CN107022563A - Genetically modified plants - Google Patents

Abstract

The genetically modified plants with increased nitrogen metabolism of the nucleic acid construct of the albumen worked as movable signal are encoded the present invention relates to expression.

Description

transgenic plant

field of invention

The present invention relates to transgenic plants expressing the mobile signal HY5 having improved properties, such as growth, and improved nitrogen metabolism, methods for producing such plants and methods for improving nitrogen metabolism and growth.

Background technique

Nitrogen (N) is fundamental to crop development as it forms the building blocks of many organic molecules, nucleic acids and proteins. N nutrition affects plant functions at all levels, from metabolism to resource allocation, growth and development. Due to the intense nitrification of applied organic and fertilizer N, the most abundant source of N acquisition for plant roots is nitrate (NO3 ⁻ ) in naturally aerobic soils.

During the past five decades, global crop production capacity has increased dramatically, mainly due to improved crop varieties and large inputs of fertilizers, especially nitrogen. However, for many crops, fertilizer N use efficiency is only about 30-50%, and most of it is lost to the environment, leading to various harmful effects such as deterioration of air and water quality and loss of biodiversity (Ju, X.T. et al. Proc Natl AcadSci USA. 106, 3041-3046 (2009)). Furthermore, nitrogen is one of the most expensive nutrients supplied and commercial fertilizers represent a major cost in plant production. It is estimated that excess N in the environment currently costs the EU between €70 billion and €320 billion per year (Sutton et al., Nature 472, 159-61 (2011). In China, the increase in grain production over the past 30 years has been accompanied by an increase in N use efficiency ( NUE) was drastically reduced from 55 to 20 kg grain/kg applied fertilizer N (Guo, et al. Science 327, 1008-1010 (2010)). Increasing NUE and limiting N fertilizer use are both protective of the environment and improving sustainable and productive An important challenge in agriculture. This challenge is particularly relevant to cereal crops that require large amounts of N fertilizer to obtain maximum yield and whose NUE is estimated to be much less than 50% (Hirel et al., Journal of Experimental Botany, Vol.58, No.9, pp.2369-2387 (2007)).

It is therefore important to identify key steps that control plant N metabolism, including N use efficiency (NUE) and N uptake. For most plant species, NUE depends primarily on how the plant extracts inorganic nitrogen from the soil, absorbs nitrate and ammonium, and cycles organic nitrogen. NUE can be defined as the grain yield per unit of N available in soil (including residual N present in soil and manure), i.e. output (total plant N, grain N, biomass yield, grain yield) and The ratio of inputs (total N applied, soil N or N-fertilizer). Thus, NUE can be divided into two processes: uptake efficiency (NupE; the ability of a plant to remove N from soil as nitrate and ammonium ions) and utilization efficiency (NutE; ability to use N to produce grain yield).

Nitrogen utilization by plants involves multiple steps, including uptake, uptake, migration, and as plants age, recycling and reactivation. To control changing nitrate concentrations in soil, plant roots have developed at least three nitrate uptake systems, two high-affinity transport systems (HATS) and one low-affinity transport system (LATS), responsible for nitrate acquisition (Crawford and Glass Trends Plant Sci 3:389-395 (1998)). Constitutive HATS (cHATS) and nitrate-inducible HATS (iHATS) operate to uptake nitrate in the external medium at low nitrate concentrations, with a saturation range of 0.2-0.5 mM. LATS played a role in nitrate acquisition with a higher external nitrate concentration than before. Uptake through LATS and HATS is mediated by nitrate transporters belonging to the NRT1 and NRT2 families, respectively. Uptake via the root is regulated by negative feedback, linking the expression and activity of nitrate uptake to the N status of the plant.

The Arabidopsis thaliana (At) protein HY5, a basic leucine zipper (bZIP) transcription factor, is known to integrate a variety of phytohormonal (e.g., auxin and abscisic acid) and environmental (e.g., low temperature) signaling and play a role in the control of plant photomorphogenic development (18, 28-30). Lateral root formation is also known to be increased in hy5 loss-of-function mutants (18, 28).

It is well established that shoot-to-root long-distance signaling induces root growth and N uptake (26, 27). The present inventors identified for the first time the molecular basis of this signal and showed that HY5 is a shoot-to-root movement signal that mediates N metabolism in plants. This provides an alternative strategy for improving nutrient use efficiency in crops.

Productive agriculture requires large quantities of expensive nitrogenous fertilizers. Improving the NUE of crop plants is therefore crucial. There is a need to provide crop plants with more nutritionally efficient genotypes to ensure sustainable crop production for global food security, and to reduce the cost of fertilizer inputs and negative environmental effects, such as those on air and water quality, and biodiversity loss of sex. The present invention aims to address this need.

Summary of the invention

The present inventors surprisingly found that HY5 is involved in N metabolism. As demonstrated in the Examples described herein, expression of AtHY5 in transgenic plants resulted in increased nitrate uptake in roots. Furthermore, the present inventors showed that HY5 functions as a mobile signal from shoots to roots in response to light and mediates root growth and nitrate uptake. Therefore, although HY5 is produced in roots, only when HY5 protein produced in shoots/leaves due to light perception moves to roots, it accumulates to effective levels and induces root development and NO3 ^- uptake.

