QTL (eQTL) for each of these components. The co-association network showed that JA and JA-related genes nucleated by the JA-regulated phenolamide master transcription factor regulator NaMYB8 (Onkokesung et al., Plant Physiol 158, 389-407 (2012)) formed a genetic center that clustered with induced phenolamides such as N-coumaroyl putrescine (CoP), N-caffeoyl putrescine (CP), N-feruloyl putrescine (FP) and malonylated 17-HGL-DTG (Figure 3). More peripheral to the center were glycosylated 17-HGL-DTG precursors such as lycium barbarum I, lycium barbarum IV, attenoside and nicotinic acid III and other special metabolites such as nicotine, acyl sugars and flavonoids (Figure 25). NaJAZi genes clustered around JA, but away from NaJAR4 and NaCOI1, implying the involvement of non-canonical JA signaling. To further analyze the components contributing to green leafhopper susceptibility in the co-association network, we performed pairwise correlation analysis of the omics datasets with green leafhopper abundance and damaged leaf area in RILs of the MAGIC population (Fig. 4). Putrescine-derived phenolamides and malonylated 17-HGL-DTG were negatively correlated with green leafhopper abundance and damage, while glycosylated 17-HGL-DTG precursors were positively correlated. JA-related genes NaMYB8, NaLOX3, NaAOC, NaOPR3, NaJAR6, and NaWIPK showed the highest negative correlation scores with green leafhopper abundance and damage. There was considerable heterogeneity in the expression of the JA-associated JAZ gene family, with expression of NaJAZa, NaJAZd, NaJAZf, NaJAZi, and NaJAZj negatively correlated with Empoasca number and damage; expression of JAZ genes known to mediate defense responses in M. Sexta, such as NaJAZh (Oh et al., Plant Physiol 159, 769-788 (2012)), did not show significant correlations. JA (but not JA-Ile, the canonical mediator of JA signaling) was negatively correlated, and negative correlations were also observed for hydroxylated and carboxylated JA (OH-JA, OH-JA-Ile, and COOH-JA-Ile) and JA-valine conjugates (JA-Val). These results imply that the JA signaling sector may be involved in the JA-dependent resistance response of Empoasca leafhoppers.

Example 2

The JA-JAZi module induced by the JA-JAZi module regulates the resistance to Empoasca spp.

To further disentangle the complexity of the JA signaling component initiated by Empoasca and the downstream metabolic features it regulates that lead to Empoasca resistance, a reverse genetics approach was used to examine the involvement of phenolamides. Using laboratory colonies of Empoasca decipiens, isogenic lines of N. attenuata plants that were RNAi-silenced (ir: inverted repeat) or overexpressed (ov) in different JAZ genes and NaMYC2 were screened in a greenhouse open selection screening experiment to evaluate the lack of JA signaling in NaMYB8, the master TF regulator of phenolamides, and in DH29 and CV86, which catalyze the spermidine conjugation step in phenolamide biosynthesis (Figure 5 and Figures 26 and 27). In contrast to nymphs and adults of E. decipiens, which preferentially selected irMYC2, irMYB8, and ovJAZi plants for feeding and reproduction, other transgenic lines were only slightly damaged by a few probing events (Figure 5). Multiple JAZ proteins allow the JA signaling cascade to regulate a range of metabolic and developmental traits in different tissues at different times (Chini et al., Curr Opin Plant Biol 33, 147-156 (2016), Major et al., New Phytol 215, 1533-1547 (2017)), thereby contextualizing responses and optimizing adaptability. The modularity of the JA-JAZi portion may provide specific responses associated with Empoasca leafhoppers. Tissue pan-transcriptomic analysis of all JAZ genes in the Nicotiana attenuata genome showed that NaJAZi was highly expressed in flower tissues and had no response to M. Sexta attacks in leaves (Li et al., Proc Natl Acad Sci U S A 114, E7205-E7214 (2017)), while NaJAZh showed the opposite pattern (Figures 6 and 28). These patterns were confirmed by exposing leaves to E. decipiens and Manduca sexta attack and monitoring the kinetics of JAZ transcript accumulation: Manduca sexta feeding triggered NaJAZh transcripts in leaves, while E. decipiens feeding triggered NaJAZi transcripts in leaves (Figure 6). The Y2H assay showed that NaJAZi interacted with NaMYC2a, while NaMYB8 interacted with NaMYC2b (Figure 29). These data suggest that the MYC2-MYB8-JAZi JA moiety is involved in E. decipiens resistance in leaves.

To identify metabolites triggered by this JA moiety, leaves of rosette plants of JA signaling defective transgenic lines ( FIG. 5 ), as well as on irAOC and irCOI1 plants, on irGGPPS lacking 17-HGL-DTG accumulation, and on irPMT plants with impaired nicotine accumulation ( FIG. 30 and 31 ), were raised with E. decipiens adults and nymphs or Manduca sexta larvae. High-resolution indifferent (data-independent) MS/MS spectra (referred to as idMS/MS) were collected from these leaf extracts using an analytical and computational workflow ( Li et al., Proc Natl Acad Sci USA 112, E4147-4155 (2015), Li et al., Proc Natl Acad Sci USA 113, E7610-E7618 (2016), Li et al., Sci Adv6, eaaz0381 (2020)). Metabolome specialization (δ index), metabolome diversity (Hj index) and metabolic specificity (Si index) of individual metabolites were quantified using information theory framework (Li et al., Sci Adv 6, eaaz0381 (2020), Li and Gaquerel, Annu Rev Plant Biol 72, 867-891 (2021)). In the dimension of metabolome specialization and diversity processed by information theory, tobacco hornworm attacks triggered overall higher metabolome plasticity, thereby producing a higher δj score than that attacked by green leafhopper (E. decipiens). Different transgenic lines show different trajectories (Fig. 7 and Figure 32) of metabolome plasticity reprogrammed by attacks of two insect species.

Focusing on the transgenic lines preferred by Empoasca, it is noted that the unique features of metabolome specialization caused by herbivore attack are weaker in irMYC2 and irMYB8 plants (Fig. 7 and Fig. 32). This means that MYC2 is the main regulator of metabolome plasticity in response to insect attack, and the herbivorous induced phenolamides of MYB8 dependence include metabolic parts that cause increased metabolome specialization. In the ovJAZi system, the metabolome change trajectory caused by Empoasca and Manduca attack is separated (rather than eliminated), which further points out that a small part of metabolites caused by Empoasca attack is regulated by NaJAZi, and may be related to Empoasca resistance.

To identify these metabolites, SI scores were ranked according to metabolite specificity calculated from each MS/MS spectrum of the E. decipiens-induced metabolome in the four transgenic lines and correlated with co-expression heatmaps derived from correlations between individual metabolites and E. decipiens numbers and damage calculated using the overall variance generated from all reverse genetics lines used in the feeding experiment (Figure 8, Figure 32). Phenolamides topped the metabolite-specific Si scores, with putrescine-derived metabolites ranking first, and these were negatively correlated with E. decipiens numbers and damage, unlike specific 17-HGL-DTG, quinate conjugates, and nicotine that showed positive correlations (Figure 8).

Putrescine-derived phenolamides CoP, CP, and FP were reduced in irAOC, irCOI1, irMYC2, and irMYB8 lines, and selectively decreased in ovJAZi plants damaged by feeding on Empoasca, but not in ovJAZi plants damaged by feeding on Manduca, whereas other spermidine-derived phenolamides showed similar responses to attack by both herbivore species in ovJAZi plants ( FIG. 33 ). Based on the lack of response of leafhoppers to irDH29 and irCV86 lines, spermidine-derived metabolites could be excluded as mediators of Empoasca non-host resistance ( FIG. 5 ). To further explore the involvement of CoP, CP, and FP, in vivo Empoasca selection assays were performed by infiltrating leaves of irMYC2 plants lacking the priming phenolamides at physiologically relevant concentrations, respectively ( FIG. 9 and FIG. 34 ). However, these infiltrations did not alter the preference of Empoasca for irMYC2 plants. In vitro Empoasca direct feeding assays using physiologically relevant concentrations of the individual compounds in glucose solution showed no significant changes in mortality of Empoasca compared to the E. decipiens controls fed on glucose (Figure 9). These data suggest that CoP, CP, and FP are not the direct cause of Empoasca resistance, but rather other, as yet unknown, putrescine-derived phenolamide metabolites.

Example 3

Multi-omics reveals defense and its three-branching pathways

Leaves of Nicotiana acuminate plants attacked by herbivores grown in greenhouses accumulated a variety of putrescine and spermidine-derived phenolamides (Li et al., Proc Natl Acad Sci US A112, E4147-4155 (2015), Onkokesung et al., Plant Physiol 158, 389-407 (2012)). 15 RILs (Figure 1) were selected from the field-based multi-omics data set of the MAGIC population, which accumulated high levels of structurally different OS-induced phenolamides to build idMS/MS and identify the structure of putrescine-derived phenolamides; This effort produced 518 non-redundant idMS/MS spectra (Figure 10). Double clustering analysis was performed to cluster spectra according to fragments (normalized dot products) and similarities based on neutral losses, which produced seven modules (Figure 10). Module 5, which is particularly rich in phenolamide-related compounds containing caffeoyl or putrescine groups, was further mapped onto a molecular network (Figure 10). The unknown compound at m/z 347.196 ([M+H] ⁺ , C ₁₉ H ₂₇ N ₂ O ₄ ⁺ ) occupied the first layer of direct connection network neighbors of the two isomers of CP (m/z 251.14) because they shared the neutral loss of putrescine of Δ88.10 Da and the fragment peak at m/z 163.04 (C ₉ H ₇ O ₃ +), which corresponds to the caffeoyl group ( Figures 10 and 35 ). Due to the loss of putrescine in the molecular ion, the idMS/MS of the fragment peak at m/z 259.09 (C ₁₅ H ₁₅ O ₄ ⁺ ) showed that it was further fragmented to m/z 163.04 with a neutral loss of 96.055 Da (C ₆ H ₈ O). This means that the unknown m/z 347.19 is a CP derivative decorated with C ₆ H ₈ O residues on the aromatic ring of the caffeoyl group ( FIG. 35 ).

To test whether the unknown m/z 347.19 metabolite is regulated by a specific JA-JAZi module, the co-association network and co-expression analysis of induced m/z 347.19 on leafhopper number and damage and JA in field-grown MAGIC populations was explored (Fig. 1). m/z 347.19 was negatively correlated with leafhopper damage and positively correlated with JA, JA-Ile, and JA-Val (Fig. 36). In the next step, the leafhopper-induced metabolome of JA-deficient transgenic lines was mined (Fig. 2). However, using a similar computational workflow, it was not possible to identify this compound in the previously described dataset (Fig. 2). Extensive experiments showed that field-grown RILs induced more of this unknown compound than greenhouse-grown RILs (Data 2), and that subtle differences in leaf sampling and extraction techniques, particularly simultaneous exposure of leaves to aluminum foil and liquid nitrogen and adverse pH conditions (Table 1), led to the loss of this labile phenolamide in plants grown and sampled under greenhouse conditions.

Empoasca was again reared on irMYC2 plants and, by optimizing extraction conditions, it was found that Empoasca feeding strongly triggered accumulation of m/z 347.19 in EV plants; these accumulations were abolished in irMYC2 lines (Figure 11). In this extraction optimization work, it was realized that the Empoasca leafhopper priming procedure could be replaced with experimentally more tractable larval oral secretions or MeJA priming. MeJA-induced production of m/z 347.19 was consistently hampered in irMYC2, but also in irMYB8 and ovJAZi lines (Figure 11). These results suggest that the unknown m/z 347.19 is partially regulated by JA-JAZi-MYC2-MYB8 signaling, which may contribute to Empoasca resistance.