The unexpected discovery of HY5, a shoot-root mobile protein that regulates light-responsive carbon and nitrogen metabolism, enables plants to coordinate shoot and root growth throughout the biological response to changes in ambient light, opens the door to enhanced crop nutrient utilization, growth and productive biotechnological approaches, especially the use of tissue-specific promoters to elevate HY5 levels for beneficial effects.

In a first aspect, the present invention relates to a method for increasing nitrogen metabolism in plants, comprising introducing and expressing in a plant a nucleic acid construct comprising a HY5 nucleic acid sequence.

In another aspect, the present invention relates to transgenic plants comprising a nucleic acid construct comprising a HY5 nucleic acid sequence and tissue-specific regulatory sequences.

In another aspect, the present invention relates to a transgenic plant comprising a nucleic acid construct comprising a HY5 nucleic acid sequence, wherein said plant is not Arabidopsis thaliana.

In another aspect, the present invention relates to a nucleic acid construct comprising a HY5 nucleic acid sequence and a tissue-specific promoter.

In a further aspect, the invention relates to a vector comprising a nucleic acid construct as described herein.

In another aspect, the invention relates to a host cell comprising a nucleic acid construct as described herein or a vector as described herein.

In a further aspect, the present invention relates to a method for producing plants with increased nitrogen uptake, comprising introducing and expressing in a plant a nucleic acid construct comprising a HY5 nucleic acid sequence and a tissue-specific promoter.

In another aspect, the present invention relates to a method for increasing the presence of HY5 protein in plant roots, comprising introducing and expressing in a plant a nucleic acid construct comprising a HY5 nucleic acid sequence operably linked to a green tissue-specific promoter.

In another aspect, the present invention relates to a method for regulating C and N metabolic balance in a plant, which comprises introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in a plant.

In another aspect, the present invention relates to a method for increasing the presence of HY5 protein in plant roots, comprising introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in the green tissue of a plant.

In a final aspect, the present invention relates to genetically altered plants, wherein said plants carry mutations in the endogenous HY5 nucleic acid sequence or endogenous HY5 promoter, or wherein said mutations introduce at least one extra copy of HY5 nucleic acid.

In summary, the present invention provides the following technical solutions:

1. A method for increasing nitrogen metabolism in plants, said method comprising introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in a plant.

2. The method according to item 1, wherein said nucleic acid construct comprises SEQ ID NO: 1 or 2 or a homologous sequence encoding SEQ ID NO: 3 of the AtHY5 protein or its functional variants defined by encoding SEQ ID NO: 3 the nucleic acid sequence of the object.

3. The method according to item 2, wherein said homologue has at least 30% sequence identity to SEQ ID NO:3.

4. The method according to item 2 or 3, wherein said homologue is selected from SEQ ID NOs: 4 to 29.

5. The method according to the preceding items, wherein the plant is selected from the group consisting of corn, rice, wheat, Brassica napus/Canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean , beans, lettuce, cotton, sugar cane, sugar beets, broccoli or other Brassica vegetables or poplar.

6. The method according to the preceding items, wherein the nucleic acid construct comprises regulatory sequences.

7. The method according to item 6, wherein the regulatory sequence is selected from a constitutive promoter or a tissue-specific promoter.

8. The method according to item 7, wherein the tissue-specific promoter is a green tissue-specific promoter.

9. A transgenic plant comprising a nucleic acid construct comprising a HY5 nucleic acid sequence and tissue-specific regulatory sequences.

10. The transgenic plant according to item 9, wherein the tissue-specific promoter is a green tissue-specific promoter.

11. The transgenic plant according to item 10, wherein said nucleic acid construct comprises the nucleic acid sequence of SEQ ID NO: 1 or 2 of the AtHY5 protein defined by SEQ ID NO: 3 or the homologue of encoding SEQ ID NO: 3 .

12. The transgenic plant according to item 11, wherein said homologue has at least 30% sequence identity to SEQ ID NO:3.

13. The transgenic plant according to item 11 or 12, wherein said homologue is from SEQ ID NOs: 4 to 29.

14. The transgenic plant according to any one of items 9-13, wherein said plant is selected from the group consisting of corn, rice, wheat, Brassica napus/Canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, Grapes, barley, peas, beans, fava beans, lettuce, cotton, sugar cane, sugar beets, broccoli or other Brassica vegetables or poplar.

15. A transgenic plant comprising a nucleic acid construct comprising a HY5 nucleic acid sequence, wherein said plant is not Arabidopsis.

16. A nucleic acid construct comprising a HY5 nucleic acid sequence and a tissue-specific promoter.

17. The nucleic acid construct according to item 16, wherein the tissue-specific promoter is a green tissue-specific promoter.

18. The nucleic acid construct according to item 16 or 17, wherein said nucleic acid construct comprises SEQ ID NO: 1 or 2 of the AtHY5 protein defined by SEQ ID NO: 3 or a homologue encoding SEQ ID NO: 3 nucleic acid sequence.

19. The nucleic acid construct according to item 18, wherein said homologue has at least 30% sequence identity to SEQ ID NO:3.