To investigate the biosynthetic origin of m/z 347.19, OS-induced leaves of the entire MAGIC RIL population grown under greenhouse conditions and phenolamide permissive conditions were extracted and mQTL analysis was performed (Figure 12). The analysis inferred a series of genes known to be involved in CP regulation and biosynthesis (P's <10-3), including NaMYC2a, NaMYB8 and NaAT1, which encode hydroxycinnamoyl-CoA: putrescine acyltransferases responsible for CP biosynthesis (Onkokesung et al., Plant Physiol 158, 389-407 (2012)), and two polyphenol oxidases NaPPO1 and NaPPO2 located on chromosomes 7 and 8, respectively (Figure 12). Co-expression analysis of a microarray dataset of irMYB8 lines using NaAT1 and NaDH29 as bait (Schafer et al., J Integr Plant Biol 59, 844-850 (2017)) revealed a cluster of genes known to be involved in CP biosynthesis in the NaAT1 grouping, including NaPAL1, NaPAL2, Na4CL1, and NaC3H. The berberine bridge enzyme-like gene NaBBL2 was highly co-expressed with NaAT1, and its induced expression was reduced in the irMYB8 lines (Figures 12 and 37). The mQTL dataset was revisited and NaBBL2, located on chromosome 3, was found to be associated with m/z 347.19 (Figure 12), although the statistical significance was reduced (P = 0.0013). The time-resolved microarray data (Kim et al., PLoS One 6, e26214 (2011)) of NaAT1, NaPPO2 and NaBBL2 expression caused by herbivory show that NaAT1 and NaPPO2 show similar induction patterns in Nicotiana acuminate WT, while NaBBL2 is highly induced at 1 hour and keeps its induction at a later time point, although the level is reduced (Figure 38). In addition, in the RNAseq transcriptome data set caused by herbivory in irMYC2 system, its induction of NaAT1, NaPPO1, NaPPO2 and NaBBL2 is reduced (Figure 39).

Although the number and order of the biosynthetic steps of m/z 347.19 accumulation are still difficult to determine, it is speculated that oxidation and acylation reactions may be required. Therefore, further steps focus on the oxidase and acyltransferase deduced jointly from multi-omics analysis. Candidate genes include three acyltransferases NaACT1/2/3, three polyphenol oxidases NaPPO1/2/3 and a BBL gene NaBBL2. The expression of the candidate genes in Nicotiana acuminate and the NaAT1 as a positive control was evaluated by using virus-induced gene silencing (VIGS). Consistent with previous analysis, the VIGS of NaAT1 eliminated the accumulation of m/z 347.19 (Li et al., Proc Natl Acad Sci U S A112, E4147-4155 (2015)), and NaPPO1, NaPPO2 and NaBBL2 intercepted the initiation of m/z 347.19 (Figure 13 and Figure 40). Untargeted metabolomics analysis of VIGS-silenced plants showed that silencing NaPPO1 and NaPPO2 truncated the accumulation of m/z 347.19, while other phenolamides did not change (Figures 13 and 41). These results indicate that NaPPO1, NaPPO2, and NaBBL2 are required for the in vivo production of m/z 347.19.

Previous analysis implied that the additional _C6H8O residue at m/z 347.19 was generated by _a fatty acid oxidation lipid cascade that converts C18 polyunsaturated fatty acids released from biofilms during stress, wounding, and herbivory (Matsui, Curr Opin Plant Biol 9, 274-280 (2006)) to produce green leaf volatiles (GLVs) rich in reactive C6 derivatives (Li et al., Proc Natl Acad Sci USA 112, E4147-4155 (2015)). However, the source of the C6 metabolites and the biochemical reactions involved in the formation of m/z 347.19 remain unknown. To elucidate this, herbivory-induced GLVs were measured in the same greenhouse-grown MAGIC RIL population for population (Figure 3) and correlated with herbivory-induced m/z 347.19 accumulation. (Z)-3-hexenal-derived volatiles, such as (Z)-3-hexenyl-propionate and (Z)-3-hexenol, were the most significantly positively correlated metabolites with m/z 347.19, while 1-hexanol, linalool, or other induced volatiles were not (Figures 14 and 42).

C6 aldehyde (whose molecular formula is _{C6H8O )} is the most reactive aldehyde produced from the GLV pathway, and it has been postulated (Li et al., Proc Natl Acad Sci US A112, E4147-4155 (2015)) that it is the missing substrate in the biosynthesis of m/z 347.19. Consistent with previous analyses (Allmann et al., Plant Cell Environ 33, 2028-2040 (2010)), stable silencing of lipoxygenase 2 (irLOX2) in Nicotiana attenuata, which controls the first key step in the GLV pathway, abolished C6 aldehyde production and total GLV emission, and stable silencing of the crossover of irLOX2 and irLOX3 (irLOX2×irLOX3) completely abolished m/z 347.19 production (Li et al., Proc Natl Acad Sci US A112, E4147-4155 (2015)) (Figure 15). Silencing NaHPL (using antisense construct: asHPL), which catalyzes the formation of the initial C6 GLV product of (Z)-3-hexenal and its isomer (E)-2-hexenal, resulted in a significant time-dependent reduction in GLV in N. acuminate (Joo et al., Plant Cell Environ 42, 972-982 (2019)), as well as reduced accumulation of m/z 347.19 (to about 1/3 of WT levels: Figure 15). From these results, it was concluded that in response to Empoasca detection, m/z 347.19 was produced by a trifurcated metabolic pathway consisting of the LOX2/HPL-GLV pathway, the LOX3-JA-regulated phenylpropanoid pathway, and the polyamine pathway, which consisted of NaPPO1, NaPPO2, and NaBBL2 using CP and (Z)-3-hexenal or (E)-2-hexenal condensation to produce m/z 347.19 (Figure 14).

In order to verify this hypothesis, the purified NaPPO1, NaPPO2 and NaBBL2 proteins (Figures 44-45) with N-terminal hexa-histidine tags after being expressed in Escherichia coli were separated. NaPPO1 or NaPPO2 (NaPPO1/2) were hatched with CP and (Z)-3-hexenal to produce m/z 347.19 peak, and its MS/MS spectrum and retention time were identical with the m/z347.19 induced in Nicotiana acuminate leaf blade (Figure 46). In addition, the by product ([M+2H] ²⁺ , C ₂₆ H ₃₆ N ₄ O ₆₂ +) (data 2) of the double-charged CP dimer at m/z250.13 place that was not detected in the Nicotiana acuminate leaf WT blade induced by oral secretions was also produced in vitro (Figure 16 and Figure 47). Only when the amount of (Z)-3-hexenal substrate was lower than the amount of external CP substrate, these double-charged CP dimers would be produced. NaPPO1/2 showed little to no activity when incubated with CP and (E)-2-hexenal (Figure 16). NaBBL2 alone cannot use CP and (Z)-3-hexenal as substrates to produce m/z 347.19, and under in vitro conditions, in the presence of NaPPO1/2, CP and (Z)-3-hexenal, the addition of NaBBL2 did not significantly increase the preparation of m/z 347.19 (Figures 48-52 and Table 2). Because PPO has a wide range of substrate specificity, it can accept hydroxybenzene and/or ortho-dihydroxylated benzene as substrates (McLarin and Leung, S Crit Rev Biochem Mol Biol 55, 274-308 (2020)), by incubating NaPPO1/2 and (Z)-3-hexenal with CoP or chlorogenic acid (CGA) containing aromatic dihydroxy as CP, the substrate specificity of NaPPO1 and NaPPO2 was explored. However, no new products were found, indicating that CoP and CGA were not accepted as substrates by NaPPO1/2 (Figure 53). Taken together, these data revealed that NaPPO1/2 accepted CP and (Z)-3-hexenal as substrates to produce m/z 347.19 in vitro.

Example 4

Reactivity m/z 347.19 Biosynthetic logic of chemical composition

In order to illustrate the chemical structure of m/z 347.19, attempt to use induced Nicotiana tabacum leaf material and enzyme assay derivative product to separate and purify m/z 347.19.But, due to the instability of m/z 347.19, several attempts all failed.Although relatively stable in ammonium acetate buffer (pH 4.8), when concentrated by rotary evaporation or freeze drying, m/z 347.19 decomposes rapidly (table 1).These observed results show that m/z 347.19 is reactive and unstable at high pH.The purification procedure of m/z 347.19 has been modified to prepare in large quantities by enzyme assay under weakly acidic conditions, and solid phase extraction is used to purify m/z 347.19 under argon atmosphere.Then NMR analysis is carried out to the purified m/z 347.19, which illustrates that its structural feature is CP-5-(Z)-3-hexenal compound (hereinafter referred to as CPH) (Figure 54). At room temperature and in the dark, in acidified methanol-d3 (0.1% formic acid), the half-life of CPH is only about 22 h (Figure 55). CPH contains both the reactive groups of the α,β-unsaturated aldehyde from (Z)-3-hexenal (which is electrophilic) and the amine features of caffeoylputrescine (which is nucleophilic).

CPH is the result of a biochemical association of so-called “direct” (CP) and “indirect” ((Z)-3-hexenal) defense metabolites, and CPH is hypothesized to be the metabolic trait underlying nonhost resistance in Empoasca. Two possible mechanisms of action have been suggested: rapid polymerization of electrophilic and nucleophilic groups blocks the probing mouthparts of Empoasca leafhoppers; and α,β-unsaturated aldehydes can act as protein cross-linkers that disable Empoasca proteins (29). A three-step biosynthetic mechanism for the preparation of CPH has been proposed: oxidation of CP by NaPPO1/2 to the corresponding caffeoylquinone derivative, activation of (Z)-3-hexenal for Michael addition, and aromatization of the product to form CPH (Fig. 17).

CPH is responsible for resistance in Empoasca

To test whether CPH is responsible for resistance to green leafhoppers, NMR-confirmed CPH was fed to green leafhoppers (E. decipiens) in vitro in a diet containing 10% glucose at a physiologically relevant concentration of 1 μM (estimated from induced leaves collected in the field). After 6 hours of feeding, CPH treatment caused almost 100% mortality of green leafhoppers (E. decipiens), which was in sharp contrast to the growth of leafhoppers under the control diet (P = ^3x10-8 , Studentt-test) (Figure 18). NaAT1 expression in Nicotiana acuminate plants was silenced using VIGS, which disrupted the production of CP and CPH (Li et al., Proc Natl Acad Sci US A112, E4147-4155 (2015)) (Figure 13). In vivo selection assays revealed that NaAT1-silenced plants received significantly greater green leafhopper damage and higher green leafhopper numbers than EV plants (Figure 18). Similarly, silencing of NaPPO1 or NaPPO2 in plants only abolished the accumulation of CPH in plants without significantly altering other phenolamide pools (Figures 13 and 41), and resulted in a clear preference for Empoa feeding (P = 3.8 x 10-6 and P = 1.5 x 10-3, Student t-test, respectively) and greater Empoa damage (P = 1.1 x 10-7 and P = 3.5 x 10-6, Student t-test, respectively) compared to EV plants (Figure 18). Taken together, these in vitro and in vivo results indicate that CPH in N. attenuata is responsible for Empoa non-host resistance in this plant.

NaBBL2 is required for engineering CPH biosynthesis in crops

The discovery of CPH and its biosynthetic pathway behind non-host resistance provides a framework for CPH biosynthetic engineering in crops as a means to optimize the endogenous metabolism of plants to defend against attacks by devastating leafhopper pests, the diseases they carry, and other non-host pests of crops. It was investigated whether CPH is widespread in Solanaceae and other plant taxa. Metabolic profiling of Nicotiana attenuata relatives revealed that 6 of 7 tobacco species induce CPH in a coordinated manner with CP under MeJA priming (Figure 56). Specifically, 13 different taxa were selected from different plant families, including several crops, and the amino acid identities of NaAT1, NaPPO1, NaPPO2 and NaBBL2 were compared with their closest homologs using BLAST searches of the NCBI sequence database (Figure 57): 5 Solanaceae taxa out of the 13 species examined contained all orthologs of the four protein sequences. In addition, MeJA-induced CP was detected in 8 species, including 7 Solanaceae species and wheat, while only 2 species, pepper and Nicotiana benthamiana, produced CPH in addition to Nicotiana attenuata (Figure 57). These results imply that CPH production may be limited to the Solanaceae family.

Synthetic biology allows metabolic pathways to be transferred between different taxa due to shared cofactors and metabolism (30-33). Attempts have been made to reconstruct the complete CPH pathway in vivo (Figure 20).

Vicia faba and Solanum tumefaciens were chosen for Agrobacterium tumefaciens-mediated transient expression of the CPH pathway for several reasons: Both species do not accumulate CPH in untreated and MeJA-treated tissues. Vicia faba is an ideal host plant for rearing Empoasca spp. CoP, CP or FP do not accumulate in Vicia faba, while MeJA treatment of Solanum tumefaciens induces CP levels, providing internal precursors for CPH production (Figure 20). In addition, both are easily transformed and are likely to produce properly folded, active proteins that can be used to test the biochemical activity of CPH biosynthetic genes in plants.

NaPPO1 or NaPPO2 were transiently co-expressed in Vicia faba with (Z)-3-hexenal and CP leaf infiltration or in Solanum oleraceum without CP infiltration. However, no CPH was detected in either species (Figure 20). PPOs are normally localized in plastids, which would physically separate them from their phenolic substrates, which are known to be localized in the vacuole (34). In Nicotiana attenuata, thylakoid translocation domains were identified in both NaPPO1 and NaPPO2 N-terminal sequences (Figure 58). Transient expression of GFP-tagged NaPPO1 and NaPPO2 in Nicotiana attenuata leaves confirmed that both NaPPO1 and NaPPO2 were localized in plastids (Figure 59).

Thus, the proposed three-branch pathway for CPH biosynthesis is challenged by the separate enzyme locations of the different components: CP (possibly in the vacuole or cytoplasm: (34, 35)); GLV, JA, and PPO (plastids: (34, 36)). This challenge is reminiscent of nicotine biosynthesis, which requires the BBL gene to link the mitochondrial-localized pyridine ring from niacin with the peroxisomal-localized pyrrolidine ring from the N-methylpyrrolinium cation to produce niacin (37). It was hypothesized that NaBBL2 is required for CPH production in vivo, and to test this hypothesis, NaBBL2 was expressed in Solanum tumefaciens plants together with NaPPO1 or NaPPO2. One day after infiltration with Agrobacterium tumefaciens, Solanum tumefaciens plants were treated with MeJA to induce CP production, and three days later, (Z)-3-hexenal was infiltrated into the leaves. Six hours later, the leaves were harvested for LC-MS analysis, which revealed that the leaves had accumulated a large amount of CPH (Figure 20). For faba bean plants, NaBBL2 was expressed together with NaPPO1 or NaPPO2; CP and (Z)-3-hexenal were infiltrated into leaves 3 days after Agrobacterium tumefaciens infiltration, and leaves were harvested 6 hours later for LC-MS analysis. CPH accumulated again (Figure 20). From these results, it is inferred that NaBBL2, although not required for in vitro synthesis, is required for CPH biosynthesis in vivo. Additional work is needed to evaluate whether NaBBL2 plays a role in solving the location challenge, which may have other possible solutions (Figures 60 and 61 and Supplementary Materials). Finally, green leafhopper feeding experiments were conducted on CPH-engineered faba bean and Solanum oleraceum plants, and it was observed that these green leafhopper host crops became lethal host plants for green leafhopper (Figure 20).

This mechanistic analysis of non-host resistance in green leafhoppers provides another example of innovative chemical solutions evolved by natural plants to solve their ecological challenges (Rajniak et al., Nature 525, 376-379 (2015)). The natural history-driven multi-omics framework used to discover CPH and its combination with synthetic biology programs highlights how easily the innovations of millions of years of natural selection can be transferred to crops to catalyze the next generation of greener and more economically meaningful revolutions in plant protection (S.M.Cook et al., Annu Rev Entomol 52, 375-400 (2007)) and domestication (Zhu et al., Cell 172, 249-261e212 (2018), Sanchez-Perez et al., Science 364, 1095-1098 (2019), Szymanski et al., Nat Genet 52, 1111-1121 (2020)). The challenges facing crops are not so different from those faced by natural plants, they are constantly tested by herbivore communities that challenge the host/non-host distinction. In a world of climate change and globally homogenized herbivore communities, opportunistic associations will dominate natural and artificial ecosystems. Understanding how natural plants cope with opportunistic associations will help design crops that are more resilient to the unknown pressures of global climate change (Xu and Weng, Advanced Genetics 1, e10022 (2020)).

Example 5

Summary of Materials and Methods

Two replicates of 650 RILs from 26 parental MAGIC populations and their 26 parental lines were planted at the WCCER field station in Prescott, Arizona, USA. To elicit a standardized herbivory response, leaves of all RILs at the early flowering stage were wounded and immediately treated with diluted Manduca sexta oral secretions (W+OS) or not treated (control) and harvested on dry ice at 1 and 72 h. One week after metabolite sampling, all plants in the field population were screened for the number and damage of natural Empoasca leafhoppers; these leafhoppers would opportunistically sample N. acuminate plants from neighboring natural cucumber host plants. A set of 646 RILs from the MAGIC population were used for mQTL and eQTL mapping between SNPs and the relative abundance of each compound or transcript using the R package GAPIT using general linear models (GLM). Multi-omics co-association networks were constructed from correlations between metabolomes, transcriptomes, phytohormones, and SNPs. For the Empoasca selection assay, transgenic lines of Nicotiana acuminate at the early rosette growth stage were randomly placed in an open-selection greenhouse environment containing Empoasca leafhoppers reared on leguminous plants in the MPI-CE greenhouse in Isserstedt, Germany. Jasmonic acid signaling genes induced by Empoasca were characterized using yeast two-hybrid and qRT-PCR. Compound-specific idMS/MS were constructed using UHPLC-ESI/qTOF-MS for idMS/MS acquisition and rule-based computational approach for idMS/MS assembly. Metabolome diversity and specialization as well as metabolic specificity were calculated using information theory by considering the Shannon entropy of idMS/MS frequency distribution. Empoasca selection in vivo and Empoasca feeding assays in vitro were performed by infiltrating synthetic caffeoylputrescine (CP), coumaroylputrescine (CoP) or feruloylputrescine (FP) into irMYC2a/2b leaves or by feeding Empoasca leafhoppers with compounds diluted in 10% glucose solution. MS/MS structural metabolomics analysis was performed using idMS/MS constructed from 15 RILs that induced putrescine-containing phenolamides and accumulated multiple known and unknown phenolamides upon OS induction. MS/MS similarity scoring, bi-clustering, and molecular networking were used to identify the unknown m/z 347.19 metabolite. OS-induced volatile emissions were collected from 650 MAGIC RILs planted in the main MPI-CE greenhouse using polydimethylsiloxane (PDMS) tubes and analyzed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). NaPPO1/2 and NaBBL2 genes were elucidated by combining herbivory-induced mQTL analysis of the unknown m/z 347.19 and transcriptomic analysis of microarray and RNAseq datasets of OS induction dynamics of WT and irMYC2a/2b and irMYB8 lines. The candidate genes were functionally validated by virus-induced gene silencing (VIGS) and in vitro enzyme assays using Escherichia coli expressed NaPPO1, NaPPO2 and NaBBL2 with CP and (Z)-3-hexenal. The chemical structure of CPH (CP-5-(Z)-3-hexenal) was characterized by NMR. The resistance function of CPH against Empoa spp. was tested by in vitro non-selective assays with synthetic CPH or by in planta selection assays using VIGS plants of EV, NaPPO1, NaPPO2 and NaAT1. The biosynthetic pathway of CPH was reconstructed in Vicia faba and Solanum oleraceum by transient co-expression of NaPPO1, NaPPO2 and NaBBL2 in the presence of leaf infiltration of CP and (Z)-3-hexenal. The non-host resistance function of CPH against Empoa spp. was further evaluated using CPH-engineered Vicia faba and Solanum oleraceum plants.

Example 6

Plants and materials Methods, metabolite extraction

Plant and material methods: Nicotiana attenuata of inbred 31 generations (originally collected at DI Ranch in southwestern Utah, USA) was used as the wild-type (WT) genetic background for all experiments and transformations. Plants were grown in a greenhouse at the Max Planck Institute for Chemical Ecology (MPI-CE) in Jena, Germany, with a diurnal cycle of 16h (26-28°C)/8h (22-24°C) as previously described (Krügel et al., Chemoecology 12, 177-183 (2002)). VIGS experiments were performed in a climate chamber (York) with growth conditions of 22/22°C 16/8h light/dark at 65% relative humidity at the start of the experiment, with a light level of approximately 100 μmol m-2s-1 for 2 days after inoculation, after which the light level returned to normal high light levels (400-1000 μmol m-2s-1 PAR) (Saedler and Baldwin, J ExpBot 55, 151-157 (2004)). Empoasca selection assays were performed in a plant growth chamber (Percival Scientific) with a constant temperature of 24°C, a light regime of 16 h day/8 h night, 70% relative humidity, and a light intensity of 100% (350 μmol s-1 m-2 PAR). To prevent Empoasca from infesting the main greenhouse facility of MPI-CE, we conducted large-scale selection assays in another greenhouse facility located in Isserstedt, Germany (approximately 7 km from the main greenhouse facility). The selection assay was carried out under natural light conditions, a constant temperature of 24°C and a relative humidity of 70%.

Metabolite extraction: Approximately 100 mg of ground leaf tissue was weighed and extracted using an extraction buffer containing 80% methanol as follows. 1 ml of extraction buffer (50 mM acetate buffer (pH 4.8) containing 80% methanol) was added per 100 mg of tissue, and the sample was homogenized in a ball mill (Genogrinder 2000; SPEX CertiPrep) at 1× speed and 1100 strokes per minute for 45 seconds. The homogenized sample was centrifuged at 16,000×g at 4°C for 30 minutes, and 800 μL of the supernatant was transferred into a 1.5-mL microcentrifuge tube and re-centrifuged as before. 600 μL of the supernatant was transferred to a 2 mL glass vial for MS-based metabolomics analysis.