20. The nucleic acid construct according to item 18, wherein the homologue is selected from SEQ ID NOs: 4 to 29.

21. The nucleic acid construct according to any one of items 16 to 20, wherein the plant is selected from the group consisting of corn, rice, wheat, Brassica napus/Canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco , grapes, barley, peas, beans, fava beans, lettuce, cotton, sugar cane, sugar beets, broccoli or other Brassica vegetables or poplar.

22. A vector comprising the nucleic acid construct according to any one of items 16 to 21.

23. A host cell comprising the nucleic acid construct according to any one of items 16 to 21 or the vector according to item 22.

24. A method for producing plants with increased nitrogen uptake, comprising introducing into a plant and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence and a tissue-specific promoter.

25. A method for increasing the presence of HY5 protein in plant roots, comprising introducing and expressing in a plant a nucleic acid construct comprising a HY5 nucleic acid sequence operably linked to a green tissue-specific promoter.

26. A method for regulating the balance between C and N metabolism in a plant, comprising introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in a plant.

27. A method for increasing the presence of a HY5 protein in plant roots, comprising introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in the green tissue of a plant.

28. A genetically altered plant, wherein said plant carries a mutation in the endogenous HY5 nucleic acid sequence or in the endogenous HY5 promoter or wherein said mutation introduces at least one additional copy of the HY5 nucleic acid into the plant genome.

The invention is further described in the following non-limiting figures.

Attached picture

Figure 1. HY5 regulates root growth and shoot-irradiation promotion of NRT2.1-dependent NO3 ^- uptake. (A) Schematic representation of differential shoot/root irradiation conditions. 5-day-old seedlings were exposed to 3 days of differential light treatment (100 μmol.s ^-1 .m ^-2 ): irradiated (S(L)) or dark (S(D)) shoots, irradiated ( R(L)) or dark (R(D)) roots. (B) Differential shoot/root irradiation effects on WT and hy5-526 primary root growth. Arrows indicate the apical position at the start of the experiment. Scale bar, 1 cm. (C) Differential shoot/root irradiation effects on primary root extension length of WT, hy5-526, hy5 and cop1-4 seedlings. (D) Differential shoot/root irradiation effects on ¹⁵ NO3 ^- uptake by seedling roots. (E) Differential shoot/root irradiation effects on primary root extension length of 10-day-old grafted plants. Grafts are indicated as scion/rootstock (eg, HY5/hy5-526 has HY5 scion and hy5-526 rootstock). (F) Differential shoot/root irradiation effects on ¹⁵ NO3 ^-uptake by roots of plants grafted as in (E). (CF) Data are expressed as mean±sem (n=30). The same lowercase letters indicate non-significant differences (P<0.05) between means.

Figure 2. Translocation of HY5 from shoots to roots regulates root growth and NO3 ^- uptake.

(A) Differential shoot/root irradiation effects on primary root extension growth of transgenic plants. (B) Distribution of HY5-GFP in pHY5; HY5-GFP hy5 roots. (C) Distribution of detectable HY5-GFP in pCAB3:HY5-GFP hy5 roots. (D) Relative myc-HY5 transcript abundance in 14-day-old plants, with transcripts set to 1 relative to the level of pCAB3:myc-HY5hy5 plants. Data are shown as mean±sem (n=3). (E) Immunological detection of myc-HY5 in shoots (S) and roots (R), HSP90 loading control. (F) Distribution of HY5-GFP in roots of shoot-irradiated pCAB3:HY5-GFP hy5/hy5 grafts. (G) Detection of HY5-GFP in roots of shoot-irradiated TEV protease-expressing pCAB3:2×GUS TEV ^re -HY5-GFP hy5 plants. (H) Differential shoot/root irradiation effects on primary root extension growth of plants expressing TEV protease. (I) Differential shoot/root irradiation effects on ^root15NO3- uptake ^of plants expressing TEV protease. (J) Differential shoot/root irradiation effects on the expression of the pHY5:GFP transgene in roots. (K) Distribution of HY5-GFP in seedling roots (grafted as indicated). (B, C, F, G, J, K) Scale bars, 50 μm. (A, D, H, I) Data are expressed as mean±sem (n=30). The same lowercase letters indicate non-significant differences (P<0.05) between means.

Figure 3. HY5 coordinates N and C metabolism. (A) NRT2.1 transcript levels in primary seedling roots (genotypes/graft chimeras as indicated), with transcripts set to 1 relative to levels in WT roots. Data are shown as mean±sem (n=3). (B) Root NO3 ^- uptake in grafted chimeras. Data are shown as mean±sem (n=30). (C) shows the NRT2.1 promoter fragment used for ChIP analysis of extracts from 14-day-old pHY5:myc-HY5hy5 plants. Data are shown as mean±sem (n=3). Arrows indicate C/G box sequence motifs. (D) Fragment 3 from (C) was incubated with MBP-HY5. Competition was performed with 10, 20, 50 or 100-fold excess of unlabeled probe. (E) Relative abundance of PSY, TPS1, SWEET11 and SWEET12 transcripts in 7-day-old seedlings. Values are expressed relative to WT levels, data are mean±sem (n=3). (F) ChIP assay. Fragments containing G-box motifs in the TPS1, SWEET11 and SWEET12 promoters were used for ChIP analysis of extracts from 14-day-old pHY5:myc-HY5hy5 plants. Data are shown as mean±sem (n=3). (G) Sucrose affects NRT2.1 transcript abundance. Transcript was set to 1 relative to the level of WT S(L)/R(D) seedlings. Data are shown as mean±sem (n=3). (H) Sucrose affects root ¹⁵ NO3 ^- uptake. Data are shown as mean±sem (n=30). (I) In vivo binding of HY5 to NRT2.1 promoter. ChIP-PCR analysis was performed using 10-day-old pHY5:myc-HY5hy5 plants. Data are shown as mean±sem (n=3). The same lowercase letters indicate non-significant differences (P<0.05) between means.