Example 7

Magic 2019 field experiment, insect collection and handling

Magic 2019 field experiment: 650 recombinant inbred lines (RILs) of the MAGIC population and two replicates of 26 parental lines used to breed the MAGIC RIL population were planted at the Walnut Creek Center for Education and Research (WCCER) in Prescott, Arizona, USA (Ray et al., Plant J. 99, 414-425 (2019)). Before planting, a drip irrigation system was installed to water the MAGIC population plants. The field included 8 main pipes with a diameter of 1 inch, each main pipe included 4 irrigation pipe branches, each branch included 18 drippers, and each dripper irrigated 4 plants. Four plants were planted at each dripper with an interval of 50 cm, and clusters of 4 plants were planted at an interval of 150 cm. Figure 21 depicts the irrigation system and the overall planting layout, where the vertical lines represent the 8 main water pipes. The tobacco seeds treated with tobacco were allowed to grow on the Plants were germinated in peat pellets that had been hydrated with natural soil extracts to provide the plants with an appropriate microbiome during germination (Santhanam et al., Proc Natl Acad Sci US A112, E5013-E5020 (2015)). To elicit a standardized herbivore-specific response in a kinetically defined manner, three rows of puncture wounds were made on each side of the leaf midrib using a fabric pattern wheel (Dritz, Spartanburg, SC) and immediately treated with 20 μL of a 1:5 dilution of tobacco hornworm oral secretions (W+OS) or left untreated (control) (McCloud and Baldwin, Planta 203, 430-435 (1997)). On each plant sampled, the first three stem leaves were treated with W+OS and sampled at 1 hour and 72 hours; control leaves were sampled at 0 hours. All leaf samples were immediately frozen on dry ice and transported by aircraft on dry ice in dry ice shipping containers and stored in a −80 °C freezer at MPI-CE. One week after metabolite sampling, the field team counted the number of green leafhoppers on each plant and visually estimated the proportion of leaf area damaged by leafhopper feeding on each plant in the MAGIC population.

Insect collection and treatment: A laboratory colony of Empoasca decipiens was established from insects collected from Buddleja (Butterfly Lilac) plants growing in the MPI-CE environment in Jena, Germany. Insects were reared in four mesh tents with broad beans in a greenhouse in Isserstedt, Germany. The tents (220 x 90 x 110 cm, Tatonka, Germany) were completely covered with mesh to allow air exchange. In order to screen for genetic and metabolic responses specific to leafhopper attack, 25 adult leafhoppers were kept in 50-mL plastic containers (Huhtamaki) on leaves of different transgenic lines at the rosette stage (Figures 30 and 31). Empty 50-mL plastic containers were placed on EV plant leaves as controls. For metabolomics analysis, the leaves were treated for 72 hours, the plastic containers were removed from the plants together with the leaves and insects, and the leafhoppers were returned to the colony. To quantify transcript abundance, samples were collected at 0, 0.5, 1, 2, 3, 6, 9, and 24 hours. Harvested leaves were immediately frozen in liquid nitrogen and stored in a -80 °C freezer until use. Control leaves were harvested and stored in a similar manner.

M. Sexta eggs were obtained from an indoor colony maintained at the Department of Evolutionary Neuroethology at the MPI-CE in Jena, Germany, as described previously (Koenig et al., Insect Biochem Mol Biol 66, 51-63 (2015)). M. Sexta feeding assays were performed by placing newly hatched M. sexta on fully expanded rosette stage leaves.

For methyl jasmonate (MeJA) treatment, MeJA (Sigma, catalog number: 392707-25ML) was dissolved in hot liquefied lanolin (50°C) at a concentration of 7.5 mg mL-1. Pure lanolin was used as a negative control. With 20 μL of lanolin paste (Lan+MeJA) containing 150 μg MeJA, with 20 μL of lanolin+wound treatment (Lan+W), or with 20 μL of pure lanolin as a control (Baldwin et al., J Chem Ecol 22, 61-74 (1996)), the axis outside of the base of the first three fully elongated leaves was treated (4 weeks after potting). Leaves were harvested 72 hours after treatment (midrib removed), quickly frozen in liquid nitrogen, and stored at -80°C until use.

Example 8

Green leafhopper selection experiment and transgenic lines

Green leafhopper selection experiment: make the tobacco plant attenuate in the main greenhouse facility of MPI-CE germinate and grow, and transfer to the Isserstedt greenhouse in the early stage of rosette stage.After plant is transferred to the Isserstedt greenhouse, make plant adapt to at least two days, then start experiment.Plant is randomly placed on the table, the distance between each other is at least 40 centimeters.Green leafhopper (E.decipiens) colony tent is placed on two tables adjacent to the tobacco plant attenuate (Figure 26), and about 400 green leafhopper (E.Decipiens) individuals are released into the greenhouse.After two weeks, the percentage of the total leaf area of each leaf is estimated by visual estimation, as previously described (Kallenbach et al., Proc Natl Acad Sci U S A109, E1548-E1557 (2012)).The average leaf damage of three different leaf types has been calculated: rosette leaf, the first (oldest) three stem leaves and all younger stem leaves. We also counted the number of green leafhopper individuals on each plant. Each genotype was represented by 10 replicate plants.

Plants with silenced PPO1, PPO2 and BBL2 were tested for green leafhopper selection in tents placed in a growth chamber (Percival Scientific) (Figure 31). Plants with silenced PPO1, PPO2 and BBL2 were transferred from the greenhouse to the tents when the plants were in the early elongation stage of growth. After the plants were transferred to the tents, the plants were adapted for at least two days and then exposed to green leafhopper (E. decipiens) attacks. Plants were randomly placed in the tents with a distance of at least 15 cm between each other; each tent included four genotypes: EV, PPO1, PPO2 and BBL2 silenced plants. 50 green leafhopper (E. decipiens) adults were released into each tent. After one week, the damaged area was visually estimated and quantified as a percentage of the total leaf area of each leaf, as described above. We also counted the number of green leafhopper individuals on each plant in the tent. Each genotype was represented by 8 replicate plants.

Generation of transgenic lines: NaMYC2 (irMYC2, NaJAZi, NaJAZc, NaJAZe, NaJAZL, NaJAZg, NaDH29, NaCV86, NaCV86, NaCV86, NaCV86) were generated by a published Agrobacterium tumefaciens-mediated transformation method (Krügel et al., Chemoecology 12, 177-183 (2002)) using a pSOL8 binary vector containing inverted repeats of NaMYC2 (LOC109232914, LOC109205493), NaJAZi (LOC109240311), NaJAZc (LOC109233155), NaJAZe (LOC109223947), NaJAZL (LOC109220335), NaJAZg (LOC109219395), NaDH29 (LOC109206371), or NaCV86 (LOC109206370). A-17-110-2-2)/JAZi (irJAZi, A-17-013-2)/JAZc (irJAZc, A-09-220-4-1)/JAZe (irJAZe, A-09-250-7)/JAZg (irJAZg, A-19-070-5)/JAZL (irJAZL, A-18-029)/DH29 (irDH29, A-06-051-1)/CV86 (irCV86, A-06-022-6) silenced lines and NaJAZi (ovJAZi, A-17-007-1)/JAZL (ovJAZL, A-18-042) overexpression lines. NaJAZi and NaJAZL overexpression lines were constructed by using pSOL9 vectors with NaJAZi (LOC109240311) and NaJAZL (LOC109220335) sequences, respectively. Homozygous _T2 diploid plants carrying a single insertion were used in all studies. Insertion copy number and fidelity of insertion (overreading and truncation) were evaluated by NanoString analysis (He et al., Proc Natl Acad Sci US A116, 14651-14660 (2019)). In short, DNA extracted from a single transgenic plant was used for NanoString The specific region of the inserted fragment was detected by the designed probe. In order to verify the single copy number insertion inferred from the selective marker resistance segregation rate, a published transformation line with a single copy insertion (irAGO8) was used as a positive control (Pradhan et al., Plant Physiol 175, 927-946 (2017)).

The following four previously characterized transgenic lines were also used in this study: irAOC (A-07-457-1) plants silenced in the expression of the NaAOC gene (Kallenbach et al., Proc Natl Acad Sci U S A 109, E1548-E1557 (2012)), irCOI1 (A-04-249-A-1) plants silenced in the expression of the NaCOI1 gene (Paschold et al., Plant J 51, 79-91 (2007)), irMYB8 (A-07-810-2) plants silenced in the expression of the NaMYB8 gene (Onkokesung et al., Plant Physiol 158, 389-407 (2012)), irJAZh (A-09-368-7) plants silenced in the expression of the NaJAZh gene (Oh et al., Plant Physiol 159, 769-788 (2012)), irPMT (A-03-108) plants silenced in the expression of the NaPMT gene (Steppuhn et al., PLoS Biol 2, E217 (2004)), irGGPPS (A-08-231) plants silenced in the expression of the NaGGPPS gene (Heiling et al., Plant Cell 22, 273-292 (2010)), asHPL (A-247) plants silenced in the expression of the NaHPL gene (Halitschke et al., Plant J 40, 35-46 (2004)), and irLOX2 (A-04-52-2) and irLOX2 and LOX3 hybrid (A07-707-2) plants silenced in the expression of the NaLOX2 and NaLOX2 and NaLOX3 genes, respectively (Allmann et al., Plant J 40, 35-46 (2004)). CellEnviron 33, 2028-2040(2010)).

Example 9

Transcriptomics data mining and microarray analysis

Transcriptomics data mining and analysis of microarray and RNAseq data sets : Microarray data were originally published in Kim et al., PloS One 6, e26214 (2011) and stored in the National Center for Biotechnology Information Gene Expression Comprehensive Database (GEO number: GSE30287). Before statistical analysis, the original intensity was log2 and baseline converted using the R software package and normalized to its 75th percentile. Raw RNAseq data were processed as described in Pertea et al., Nat Protoc 11, 1650-1667 (2016). In short, the original RNA-seq readings were first converted to fastQ format. HISAT2 converts fastQ to sam, and SAMtools converts sam files to sorted bam files. StringTie was used to calculate gene expression with the number of fragments (FPKM) per kilobase transcript per million sequencing reads.

Association mapping: Using the R package software GAPIT (Genome Association and Prediction Integrated Tool), a set of 646 RILs from the MAGIC population were used to perform mQTL and eQTL mapping between SNPs and the relative abundance of each compound or transcript (Lipka et al., Bioinformatics 28, 2397-2399 (2012)). General linear models (GLM) were used for association analysis.

Example 10

Volatile substance measurement

Volatile substance measurement in MAGIC RIL population : To characterize the volatile substance emission induced by herbivores, 650 MAGIC RILs were planted in a single replicate in the greenhouse of MPI-CE. As described above for metabolite analysis, the third oldest stem and leaf of each plant was treated with wounds and tobacco hornworm (M. Sexta) oral secretions (W+OS), and the treated leaves were individually packaged in 500-mL transparent (PET) cups. Volatiles were collected for 24 hours on two sections of 5-mm polydimethylsiloxane (PDMS) tubes suspended in the head space above the leaves in the collection cup (Kallenbach et al., Plant J78, 1060-1072 (2014)). PDMS tubes were collected and stored at -20°C until analysis.

Volatiles were analyzed by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS). GC-MS (Shimadzu, GCMS-QP2010Ultra) was equipped with a TD autosampler (Shimadzu TD-20) and a semipolar capillary column (Phenomenex ZB-WAXplus, 30m x 0.25mm ID, 250μm film thickness). The compound was desorbed as previously described (Kallenbach et al., Plant J 78, 1060-1072 (2014)), and separated by applying the following column temperature gradient: 0 to 5 minutes constant temperature at 40°C, 5 to 34 minutes linear temperature increase to 185°C, 34 to 35.5 minutes linear temperature increase to 230°C, 35.5 to 36 minutes constant temperature at 230°C. The MS detector was run in full scan mode from m/z 33 to 400. Relative quantification of green leaf volatiles and terpenoids was based on peak integration of extracted ion chromatograms of characteristic fragment ions. Compounds were identified based on retention times and comparison of mass spectra with authentic standards and an in-house MS library.

Embodiment 11

Protein localization and qRT-PCR analysis

Protein localization : Agrobacterium tumefaciens strain GV3101 carrying 35S::NaPPO1-GFP or 35S::NaPPO2-GFP was infiltrated into N. attenuata leaves. Three days after infiltration, confocal images of the epidermal cells outside the axis of the infiltrated leaves were acquired using a CLSM 510 confocal scanning microscope (Zeiss).

qRT-PCR analysis of JAZ and m/z 347.19 8CHP biosynthetic genes : Wild-type leaves of N. attenuata were cut from the stem with scissors and tissue samples were collected at the 0 hour time point (control), snap frozen in liquid nitrogen and stored at -80°C until use. For treated leaves, one leaf from each plant was exposed to feeding by E. Decipiens or M. Sexta in a growth chamber (Figures 30 and 31). Leaf samples were collected at 0.5, 1, 2, 6, 9, 12 and 24 hours after treatment, snap frozen in liquid nitrogen and stored at -80°C until use.

Primers were designed for specific regions of the targeted gene using Primer3 (Untergasser et al., Nucleic Acids Res 40, e115 (2012)), with amplicon lengths between 70 and 200 bp. After grinding the leaf tissue samples in liquid nitrogen, total RNA was extracted from aliquots of 100 mg of powdered leaf material according to the standards of the NucleoSpin RNA Plant Kit (MACHEREY-NAGEL, catalog number: 740949.50). RNA samples were measured on a Nanodrop spectrophotometer (Peqlab, ND-1000). 1 μg RNA was used to synthesize cDNA according to the standards of the PrimeScript ^TM RT Kit (Perfect Real Time, Takara, catalog number: RR037B). Taykon ^TM No ROX was used on a Stratagene MX3005P instrument. MasterMix (Eurogentec, catalog number: UF-NSMT-B0701) was used for qPCR experiments. The Nicotiana attenuata elongation factor 1-α gene (EF1-α; accession number GBGF01000210.1) was used as a housekeeping gene to standardize the qPCR results. All qPCR primers are listed in Table 3.

Example 12

Yeast two-hybrid assay and VIGS

Yeast two-hybrid assay : Y2H assay was performed using the Matchmaker Gold yeast two-hybrid system (Clontech) according to the manufacturer's instructions. Briefly, constructed AD and BD fusions were transformed into freshly prepared Y2Hgold competent cells together with their own empty vector (as a negative control) using the Yeastmaker ^TM yeast transformation kit (Clontech, catalog number: 630304) and plated on selective dropout medium (SD-Leu/-Trp). Transformants were recorded after incubation at 30°C for 5-7 days on QDO (SD-Leu/-Ade/-His/-Trp) medium in the presence of 2mM 3-AT.

VIGS : A vector (pTV00) containing fragments of NaAT1 and candidate genes for biosynthesis of CPH metabolites was generated, and plant growth and VIGS inoculation were performed as previously described (Saedler and Baldwin, J Exp Bot 55, 151-157 (2004)). Briefly, to silence the NaAT1 gene and m/z 347 biosynthesis candidate genes (including NaPPO1, NaPPO2, NaBBL2), 150-400 bp antisense fragments of these genes were cloned into the polylinker of the pTV00 vector (provided by Sir David Baulcombe's laboratory) and transformed into Agrobacterium tumefaciens GV3101. The empty cloning vector pTV00 was used as a negative control for the non-specific phenotypic effects of VIGS. The positive control vector pTVPD was prepared by cloning a 206 bp fragment of the Nicotiana benthamiana phytoene desaturase gene (NtPDS, Niben101Scf01283g02002.1) in the antisense direction into the polylinker of pTV00. Plants were treated by the needleless syringe infiltration method described previously (Saedler and Baldwin, J Exp Bot 55, 151-157 (2004)) 23-25 days after germination. Four weeks after potting, silent leaves were induced by W+OS treatment. After three days, the treated leaves were quickly frozen in liquid nitrogen and stored at -80°C until use, and untreated leaves were collected in a similar manner as controls. The VIGS experiment was repeated at least three times.

Example 13

Cloning of candidate CHP biosynthetic genes and reconstruction of the CHP pathway

Cloning of candidate CHP biosynthetic genes : According to the manufacturer's instructions, all PCR amplification steps were performed using Phusion high-fidelity DNA polymerase (New England Biolabs, catalog number: M0530L). Oligothymidine primers were purchased from Integrated DNA Technologies. DNA fragments were purified from agarose gel using NucleoSpin Gel and PCR clean-up kit (Macherey-Nagel, catalog number: 740609.50). Before transformation into other heterologous hosts, plasmid separation was performed using One Shot ^TM TOP10 chemically competent Escherichia coli (Invitrogen, catalog number: C404006). Plasmid DNA was isolated from Escherichia coli culture using NucleoSpin plasmid kit (Macherey-Nagel, catalog number: 740588.50). After the target gene was amplified by PCR, the sequence was confirmed by Sanger dideoxy sequencing. The PCR fragment was purified using DyeEx 2.0 spin kit (250) (QIAGEN, catalog number: 63206). Sequencing was performed on an ABI 3130 gene analyzer at the MPI-CE molecular ecology department in Jena, Germany. For a list of primers used for cloning, see Table 3.

Reconstruction of the CHP pathway in bioassays of broad bean, Solanum chilensis and Empoasca spp.: Sequences corresponding to the full-length open reading frames of all selected candidate genes were obtained from the Nicotiana acuminate genome release 2.0 (Xu et al., Proc Natl Acad Sci USA 114, 6133-6138 (2017)). The gene sequences were PCR amplified (for primers, see Table 3) and recombined into the donor vector pDONR207. The sequence-verified entry clones of the candidate genes (including NaPPO1, NaPPO2 and NaBBL2) were recombined into the pEAQ-HT-DEST vector by Gateway and sequence-verified. The resulting pEAQ-HT construct was transformed into Agrobacterium tumefaciens (GV3101) using electroporation. Transformants were grown on yeast extract broth plates supplemented with 20 mg mL ^-1 gentamicin, 100 mg mL ^-1 rifampicin and 25 mg mL ^-1 kanamycin. Single colonies were inoculated into 5 mL of yeast extract broth supplemented with 20 mg mL ^- 1 gentamicin, 100 mg mL ^-1 rifampicin and 25 mg mL ^-1 kanamycin. After overnight incubation, the bacteria for transient coexpression were mixed, precipitated by centrifugation, and resuspended in infiltration buffer (100 μM acetosyringone, 10 mM MgCl ₂ , and 10 mM MES, pH 5.7). After 2 hours of incubation at room temperature, the bacterial mixture (OD=0.25 for each strain) was infiltrated into the outer side of the axis of the fully expanded leaves of 3-4 strains of 6-week-old broad beans or Chilean nightshade plants grown in a growth chamber maintained under VIG conditions. The leaves of Chilean nightshade plants were treated with MeJA. The infiltrated and MeJA-treated plants were incubated under normal growth conditions for 3 days before metabolite analysis. Biological replicates consisted of several leaves from different plants. For substrate infiltration experiments, 500 μM (E)-N-caffeoyl putrescine (BOC Sciences, catalog number: B0005-053482) and 1 mM cis-3-hexenal (Sigma, catalog number: W256102-sample-K) dissolved in 50 mL acetate buffer (20 mM, pH 4.8) containing 0.01% DMSO aqueous solution were infiltrated into the outer surface of the axis of leaves previously infiltrated with PPO1, PPO2 and BBL2 Agrobacterium tumefaciens using a needleless 1-mL syringe. One day after Agrobacterium tumefaciens infiltration, 1 mL (E)-N-caffeoyl putrescine (500 μM) or cis-3-hexenal (1 mM) was infiltrated in EV plant leaves as a negative control. Leaf disks were harvested after one day, snap frozen and extracted for CPH metabolite profiling on LC-MS. Four CPH-accumulating broad bean or eggplant leaves were selected for Empoasca bioassay, and 25 adult leafhoppers were housed in 50-mL plastic containers (Huhtamaki) on each leaf previously infiltrated with Agrobacterium tumefaciens. Empoasca survival was recorded every 1 hour overnight.

Embodiment 14

UHPLC-ESI/gTOF-MS attribute analysis mode analysis conditions and MS/MS data acquisition conditions

UHPLC-ESI/gTOF-MS attribute analysis mode analysis conditions and MS/MS data acquisition conditions : An Acclaim column (150×2.1 mm, particle size 2.2 μm, ThermoFisher Scientific) equipped with a UHPLC SecurityGuard ^™ ULTRA cartridge (Phenomenex, catalog number: AJO-8782) was used for analysis. The following binary gradient was performed using a Dionex Ultimate3000 UHPLC system: 0 to 0.5 min, isocratic 90% A (deionized water, 0.1% (v/v) acetonitrile and 0.05% formic acid), 10% B (acetonitrile and 0.05% formic acid); 0.5 to 23.5 min, gradient step to 10% A, 90% B; 23.5 to 25 min, isocratic 10% A, 90% B. The flow rate was 400 μL min ^-1 . For all MS analyses, the column eluent was injected into an Impact II or Compact (Bruker Daltonics, Bremen, Germany) quadrupole time-of-flight (qTOF) mass spectrometer equipped with an electrospray source operated in positive ionization mode (capillary voltage 4500 V, capillary outlet 130 V, drying temperature 200 °C, dry gas flow 10 L min ⁻¹ ).

The indiscriminate MS/MS mode was achieved by operating the quadrupole with a very large mass isolation window, which made all m/z signals considered as fragments. To this end, several independent analyses were performed with increasing CID collision energy values, because neither Impact II nor Compact Bruker instruments could perform CE gradients. In brief, the sample was first analyzed by UHPLC-ESI/qTOF-MS, where a single MS mode (low fragmentation conditions from in-source fragments) was used, by scanning from m/z 50 to 1500 with a repetition rate of 5 Hz. Indiscriminate MS/MS analysis was performed using nitrogen as the collision gas, and included independent measurements at 4 collision-induced dissociation voltages: 20, 30, 40 and 50 eV. Throughout the measurement, the quadrupole was operated with the maximum mass isolation window, from m/z 50 to 1500. When the precursor m/z and isolation width parameters were experimentally set to 200 and 0 Da, respectively, this mass range was automatically activated by the instrument's operating software. Scan the mass fragments in the manner described for the single MS mode. Mass calibration was performed using sodium formate (50 mL isopropanol, 200 μL formic acid, 1 mL 1 M NaOH in water). Post-run calibration of the data files was performed using the Bruker HPC (High Precision Calibration) algorithm based on the average spectrum of the calibration segment at the beginning of each run. Raw data files were converted to netCDF format using the export function of the Data Analysis v4.0 software (Bruker Daltonics, Bremen, Germany).

IdMS/MS assembly and similarity scoring : Data-independent or indiscriminate MS/MS fragment analysis (hereinafter referred to as idMS/MS) is performed to obtain structural information about overall detectable metabolic properties. IdMS/MS assembly is achieved by correlation analysis between MS1 and MS/MS mass signals for low and high collision energies and newly implemented rules. Correlation analysis of precursor to product distribution is implemented using R scripts, and rules are implemented using C# scripts ( https://github.com/MPI-DL/indiscriminant-MS-MS-assembly-pipeline ) (Li et al . , Sci Adv 6, eaaz0381 (2020)).

For MS/MS similarity scoring, idMS/MS spectra were aligned in pairs and their similarity was calculated based on two scores. First, the fragment similarity between spectra was scored using the standard normalized dot product (NDP), also known as the cosine correlation method, using the following formula:

where S1 and S2 correspond to spectrum 1 and spectrum 2, respectively, and W _S1,i and W _S2,i represent the peak intensity-based weights given to the ith common peak that differs less than 0.01 Da between the two spectra.