Figure 4. HY5 coordinates plant growth and nutrition in response to fluctuating light environments. (A) Primary root extension length of seedlings grown at the indicated fluence rates. Data are shown as mean±sem (n=30). (B) ¹⁵ NO3 ^-uptake by seedlings grown at the indicated fluence rates. Data are shown as mean±sem (n=30) (C) Seedling shoot biomass at different fluence rates. Data are shown as mean±sem (n=30). (D) Whole plant biomass of plants grown in soil for 21 days (16 h photoperiod) at different light fluence rates. (E) C content of plants shown in D. (F) N content of plants shown in D. (G) C/N content ratios of 21-day-old plants grown at the indicated fluence rates. (DG) Data are shown as mean±sem (n=16). The same lowercase letters indicate non-significant differences between means (P<0.05).

Figure 5. Expression of PpHY5 in Arabidopsis and expression of OsHY5 in Arabidopsis. Conserved function of HY5 homologues from Arabidopsis thaliana, rice (Oryza sativa) and Physcomitrella patens. (A-C) Hypocotyl length (A), primary root meristem cell number ( B) and root NRT2.1 transcript levels (C). Data are shown as mean ± s.e.m (n=30). (D) Distribution of GFP fused to AtHY5, OsHY5 or PpHY5 in rootstock parts (WT roots) of grafted plants.

Figure 6. a) Alignment of AtHY5 and homologues; b) Alignment of AtHY5 and AtHYH sequences, c) homologue tree.

Figure 7. Effect of differential shoot/root irradiation on lateral root development. (A) Effect of differential shoot/root irradiation on lateral root development. 3-day-old WT seedlings were transferred to new plates and then exposed to differential light treatment (100 μmol.s ⁻¹ .m ⁻² ) for 10 d. Scale bar, 1 cm. (B) Lateral root development in different treatments. Data are shown as mean±sem (n=30). The same lowercase letters indicate non-significant differences (P<0.05) between means.

Figure 8. Allelic variation of the HY5 sequence. (A) Splice site mutation in hy5-526. Dark gray boxes indicate exons, black lines indicate introns, and numbers indicate exon size (bp). (B) Protein sequence comparison between HY5 and mutant hy5-526. Numbers on the right indicate residue positions in the whole protein. Identical residues are indicated by dark gray boxes and variant residues are indicated by light gray boxes.

Figure 9. Hypocotyl length of light-grown 6-day-old seedlings (WT, hy5, and hy5 containing transgenes expressing HY5-GFP or myc-HY5, as indicated). Data are shown as mean ± s.e.m. (n=30). The same lowercase letters indicate non-significant differences (P<0.05) between means.

Figure 10. Distribution of detectable HY5-GFP in grafted plant pCAB3:HY5-GFPhy5 scions. (A) Experiments using hypocotyl grafted chimeras. (B) HY5-GFP distribution in scion leaves of 10-day-old grafted plants.

Figure 11. Distribution of HY5-GFP in roots of 10-day-old grafted plants. (A) Distribution of detectable HY5-GFP in pHY5:HY5-GFPhy5 rootstock roots of grafted plants. Scale bar, 50 μm. (B) Relative HY5-GFP transcript abundance in shoots and roots of grafted plants, as expressed in (A), relative to pHY5:transcript levels of myc-HY5hy5 scion leaves of grafted plants set to 1 . Data are shown as mean ± s.e.m. (n=3). (C) Immunological detection of HY5-GFP in scion (shoot) and rootstock (root), using HSP90 loading control.

Figure 12. Effect of differential shoot/root irradiation on the distribution of HY5-GFP in leaves. (A) Distribution of HY5-GFP in leaves of shoot-irradiated pCAB3:2×GUS-TEV ^re -HY5-GFP hy5 plants. (B) Distribution of HY5-GFP in leaves of shoot-irradiated pCAB3:2×GUS-TEV ^re -HY5-GFP hy5 plants expressing TEV protease.

Figure 13. HYH is not a light-regulated shoot-to-root movement signal that regulates root growth. (A) Effect of shoot-irradiation on primary root growth of 6-day-old Ws, hyh-1 and hy5hyh-1 seedlings. (B) Differential shoot/root irradiation effects on primary root extension length of Ws, hyh-1 and hy5hyh-1 seedlings. Data are shown as mean ± s.e.m. (n=30). The same lowercase letters indicate non-significant differences (P<0.05) between means. (C) HYH-GFP undetectably moves from shoots to roots. The distribution of HYH-GFP in the scions (leaves) and stock (roots) of 10-day-old grafted plants consisting of pSUC2:HYH-GFP hyh-1 scions and HYH(Ws) rootstocks was compared with that of pSUC2:HY5-GFP hy5 scions. Comparison of HY5-GFP distribution in scions (leaves) and rootstocks (roots) of 10-day-old grafted plants constructed from HY5(Col) rootstocks.