The weights are calculated as follows:

W = [peak intensity] ^m [mass] ⁿ where m = 0.5 and n = 2 (Li et al., Sci Adv 6, eaaz0381 (2020)).

Implemented the second scoring method, it relates to the shared neutral deletions between each MS/MS.For this reason, we used the list of 52 neutral deletions (NL) often encountered in the tandem MS fragment process, and the more specific neutral deletions (Li et al., Sci Adv 6, eaaz0381 (2020)) previously annotated for MS/MS spectra of the Nicotiana acuminate secondary metabolite class, Li et al., Proc Natl Acad Sci U S A 113, E7610-E7618, (2016), Li et al., Proc Natl Acad Sci U S A 112, E4147-E4155 (2015)). Create a binary vector of 1 and 0 for each MS/MS, corresponding to the presence and absence of a certain NL, respectively.Based on the Euclidean distance similarity, calculate the NL similarity score of every pair of binary NL vectors.

Assembly rules for compound-specific idMS/MS: Due to the fact that some m/z features were detected in only a few samples, in order to reduce false positive errors arising from spurious correlations with background noise, we compared the data processing results obtained with and without the "Fill Peaks" function of XCMS (for background noise correction), and calculated background noise values from the average correction estimate used by this function to replace the "NA" intensity values of undetected peaks. When the "Fill Peaks" function was used, many "0" intensity values would still remain in the data set, which would affect the calculation of correlations, and these values would be replaced by the calculated background values. We also considered only features whose intensity was more than 3 times the background value and regarded these as "true peaks". For PCC calculations, only m/z signals with at least eight times "true peaks" in the precursor (MS1) and fragment (MS/MS) data sets were considered.

A precursor mass feature was further defined if its intensity in the sample correlated significantly with the decreased intensity of the same mass feature at low or high collision energies and the feature was not annotated as an isotopic peak by CAMERA. Correlation analysis was then performed by calculating all possible precursor-to-product pairs within a 3-s retention time window. An m/z value was considered a fragment only if its m/z value was lower than that of the precursor and the MS/MS fragment appeared in the data set at the same sample position as the precursor from which it was derived.

Many mass features of in-source generated fragments produced in MS1 mode can also be selected as candidate precursors, leading to redundant compound idMS/MS. To reduce this data redundancy, we merged spectra if their NDP similarity exceeded 0.6 and they belonged to the same chromatogram “pcgroup” annotated by CAMERA. Finally, all results from the 4 collision energies used for precursor-to-fragment association were merged into the final deconvoluted composite spectrum by selecting the highest intensity peak among all candidate peaks with the same m/z value at different collision energies.

Embodiment 15

MS/MS molecular network analysis by biclustering

MS/MS molecular network analysis by biclustering : For clustering, the R package DiffCoEx was used, which is an extension of weighted gene co-expression analysis (WGCNA). Using the NDP and NL score matrices of the MS/MS spectra, the contrast correlation matrix was calculated with DiffCoEx, where the parameters of "cu-treeDynamic" were set to method = "hybrid", cutHeight = 0.9999, deepSplit = 3, minClusterSize = 10. The R source code of DiffCoEx was downloaded from Additional file 1 in Tesson et al., BMC bioinformatics 11, 1-9 (2010), and the required R WGCNA package can be found at https://horvath.genetics.ucla.edu/html/CoexpressionNetwork/Rpackages/WGCNA/ (accessed on December 21 , 2021).

Information-theoretic computation of metabolome diversity and specialization and metabolic specificity

Metabolome diversity was calculated using the Shannon entropy of the MS/MS frequency distribution by the following equation described by Martinez et al., Proc Natl Acad Sci USA 105, 9709-9714 (2008):

where _Pij corresponds to the relative frequency of the i-th MS/MS (i=1, 2, ..., m) in the j-th sample (j=1, 2, ..., t).

The average frequency of the i-th MS/MS in the sample is calculated as:

MS/MS specificity is calculated as:

The metabolome specialization index δj for each sample j was calculated as the average of the MS/MS specificities using the following formula:

Selection of 15 RILs for idMS/MS analysis : Fifteen RILs were selected based on the following two criteria: 1) those that induced the highest phenolamide levels in the MAGIC population, especially for putrescine-containing metabolites; and 2) those that produced multiple sets of known phenolamides as well as unknowns consisting of typical phenolamide fragments (e.g., m/z 163.04 with an anomalous retention time), as assessed from previous annotation of phenolamides and manual inspection of in-source or MS/MS fragments.

Example 16

Heterologous expression and purification

Heterologous expression and purification of NaPPO1, NaPPo2 and NaBBL2 : 50ng of pET28a:NaPPO1, pET28a:NaPPO2 and pET28a:NaBBL2 plasmids were transformed into Rosetta ^TM (DE3) competent cells (Sigma-Aldrich, catalog number: 70954) by heat shock in a 42°C water bath for 90 seconds. The transformants were cultured at 37°C on LB agar plates containing 50μg mL ^-1 kanamycin. Five independent colonies were inoculated into 20mL LB medium containing 50μg mL ^-1 kanamycin and cultured at 37°C for 16 hours. The selected overnight culture was inoculated into 500mL LB liquid medium containing 50μg mL ^-1 kanamycin and cultured at 37°C to OD ₆₀₀ =0.4, at which time the culture was cooled on ice for 10 minutes. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1 mM. After incubation at 16°C and 160 rpm shaker speed for 12 hours, the cells were collected by centrifugation at 5000 x g for 10 minutes at 4°C. The supernatant was discarded and the pellet was resuspended in 9 mL of extraction/lysis buffer (50 mM sodium phosphate (pH 8.0), 500 mM NaCl, 10 mM imidazole, lysozyme (ThermoFisher, catalog number: 89833), cOmplete ^TM protease inhibitor cocktail (Merck, catalog number: 11697498001)), cooled on ice for 1 hour, and then disrupted by sonication on ice. The resulting homogenate was centrifuged at 15,000 rpm for 30 minutes at 4°C. The supernatant was incubated with 1 mL of Ni-NTA agarose resin (Qiagen, catalog number: 70666-4) pre-equilibrated with 10 mL of lysis buffer at 4°C on a rotator for 2 hours. The protein was eluted with a wash buffer containing increasing imidazole concentrations from 40 mM to 500 mM. The fractions containing the purified protein were confirmed by SDS-PAGE gel analysis. Bradford measurements of protein concentration were performed on a Tecan Infinite M200 plate reader. A calibration curve for protein concentration quantification was constructed by measuring 0, 10, 20, 40, 60, 80, 100 μg/mL of bovine serum albumin (BSA) at 595 nm with an optical path of 0.5 cm. Protein concentration was calculated using a standard curve (y = 0.0024x + 0.4558, R ² = 0.99).

Steady-state kinetics of NaPPO1, NaPPo2 and NaBBL2 enzymes : For steady-state kinetic analysis, the ability to oxidize N-caffeoyl putrescine was tested in 84 mM acetate buffer (pH 4.8) containing 1 mM (Z)-3-hexanal, 1 mM FAD, 32 μg NaPPO1/NaPPO2, 30 μg/90 μg NaBBL2 (total volume 100 μL) in a 22°C water bath; the assay lacking NaBBL2 enzyme served as a negative control. The following eight final concentrations of N-caffeoyl putrescine were tested in three independent experiments: 10, 20, 40, 60, 80, 120, 140 and 160 μM. Similarly, the ability to catalyze (Z)-3-hexanal was tested at 22°C in 84 mM acetate buffer (pH 4.8) containing 80 μM N-caffeoylputrescine, 1 mM FAD, 32 μg NaPPO1/NaPPO2, 30 μg/90 μg NaBBL2 (total volume 100 μL); the assay lacking the NaBBL2 enzyme was again used as a negative control. The following eight (Z)-3-hexanal final concentrations were tested in three independent experiments: 500, 600, 700, 800, 1000, 1200, 1600 and 2000 μM. The reaction was initiated by adding the corresponding recombinant enzyme, and the components were mixed by briefly vortexing and immediately returned to a 22°C water bath for incubation. After 20 minutes, all reactions were vortexed briefly and 100 μL aliquots of each reaction were transferred to cold Eppendorf tubes (-20°C) containing 100 μL of MeOH (with 600 ng/mL testosterone as internal standard) to stop enzyme activity. The samples were centrifuged at 16,000 g, 4°C for 30 minutes, and 150 μL of the supernatant was transferred to a 2 mL glass bottle for LC/MS (1 μL injection volume) analysis in the same manner as described previously. A standard curve (Figure 34) of caffeoyl putrescine using an authentic standard (with 600 ng/mL testosterone as internal standard) was used to calculate the concentration of substrate and product in each assay sample. The kinetic constants for caffeoyl putrescine and (Z)-3-hexanal were inferred using nonlinear regression in GraphPad Prism 9: apparent Km and Vmax (Figure 34).

Embodiment 17

In vitro assays and preparation and purification of CHP

In vitro assays for NaPPO1, NaPPO2, and NaBBL2: NaPPO1, NaPPO2, and NaBBL2 enzyme activity assays were performed by incubating 100 μg of purified recombinant protein in 84 mM acetate buffer (pH 4.8) containing 20 mM (E)-N-caffeoylputrescine (BOC Sciences, catalog number: B0005-053482) and 1 M cis-3-hexenal (Sigma, catalog number: W256102-sample-K) or trans-2-hexenal (Sigma, catalog number: W256005-1KG-K). During the incubation of the enzyme reactions at 8°C, 1 μL of the reaction buffer was directly injected every hour and analyzed by LC-MS to detect the products. Assays lacking enzyme or (E)-N-caffeoylputrescine served as negative controls.

Preparation and purification of CHP: 100 μg of purified recombinant protein was incubated at 8 ° C for 2 days in 84 mM acetate buffer (pH 4.8) containing (E)-N-caffeoyl putrescine and 1 M cis-3-hexenal, 3 mL enzyme reaction was performed, and 10 repetitions were combined. In order to remove salt, 30 mL enzyme reaction was loaded onto an SPE column (CHROMABOND HR-X, 45 μm, 3 mL/200 mg, Macherey-Nagel, catalog number: 730931P45). The SPE column was dried using argon. 3 mL of 100% methanol was used to elute the compound mixture from the SPE column. Further fractionation was performed on an Agilent 1100HPLC system equipped with a Nucleodur Sphinx RP18 column (150 × 4.6 mm, 5 μm particle size, Macherey-Nagel, Germany). The following binary mobile phase gradient was applied: 0 to 30 min, gradient step from 70% A (Milli-Q water with 0.05% formic acid), 30% B (MeOH with 0.05% formic acid) to 50% A, 50% B; 30 to 35 min, gradient step from 50% A, 50% B to 100% B; 30 to 35 min, isocratic at 100% B. The flow rate was 900 μL min ^-1 . Fractions were collected throughout the fractionation procedure with a collection window of 1 min; each fraction was checked by LC-MS. Fractions containing the target molecule were pooled, diluted with 5-fold amount of water, and loaded onto an SPE column (CHROMABOND HR-X, 45 μm, 3 mL/200 mg). After drying with argon, the SPE column was eluted with 700 μL of MeOH-d ₃ containing 0.05% formic acid. The eluate was immediately subjected to NMR analysis.

Embodiment 18

Green leafhopper feeding assay

Green leafhopper feeding assay : Survival of green leafhoppers fed on an artificial diet (10% sucrose in water) supplemented with caffeoylputrescine (CP, 100 μM), coumaroylputrescine (CoP, 7 μM), feruloylputrescine (FP, 10 μM), or the m/z 347.19 hexenyl derivative of CP (CPH, 1 μM) was recorded every 1 h overnight. The artificial feeding apparatus consisted of a 50-mL Falcon tube with a 7-mm hole drilled in the conical bottom through which leafhoppers were aspirated into the tube, which was then covered with parafilm and punctured with a needle to create ventilation holes. The large opening of the Falcon tube was covered with a double layer of parafilm, which contained liquid diet (100 μL). The artificial feeding apparatus was inverted, with the liquid diet side facing down, on a white light box (STRATAGENE) that provided bottom-light illumination.

Embodiment 19

Phylogenetic and statistical analyses

Phylogenetic analysis : Phylogenetic trees of different species were constructed using the Taxonomy Common Tree tool from NCBI. In brief, the species taxonomic names were uploaded to the Taxonomy Common Tree to generate a taxonomic tree for the selected biological group. The taxonomic tree was saved as a phylip tree file (phy format). The phylogenetic tree was visualized using the R package “treeio”.

Statistical analysis : Data were statistically analyzed using R. Statistical significance between multiple groups (≥3) of data was evaluated using analysis of variance (ANOVA), followed by Tukey's honestly significant difference (HSD) post hoc test. To analyze differences between two groups of data, two-tailed distributions of two groups of samples with equal variance were analyzed using Student's t test. For the green leafhopper choice assay test, a blocking term was included in the ANOVA to resolve contrasts in separate replicates, and the Freidman test was used, followed by a Wilcoxon post hoc test.

Embodiment 20

NMR and Chemicals

NMR : NMR spectra were recorded at 298K on a 500MHz Bruker Avance III HD spectrometer (Bruker Biospin GmbH, Rheinstetten, Germany) equipped with a cryogenic platform and a 5mm TCI cryoprobe. The spectra were referenced to residual solvent signals at δ _C 49.15 and δ _H 3.31. For spectrometer control and data processing, Bruker TopSpin ver. 3.2 was used. Standard pulse programs implemented in Bruker TopSpin were used.

Chemicals : N-caffeoylputrescine was purchased from BOC Sciences, Shirley, NY, USA. The synthesis of N-coumaroylputrescine and N-feruloylputrescine was performed as previously described (Kyselka et al., J Agric Food Chem 66, 11018-11026 (2018). The purity of the compounds was determined by LC-MS.

Embodiment 21

Role of BBL2 in CHP biosynthesis in vitro but not in vivo

Studies have found that although NaBBL2 is not necessary for in vitro synthesis, it is necessary for CPH biosynthesis in vivo. Although it is believed that NaBBL2 plays a role in solving positioning challenges, it has other possible solutions. For example, during stress-induced autophagy, plastids and their contents are regularly transferred to vacuoles (Izumi et al., Plant Cell 29, 377-394 (2017)), and if the biochemical function of PPO is still effective after transport to the vacuole, then the components required for CPH biosynthesis are all present in the same organelle (Figure 60).

Most members of the BBE-like protein family contain a double covalently linked FAD cofactor, and the physical binding of the FAD cofactor to the BBL protein occurs through specific His and Cys residues (Daniel et al., Arch Biochem Biophys 632, 88-103 (2017), Winkler et al., J Biol Chem 281, 21276-21285 (2006)). Studies have found that NaBBL2 lacks the Cys residue involved in the covalent binding of the FAD cofactor; the C to G mutation of BBL2 at the substrate binding site implies different biochemical functions of NaBBL2 in Nicotiana attenuata plants (Figure 61).

It has been suggested that BBL2 may play a role as a non-catalytic protein in the CPH pathway of Nicotiana attenuata, just like the non-catalytic chalcone isomerase (CHIL) in flavonoid metabolism reported previously (Ban et al., PNatl AcadSci USA115, E5223-E5232 (2018)). In this case, BBL2 can interact with the CP activated by the reactive PPO, making it stable in the cell environment and avoiding conversion into a byproduct that cannot react with (Z)-3-hexenal to form CPH. Therefore, BBL2 can allow substrates to be more effectively conducted between active enzymes. Many pathways involved in specific metabolism are considered to be provided in protein complexes or metabolites. Metabolites promote the conduction of unstable and toxic intermediates and increase local substrate concentrations to prevent undesirable metabolic crosstalk (Laursen et al., Science, 354, 890-893 (2016); Gou et al., Nature Plants 4, 299-310 (2018)). Such dynamic assembly and disassembly allows for rapid reconfiguration of metabolic signatures in response to environmental challenges and is thought to involve scaffold proteins ( et al., Current opinion in plant biology 8, 280-291 (2005)). Since CPH was observed to be highly induced in field-grown plants, the scaffolding function that contributes to BBL2 may allow for the dynamic assembly and disassembly of metabolite channel reaction intermediates and maximize the catalytic efficiency toward CPH production. Consistent with such a mechanism, it was observed that CPH was formed in plants only when the local CP concentration exceeded 50 μM. In the in vitro CPH assay, the lack of possible BBL2-dependent stabilization/conduction may not be a big problem because (Z)-3-hexenal can directly access the PPO enzyme. However, when only a small portion of (Z)-3-hexenal is accessible to the CP microenvironment of PPO catalysis in vivo, the stabilization/conduction of BBL2 is more critical for the efficient production of CPH.

Example 22

surface

Table 1. Stability of m/z 347.19 during sample extraction and purification

Leaf extraction conditions stability Leaf sample collection using aluminum foil in liquid nitrogen no Leaf sample collection using Eppendorf tubes without direct contact with liquid nitrogen yes Sample extraction buffer pH 4.8 yes Sample extraction buffer pH 10 no Purification conditions of metabolites stability Purify m/z 347.19 using SPE column with argon flow yes Purification by freeze drying m/z 347.19 no Purify m/z 347.19 using rotary evaporator no

Table 2. Michaelis-Menten kinetics of PPO1, PPO2 and BBL2 for caffeoylputrescine and (Z)-3-hexenal

Note. Calculated Vmax values are given in [μM mg ^-1 min ^-1 ]. Km values are given in [μM]. Values are mean ± SEM

Table 3. List of oligonucleotides used in the examples

Claims (42)

1. A compound of formula (I):

wherein:

R ¹、R²、R³ and R ⁴ are each, independently of one another, H, OH, (C ₁-C₆) -alkyl or (C ₁-C₆) -alkoxy;

R ⁵、R⁶ and R ⁷ are each, independently of one another, H or (C ₁-C₆) -alkyl;

X is a linear or branched (C ₁-C₈) -alkyl or linear or branched (C ₂-C₈) -alkenyl group,

Y is selected from -(CH₂)_m-NH₂、-(CH₂)_n-NH-(CH₂)_o-NH₂、-(CH₂)_p-NH-(CH₂)_q-NH-(CH₂)_r-NH₂ NH-(CH₂)_m-NH₂、NH-(CH₂)_n-NH-(CH₂)_o-NH₂ and NH- (CH ₂)_p-NH-(CH₂)_q-NH-(CH₂)_r-NH₂) wherein m, n, o, p, q and r are each an integer between 1 and 10, or tyramine ester, and

Z is a linear or branched (C ₁-C₈) -alkyl or linear or branched (C ₂-C₈) -alkenyl group,

Or an enantiomer, diastereomer, stereoisomer or salt thereof.

2. A compound according to claim 1, wherein:

Two of R ¹、R²、R³ and R ⁴ are OH;

R ⁵、R⁶ and R ⁷ are each H; and is also provided with

X is a straight chain (C ₂-C₈) -alkenyl group;

or an enantiomer, diastereomer, stereoisomer or salt thereof.

3. The compound according to claim 2, wherein:

R ¹ and R ⁴ are H,

R ² and R ³ are OH, and

X is-ch=ch-,

Or an enantiomer, diastereomer, stereoisomer or salt thereof.

4. A compound according to any one of claims 1 to 3, having the following formula (II):

wherein R ⁵、R⁶、R⁷ and Z are as defined above;

or an enantiomer, diastereomer, stereoisomer or salt thereof.

5. The compound according to any one of claims 1 to 4, having the following formula (III):

or an enantiomer, diastereomer, stereoisomer or salt thereof.

6. A method of preparing the compound of any one of claims 1 to 5.

7. The method of claim 6, wherein the method is an enzymatic preparation method using at least a BBL2 (berberine bridge enzyme 2) polypeptide.

8. The method of claim 7, wherein the BBL2 (berberine bridge enzyme 2) polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 1;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 1;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 1;

(d) Encoded by a polynucleotide which is a species homolog of SEQ ID NO. 1;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO. 2;

(h) Represented by a polypeptide fragment of SEQ ID NO:2 having BBL2 (berberine bridge enzyme 2) function;

(i) Represented by the polypeptide domain of SEQ ID NO:2 having BBL2 (berberine bridge enzyme 2) function;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 2 and having BBL2 (berberine bridge enzyme 2) function; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

9. The method according to claim 7 or 8, wherein the enzymatic preparation method additionally uses PPO (polyphenol oxidase) activity or polypeptide, wherein preferably the PPO (polyphenol oxidase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 3 or 5;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 3 or 5;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO.3 or 5;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 3 or 5;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO.4 or 6;

(h) Represented by a polypeptide fragment of SEQ ID NO. 4 or 6 having PPO (polyphenol oxidase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO 4 or 6 having PPO (polyphenol oxidase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 4 or 6 and having PPO (polyphenol oxidase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

10. The method according to any one of claims 7 to 9, wherein the enzymatic preparation method additionally uses AT1 (polyamine hydroxycinnamoyl transferase 1) activity or polypeptide, wherein preferably the AT1 (polyamine hydroxycinnamoyl transferase 1) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 7;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 7;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 7;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 7;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO.8;

(h) Represented by the polypeptide fragment of SEQ ID NO. 8 having AT1 (polyamine hydroxycinnamoyl transferase 1) activity;

(i) Represented by the polypeptide domain of SEQ ID NO. 8 having AT1 (polyamine hydroxycinnamoyl transferase 1) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having AT least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 8 and having AT1 (polyamine hydroxycinnamoyl transferase 1) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f).

11. The method according to any one of claims 7 to 10, wherein the enzymatic zhib method additionally uses ODC (ornithine decarboxylase) activity or polypeptide and/or HPL (hydroperoxide lyase) activity or polypeptide, wherein preferably (a) the ODC (ornithine decarboxylase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 9 or 11;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 9 or 11;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 9 or 11;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 9 or 11;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO. 10 or 12;

(h) Represented by a polypeptide fragment of SEQ ID NO. 10 or 12 having ODC (ornithine decarboxylase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO. 10 or 12 having ODC (ornithine decarboxylase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 10 or 12 and having ODC (ornithine decarboxylase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in (a) to (f); and is also provided with

(B) The HPL (hydroperoxide lyase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 13, 15 or 17;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 13, 15 or 17;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 13, 15 or 17;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 13, 15 or 17;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in (B) (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO 14, 16 or 18;

(h) Represented by a polypeptide fragment of SEQ ID NO 14, 16 or 18 having HPL (hydroperoxide lyase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO 14, 16 or 18 having HPL (hydroperoxide lyase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 14, 16 or 18 and having HPL (hydroperoxide lyase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in (B) (a) to (f).