Figure 14. HY5 binds to the HY5 promoter. (A) ChIP assay. The diagram depicts the putative HY5 promoter and fragments (1-7) used for ChIP analysis. ChIP-PCR was performed using 14-day-old pHY5:myc-HY5hy5 plants. Data are shown as mean ± s.e.m (n=3). (B) EMSA assay. The HY5 promoter fragment containing the T/G-box-motif shown in A was incubated with the indicated MBP-HY5. Competition for HY5 binding was performed with 10x, 20x, 50x and 100x unlabeled probes containing the T/G-box motif, respectively.

Figure 15. Effect of glucose on root NRT2.1 transcript abundance and ¹⁵ NO3 ^- uptake. (A) NRT2.1 transcript abundance in roots of WT and hy5 seedlings. Transcript levels are expressed relative to the abundance of Arabidopsis actin2. Data are shown as mean±sem (n=3). (B) ¹⁵ NO3 ^- uptake in roots of 7-day-old WT and hy5 seedlings. Data are shown as mean±sem (n=10).

Figure 16. Effect of sucrose on HY5 promoter activity, HY5 transcript and binding affinity of HY5 to HY5 promoter. (A) Effect of sucrose levels on HY5 transcription (as visualized by GFP expression driven by pHY5:GFP transgene) and HY5 stability (HY5-GFP expressed from pHY5:HY5-GFP) in roots. Scale bar, 50 μm. (B) Effect of sucrose level on root HY5 transcript abundance. Transcript levels are expressed relative to the abundance of Arabidopsis actin2. Data are shown as mean ± s.e.m. (n=3). (C) ChIP-PCR analysis using 10-day-old pHY5:myc-HY5hy5 plants grown on 1/2 MS medium containing 1% or 3% sucrose. Data are shown as mean ± s.e.m. (n=3). P values were generated using Student's t-test.

Figure 17. Expression of myc-HY5 restores hy5 sucrose sensitivity to WT levels. (A) NRT2.1 transcript levels in sucrose-treated hy5 and pHY5:myc-HY5hy5 roots. Transcript levels are expressed relative to the abundance of Arabidopsis actin2. Data are shown as mean±sem (n=3). (B) ¹⁵ NO3 ^-uptake rates in sucrose-treated hy5 and pHY5:myc-HY5hy5 roots. Data are shown as mean±sem (n=30). The presence of identical lowercase letters indicates non-significant differences (P<0.05) between means.

Figure 18. Effect of increasing light fluence rate on plant growth. Shoot (top) and root (bottom) systems of WT plants grown in soil at different light fluence rates for 21 days (16 h photoperiod). Scale bar, 1 cm.

detail

The present invention will now be further described. In the following paragraphs, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as preferred or advantageous may be combined with any other feature or features indicated as preferred or advantageous.

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry and recombinant DNA techniques, bioinformatics, within the skill of the art. Such techniques are explained fully in the literature.

As used herein, the terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g. , mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA produced using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not code for mRNAs or protein products. These terms also include genes. The term "gene" or "gene sequence" is used broadly to refer to a DNA nucleic acid associated with a biological function. Thus, a gene may include introns and exons as in the genomic sequence, or may include only the coding sequence as in cDNA, and/or may include cDNA in combination with regulatory sequences.

The terms "peptide", "polypeptide" and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length linked together by peptide bonds.

As used herein, the term "genetically altered" includes, but is not limited to, transgenic plants and mutant plants.

For the purposes of the present invention, "transgenic", "transgenic" or "recombinant" means, for example, a nucleic acid sequence, an expression cassette, a gene construct or a vector comprising a nucleic acid sequence or transformed with a nucleic acid sequence, an expression cassette according to the present invention or vector organisms, all those constructs produced by recombinant methods in which

(a) a nucleic acid sequence encoding a protein used in the methods of the present invention, or

(b) a genetic control sequence, such as a promoter, operably linked to the nucleic acid sequence of the present invention, or

(c) a) and b) are not in their natural genetic environment or have been modified by recombinant methods, for which modification, for example, may take the substitution, addition, deletion, inversion or insertion of one or more nucleotide residues form. The natural genetic environment is understood to mean the presence in the natural genome or chromosomal loci or genomic libraries in the original plant. In the case of a genomic library, preferably, the natural genetic environment of the nucleic acid sequence is at least partially preserved. The environment flanks the nucleic acid sequence on at least one side and has a sequence length of at least 50 bp, preferably at least 500 bp, particularly preferably at least 1000 bp, most preferably at least 5000 bp. When the expression cassette is modified by non-natural, synthetic ("artificial") methods such as, for example, mutagenesis treatment, the natural promoter of the naturally occurring expression cassette - for example a nucleic acid sequence as defined above, is associated with the Naturally occurring combinations of corresponding nucleic acid sequences of polypeptides of the method-become transgenic expression cassettes. Suitable methods are described, for example, in US 5,565,350 or WO 00/15815, which are incorporated by reference.

In certain embodiments, transgenic plants for the purposes of the present invention are thus understood to mean, as described above, that the nucleic acids used in the methods of the present invention are not at their natural loci in the genome of said plants, for Nucleic acid, which may be expressed homologously or heterologously. Thus, the plants express the transgene. However, as mentioned, in certain embodiments, transgenic also means that when the nucleic acid according to the various embodiments of the present invention is in its natural position in the genome of a plant, the sequence has been replaced with respect to the native sequence. Modification, and/or regulatory sequences of the native sequence are modified, for example by mutagenesis.

Transgenic is preferably understood to mean the expression of a nucleic acid according to the invention at an unnatural locus of the genome, ie a homologous or preferably heterologous expression of the nucleic acid takes place. According to the invention, the transgene is stably integrated into the plant, and the plant is preferably homozygous for the transgene.

Aspects of the invention relate to recombinant DNA technology, and in preferred embodiments exclude embodiments based solely on the production of plants by conventional breeding methods.

For the purposes of the present invention, a "mutant" plant is a plant that is genetically altered compared to a naturally occurring wild-type (WT) plant. In one embodiment, a mutant plant is a plant that has been altered using a mutagenesis method, such as those described herein, compared to a naturally occurring wild-type (WT) plant. In one embodiment, the mutagenesis method targets genome modification or genome editing. In one embodiment, mutagenesis methods are used to alter the endogenous HY5 nucleic acid or HY5 promoter sequence compared to the wild-type sequence. These mutations may result in activation or otherwise enhance the activity of the HY5 promoter or a functional homologue or variant thereof or may enhance the expression level of the HY5 nucleic acid or functional homologue or variant thereof compared to wild type plants. Such plants have an altered phenotype as described herein, such as increased nitrogen metabolism, compared to wild-type plants. Thus, in this example, the presence of a mutated endogenous HY5 gene or HY5 promoter sequence in the plant genome confers increased nitrogen metabolism. In preferred embodiments, the endogenous HY5 gene or HY5 promoter sequence is specifically targeted using targeted genomic modifications, and the presence of a transgene expressed in a plant does not confer the presence of a mutated HY5 gene or HY5 promoter sequence. In an alternative embodiment there is provided a mutant plant expressing a nucleic acid as defined in SEQ ID NO: 1, 2 or 3, or a functional homologue or variant thereof. Furthermore, such plants have an altered phenotype and show increased nitrogen metabolism compared to wild-type plants. Furthermore, again, in one embodiment, the phenotype of such plants is not conferred by the presence of one or more transgenes.

The inventors found that HY5 is a shoot-to-root movement signal that regulates shoot growth and C uptake coupled with photoregulation of root growth and N uptake. HY5 is transferred from shoots to roots and then activates root HY5 expression through a self-regulatory feedback loop. Furthermore, AtHY5 directly regulates the expression of the nitrate transporter AtNRT2.1, as shown in Arabidopsis. Thus, shoot-to-root migration of HY5 is required for light-activated AtNRT2.1 transcription and N uptake ( ⁱⁿ the form of NO3-) in roots. In other words, although HY5 is produced in roots, only when HY5 protein produced in shoots/leaves due to light perception moves to roots, it accumulates effective levels and induces root development and N-uptake.

Therefore, in one aspect, the present invention relates to a method for increasing nitrate metabolism in a plant, which comprises introducing and expressing in a plant a nucleic acid construct comprising a plant HY5 nucleic acid sequence.

HY5 is highly conserved in terrestrial plants. The conserved function was demonstrated by experiments described herein, and the inventors showed that rice HY5, OsHY5, when expressed in transgenic Arabidopsis, could induce AtNRT2.1, and that HY5 from moss, Physcomitrella AtNRT2.1 could also be induced (Figure 5).

According to various aspects of the invention, including the methods described above, the plant HY5 protein encoded by the plant HY5 nucleic acid is characterized by the presence of a COP1 interaction domain and a basic leucine zipper domain (Holme et al., EMBO journal, Vol.20, p. pp. 118-127, 2001). The sequences of these domains for AtHY5 are shown below. However, these sequences are highly conserved among other HY5 proteins (see eg Figure 6), and the skilled person will thus be able to readily identify HY5 proteins of plants other than Arabidopsis.

Thus, plant HY5 proteins according to various aspects of the invention comprise one or more of the conserved domains identified below or have at least 70%, 71%, 72%, 73%, 74%, 75% of the motifs shown below , 76%, 77, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98% or at least 99% overall sequence identity domains.

The AtHY5 protein includes the following COP1 interacting domain: ESDEEIRRVPEF (SEQ ID NO: 30, see Figure 6). This includes the putative CKII site for phosphorylation (ESDEE SEQ ID NO: 31). The core V-P-E/D-X-G motif (SEQ ID NO: 32, X is a hydrophobic residue) of this domain (including the COP1 interaction domain) is a highly conserved structural motif that is essential for the interaction with COP1.