12. The method of any one of claims 7 to 11, wherein the enzymatic preparation method additionally uses PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or C4H (trans-cinnamic acid 4-hydroxylase) activity or polypeptide and/or 4CL (4-coumarate: coa ligase) activity or polypeptide and/or HCT (hydroxycinnamoyl-transferase) activity or polypeptide and/or C3H (coumarate 3-hydroxylase) activity or polypeptide, wherein preferably

(A) The PAL (L-phenylalanine ammonia lyase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 19, 21, 23 or 25;

(b) Encoded by a polynucleotide that is a variant of SEQ ID NO. 19, 21, 23 or 25;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 19, 21, 23 or 25;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 19, 21, 23 or 25;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any of a (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in a (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO. 20, 22, 24 or 26;

(h) Represented by a polypeptide fragment of SEQ ID NO. 20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO. 20, 22, 24 or 26 having PAL (L-phenylalanine ammonia lyase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 20, 22, 24 or 26 and having PAL (L-phenylalanine ammonia lyase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in a (a) to (f); and is also provided with

(B) The C4H (trans-cinnamic acid 4-hydroxylase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 27

(B) Encoded by a polynucleotide that is a variant of SEQ ID NO. 27;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 27;

(d) Encoded by a polynucleotide that is a homolog of the species of SEQ ID NO. 27;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any of B (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in B (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO. 28;

(h) Represented by the polypeptide fragment of SEQ ID NO. 28 having C4H (cinnamic acid 4-hydroxylase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO. 28 having C4H (cinnamic acid 4-hydroxylase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 28 and having C4H (cinnamic acid 4-hydroxylase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in B (a) to (f); and is also provided with

(C) The 4CL (4-coumarate: coa ligase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID No. 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129 or 131;

(b) Encoded by a polynucleotide that is a variant of SEQ ID No. 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131;

(c) Encoded by a polynucleotide that is an allelic variant of SEQ ID NO. 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID No. 29, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, or 131;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any of C (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in C (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132;

(h) Represented by a polypeptide fragment of SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumarate: coA ligase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO:30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 having 4CL (4-coumarate: coA ligase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 30, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130 or 132 and having 4CL (4-coumarate: coa ligase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in C (a) to (f); and is also provided with

(D) The HCT (hydroxycinnamoyl-transferase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 31, 33, 35, 37, 39, 41 or 43;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 31, 33, 35, 37, 39, 41 or 43;

(c) 31, 33, 35, 37, 39, 41 or 43;

(d) Encoded by a polynucleotide that is a species homolog of SEQ ID NO. 31, 33, 35, 37, 39, 41 or 43;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any of D (a) to (D);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in D (a) to (D);

(g) Represented by a polypeptide having SEQ ID NO. 32, 34, 36, 38, 40, 42 or 44;

(h) Represented by a polypeptide fragment of SEQ ID NO. 32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO:32, 34, 36, 38, 40, 42 or 44 having HCT (hydroxycinnamoyl-transferase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID No. 32, 34, 36, 38, 40, 42 or 44 and having HCT (hydroxycinnamoyl-transferase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in D (a) to (f); and is also provided with

(E) The C3H (coumarate 3-hydroxylase) activity or polypeptide:

(a) Encoded by a polynucleotide having the nucleotide sequence of SEQ ID NO. 45;

(b) Encoded by a polynucleotide which is a variant of SEQ ID NO. 45;

(c) Encoded by a polynucleotide which is an allelic variant of SEQ ID NO. 45;

(d) Encoded by a polynucleotide that is a homolog of the species of SEQ ID NO. 451;

(e) Encoded by a polynucleotide having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to a polynucleotide as defined in any one of E (a) to (d);

(f) Encoded by a polynucleotide capable of hybridizing under stringent conditions to any one of the polynucleotides specified in E (a) to (d);

(g) Represented by a polypeptide having SEQ ID NO. 46;

(h) Represented by the polypeptide fragment of SEQ ID NO. 46 having C3H (coumarate 3-hydroxylase) activity;

(i) Represented by the polypeptide domain of SEQ ID NO. 46 having C3H (coumarate 3-hydroxylase) activity;

(j) Represented by a polypeptide comprising an amino acid sequence having at least 75%, 80%, 90%, 95%, 97%, 98% or 99% identity to the amino acid sequence of SEQ ID NO. 46 and having C3H (coumarate 3-hydroxylase) activity; or (b)

(K) Represented by a polypeptide encoded by any one of the polynucleotides specified in E (a) to (f).

13. The method according to any one of claims 7 to 12, wherein the enzymatic preparation is performed with an activity or polypeptide provided in vitro.

14. The method according to claim 13, wherein the method is carried out at a pH of 4.8 or less, preferably by solid phase extraction using an argon stream.

15. The method according to any one of claims 7 to 12, wherein the enzymatic preparation is performed with an activity or polypeptide provided in a living cell, tissue or organism.

16. The method according to claim 15, wherein the enzymatic preparation is performed with and in a genetically modified cell, tissue or organism, wherein the genetic modification allows for heterologous expression of one or more activities or polypeptides as defined in any one of claims 6 to 11.

17. An organism, tissue or cell for the preparation of a compound according to any one of claims 1 to 5, which is genetically modified, wherein the genetic modification allows for heterologous expression of one or more activities or polypeptides as defined in any one of claims 6 to 11.

18. The method according to claim 16 or the organism, tissue or cell according to claim 17, wherein the genetic modification results in at least the expression of a BBL2 (berberine bridge enzyme 2) polypeptide as defined in claim 7.

19. The method, organism, tissue or cell according to claim 18, wherein said genetic modification additionally results in the expression of a PPO (polyphenol oxidase) activity or polypeptide as defined in claim 9.

20. The method, organism, tissue or cell according to claim 18 or 19, wherein said genetic modification additionally results in the expression of an AT1 (polyamine hydroxycinnamoyl transferase 1) activity or polypeptide as defined in claim 9.

21. The method, organism, tissue or cell according to claim 18, 19 or 20, wherein said genetic modification additionally results in the expression of an ODC (ornithine decarboxylase) activity or polypeptide and/or an HPL (hydroperoxide lyase) activity or polypeptide as defined in claim 11.

22. The method, organism, tissue or cell according to any one of claims 18 to 21, wherein said genetic modification additionally results in the expression of PAL (L-phenylalanine ammonia lyase) activity or polypeptide and/or C4H (trans-cinnamic acid 4-hydroxylase) activity or polypeptide and/or 4CL (4-coumarate: coa ligase) activity or polypeptide and/or HCT (hydroxycinnamoyl-transferase) activity or polypeptide and/or C3H (coumarate 3-hydroxylase) activity or polypeptide as defined in claim 11.

23. The method of any one of claims 16 or 18 to 22, or the organism, tissue or cell of any one of claims 17 to 22, wherein said expression is delivered by a native, regulated, tissue-specific or constitutive promoter.

24. The method, organism, tissue or cell of claim 23, wherein the promoter allows for (i) polycistronic expression of an activity or polypeptide as defined in any one of claims 18 to 22, (ii) expression of an activity or polypeptide as defined in any one of claims 18 to 22 alone; or (iii) a group expression of at least two active groups selected from the activities defined in any one of claims 18 to 22.

25. The method, organism, tissue or cell of any one of claims 16-24, wherein said expression is over-expression.

26. The method, organism, tissue or cell of claim 25, wherein said overexpression is transmitted by a strongly regulated or a strongly constitutive promoter and/or by providing at least a second copy of a genetic element encoding said activity or polypeptide.

27. The method according to any one of claims 7 to 13 or 18 to 26, or the organism, tissue or cell according to any one of claims 17 to 26, wherein the enzymatic activity or polypeptide is from an organism belonging to the genus nicotiana, preferably the species nicotiana sanguinea.

28. The method of any one of claims 8 to 12, 15, 16 or 18 to 27, or the organism, tissue or cell of any one of claims 17 to 27, wherein the polynucleotide is comprised in one or more extrachromosomal vectors or plasmids, and/or integrated in the genome of the organism.

29. The method of any one of claims 16 or 18 to 28, or the organism, tissue or cell of any one of claims 17 to 28, wherein the genetically modified organism, tissue or cell is eukaryotic.

30. The method, organism, tissue or cell of claim 29, wherein the genetically modified organism is a plant, or wherein the tissue is a plant tissue, or wherein the cell is a plant cell.

31. The method, organism, tissue or cell according to claim 30, wherein the genetically modified organism belongs to, or the tissue or cell is from a higher plant challenged by an insect herbivore, preferably a higher plant challenged by an insect feeding in a tearing and flushing and/or piercing manner, more preferably a higher plant of the genus nicotiana, solanum, oryza, zea, bean or camellia.

32. An expression cassette for heterologous expression in a eukaryotic host cell, preferably a plant cell, wherein the expression cassette comprises a polynucleotide as defined in any one of claims 8 to 12.

33. A vector or insert construct comprising a polynucleotide as defined in any one of claims 8 to 12 or an expression cassette according to claim 32.

34. Use of a polypeptide or polynucleotide as defined in any one of claims 8 to 12, an expression cassette according to claim 32 or a vector or insertion construct according to claim 33 for the preparation of a compound according to any one of claims 1 to 5.

35. Use of an organism, tissue or cell according to any one of claims 17 to 31, or an organism, tissue or cell comprising an expression cassette according to claim 32, or a vector or insertion construct according to claim 33, for the preparation of a compound according to any one of claims 1 to 5.

36. A composition comprising a compound according to any one of claims 1 to 5, or a compound prepared by a method according to any one of claims 6 to 16 or 18 to 31, or a compound prepared from an organism, tissue or cell as defined in any one of claims 17 to 31, optionally additionally comprising an acceptable carrier, stabilizer and/or spreading agent.

37. Use of a compound according to any one of claims 1 to 5, or a compound prepared by a method according to any one of claims 6 to 16 or 18 to 31, or a compound prepared from an organism, tissue or cell as defined in any one of claims 17 to 31, or a composition according to claim 35, for plant protection against herbivores.

38. The use of claim 37, wherein the herbivore is an insect.

39. The use according to claim 38, wherein the insect is an insect that feeds in a tearing and flushing and/or piercing manner.

40. The use according to claim 38 or 39, wherein the insect is a leafhopper or plant hopper, preferably a leafhopper (Aphrodinae), bathymatopohorinae, a large leafhopper (CICADELLINAE), an isolated leafhopper (Coelidiinae), a leafhopper (Coelidiinae), coelidiinae, a subidae of tip leafhopper (Coelidiinae), a wide head leafhopper (Coelidiinae), a transverse leafhopper (Coelidiinae), a stem leafhopper (Coelidiinae), a subidae of leafhopper (Coelidiinae), coelidiinae, an ear leafhopper (Coelidiinae), a window leafhopper (Coelidiinae), coelidiinae, a multicolor leafhopper (Coelidiinae), a new isolated leafhopper (Coelidiinae), coelidiinae (Coelidiinae), a flange leafhopper (2), coelidiinae, a long chest leafhopper (Coelidiinae), a small leafhopper (Coelidiinae) or a narrow leafhopper (2) family, more preferably a small leafhopper (Coelidiinae), a brown leafhopper (Coelidiinae), a plant hopper (Coelidiinae) or a plant hopper (Coelidiinae).

41. Use of a compound according to any one of claims 1 to 5, or a compound prepared by a method according to any one of claims 6 to 16 or 18 to 31, or a compound prepared from an organism, tissue or cell as defined in any one of claims 17 to 31, or a composition according to claim 36, as an insecticide, preferably against an insect as defined in claim 39 or 40.

42. A method of plant protection comprising contacting a plant or part of a plant with a compound according to any one of claims 1 to 5 or a composition according to claim 36.