The AtHY5 protein also includes a basic leucine zipper domain shown below:

Basic motif: KRLKRLLRNRVSAQQARERKK (SEQ ID NO: 33)

Leucine zipper: LENRVKDLENKNSELEERLSTLQNENQML (SEQ ID NO: 34)

In one embodiment, the HY5 nucleic acid construct comprises SEQ ID NO: 1 or 2 encoding the AtHY5 protein defined by SEQ ID NO: 3 or a functional variant or homologue thereof.

The term "functional variant of a nucleic acid sequence" as used herein, with reference to SEQ ID NO: 1 or another sequence, refers to a variant gene sequence or part of a gene sequence that retains the biological function of the complete non-variant sequence, e.g. Confers increased growth or yield when expressed in transgenic plants. Functional variants also include variants of the gene of interest that have sequence changes that do not affect function, such as changes in non-conserved residues. Also included are variants that are substantially identical, ie, have only some sequence changes, such as changes in non-conserved residues, compared to the wild-type sequence shown herein, and are biologically active.

Therefore, as understood by those skilled in the art, it is to be understood that aspects of the present invention, including methods and uses, not only include the nucleic acid sequence comprising or consisting of SEQ ID NO: 1, but also include those that do not affect the biological activity of the resulting protein. and functional variants or parts of SEQ ID NO: 1. Alterations in a nucleic acid sequence that result in a different amino acid at a given position that do not affect the functional properties of the encoded polypeptide are well known in the art. For example, a codon for the amino acid alanine, a hydrophobic amino acid, could be coded for another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine Acid codon substitutions. Similarly, changes that result in the substitution of one negatively charged residue for another, such as aspartate for glutamate, or one positively charged residue for another, such as lysine for asparagine , can also be expected to yield functionally equivalent products. Nucleotide changes that result in changes to the N-terminal and C-terminal portions of the polypeptide molecule would not be expected to alter the activity of the polypeptide. Each suggested modification is well within the routine skill in the art, which is a determinant of the retention of biological activity of the encoded product.

The functional variant has at least 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54% , 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity.

The skilled person will appreciate that the invention is not limited to aspects using AtHY5. Thus, in one embodiment of the aspects of the invention, the nucleic acid sequence encodes a homologue of SEQ ID NO:3.

The term homologue as used herein also means AtHY5 homologues from other plant species. The homologues of the AtHY5 polypeptide or the AtHY5 nucleic acid sequence share at least 25%, 26%, and 27% with the amino acid represented by SEQ ID NO: 3 or with the nucleic acid sequence shown in SEQ ID NO: 1 or 2, respectively, in order of increasing preference , 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44 %, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or at least 99% overall sequence identity. In one embodiment, the overall sequence identity is at least 37%. In one embodiment, the overall sequence identity is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, most preferably 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99%.

Functional variants of HY5 homologues are also within the scope of the invention.

Two nucleic acid sequences or polypeptides are said to be "identical" if the sequence of nucleotides or amino acid residues, respectively, in the two sequences is identical when aligned for maximum identity, as described below. The term "identical" or percent "identity" in the context of one or two nucleic acid or polypeptide sequences means that the comparison window corresponds to the maximum Identity When compared and aligned, two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same. When percentages of sequence identity are used with respect to proteins or peptides, residue positions that are considered non-identical often differ by conservative amino acid substitutions, in which an amino acid residue substitutes for another amino acid residue of similar chemical properties (e.g., charge or hydrophobicity) groups, and thus do not alter the functional properties of the molecule. Where the sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. The manner in which this adjustment is made is well known to those skilled in the art. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequent coordinates are designated, and, if necessary, sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters. Non-limiting examples of algorithms suitable for determining sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

Examples of homologues are shown in Figure 6, Table 1 and SEQ ID NOs: 4-29. The inventors have shown that a homologue from Physcomitrella patens with 38.6% sequence identity to AtHY5 and OsHY5 with 65.5% sequence identity to AtHY5 both induce AtNRT2.1 when expressed in Arabidopsis (Fig. 5), and rescued the hy5 loss of function mutants.

Suitable homologues can be identified by sequence comparison and identification of conserved domains. Predictors exist in the art that can be used to identify such sequences. The function of a homolog can be identified as described herein, and the skilled person can thus confirm the function, eg when overexpressed in plants or when expressed in a hy5 loss-of-function mutant by rescuing the mutant phenotype.

Accordingly, the HY5 nucleotide sequences of the invention and described herein may also be used to isolate corresponding sequences from other organisms, in particular other plants, such as crop plants. In this manner, methods such as PCR, hybridization, etc. can be used to identify such sequences based on their sequence homology to the sequences described herein. When identifying and isolating homologues, sequence topology and characteristic domain structures can also be considered. Sequences can be isolated based on their sequence identity to the entire sequence or to fragments thereof. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe with other DNA fragments present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., a genomic or cDNA library) from a selected plant. Corresponding nucleotide sequences hybridize selectively. Hybridization probes can be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and can be labeled with a detectable group, or any other detectable marker. Methods for the preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and described in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2nd Edition, Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences can be performed under stringent conditions. "Stringent conditions" or "stringent hybridization conditions" mean conditions under which a probe hybridizes to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold above background) . Stringent conditions are sequence-dependent and will be different in different circumstances. By controlling the stringency of hybridization and/or wash conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatches in the sequences, thereby detecting lower degrees of similarity (heterologous probing). Typically, probes are less than about 1000 nucleotides in length, preferably less than 500 nucleotides in length.

Typically, stringent conditions will be those in which the salt concentration is less than about 1.5M Na ion concentration at pH 7.0 to 8.3, usually about 0.01 to 1.0M Na ion concentration (or other salt), and for short probes (e.g., 10 to 50 nucleotides), the temperature is at least about 30°C, and for long probes (eg, greater than 50 nucleotides), the temperature is at least about 60°C. Duration of hybridization is usually less than about 24 hours, usually about 4 to 12 hours. Stringent conditions can also be achieved by the addition of destabilizing agents such as formamide.

According to the invention, preferred AtHY5 homologues are selected from the group consisting of maize, rice, wheat, Brassica napus/Canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, broad bean, lettuce, cotton , sugar cane, sugar beet, broccoli or other brassica vegetables or poplar.

According to the methods described herein, a plant expresses a polynucleotide that is "foreign" to the individual plant, said "exogenous" polynucleotide being a polynucleotide introduced into the plant by any means other than sexual crossing . Examples of ways by which this can be achieved are described below. In one embodiment of the method, an exogenous nucleic acid is expressed in a transgenic plant, which is not endogenous to said plant, but is derived from a plant HY5 nucleic acid sequence of another plant species. For example, AtHY5 can be expressed in another plant than Arabidopsis. In one embodiment of the method, an endogenous nucleic acid is expressed in a transgenic plant, which is a nucleic acid construct comprising an endogenous HY5 nucleic acid or a sequence derived therefrom. For example, OsHY5 can be expressed in rice.

Plants, including transgenic plants, according to the various aspects of the invention, methods and uses described herein, may be monocots or dicots.

Dicotyledonous plants may be selected from families including but not limited to Asteraceae, Brassicaceae (e.g. Brassica napus), Chenopodiaceae, Cucurbitaceae, Leguminosae (Caesalpiniaceae, Aesalpiniaceae, Mimosaceae, Papilionaceae or Fabaceae), Malvaceae, Rosaceae or Solanaceae. For example, the plant may be selected from lettuce, sunflower, Arabidopsis, broccoli, spinach, watermelon, winter squash, kale, tomato, potato, yam, pepper, tobacco, cotton, okra, apple, rose, strawberry, alfalfa, bean , soybeans, beans, peas, lentils, peanuts, chickpeas, apricots, pears, peaches, vines, bell peppers, peppers or citrus species.

The monocot may be, for example, selected from the families Arecaceae, Amaryllidaceae or Poaceae. For example, the plant may be a cereal crop such as corn, wheat, rice, barley, oats, sorghum, rye, millet, buckwheat, or a forage crop such as Lolium species or Festuca species, or a crop Such as sugar cane, onions, leeks, yams or bananas.

Also included are biofuel and bioenergy crops such as canola/canola, sugar cane, sweet sorghum, switchgrass (Panicum virgatum) (switchgrass), flaxseed, lupine and willow, poplar, poplar hybrids, miscanthus (Miscanthus) or gymnosperms such as loblolly pine. Also included are those used for silage (corn), grazing or forage (grass, clover, sanfoin, alfalfa), fibers (e.g. cotton, flax), building materials (e.g. pine, oak), pulping (e.g. poplar ), feed stock for the chemical industry (e.g. high erucic acid oilseed rape, linseed) and for amenity purposes (e.g. turfgrass for golf courses), ornamental plants for public and private gardens (e.g. snapdragon, Petunias, roses, geraniums, Nicotiana) and household plants and cut flowers (African violets, Begonias, chrysanthemums, geraniums, Coleus spider plants, Dracaena ( Dracaena), rubber plant) crops.

Preferably, the plants are crop plants. By crop plant is meant any plant grown on a commercial scale for human or animal consumption or use. In a preferred embodiment, the plant is a cereal.

Most preferred plants are corn, rice, wheat, rape/canola, sorghum, soybean, sunflower, alfalfa, potato, tomato, tobacco, grape, barley, pea, bean, broad bean, lettuce, cotton, sugar cane, sugar beet , broccoli or other brassica vegetables or poplar.

The term "plant" as used herein includes whole plants, ancestors and descendants of plants and plant parts, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues and organs, wherein each of the foregoing One comprising a nucleic acid construct as described herein. The term "plant" also includes plant cells, suspension cultures, callus, embryos, meristematic zones, partners, sporophytes, pollen and microspores, likewise wherein each of the foregoing comprises a nucleic acid construct as described herein body.

The method may also comprise screening for those plants comprising a polynucleotide construct described herein and/or having any of the phenotypes described herein (such as increased nitrogen metabolism) from the plants, and selecting those plants having said phenotype type (such as increased nitrogen metabolism) in plants. In another embodiment, a further step comprises measuring nitrogen metabolism in progeny of said plants or parts thereof, and comparing nitrogen metabolism to control plants. Preferably, progeny plants are stably transformed and comprise the exogenous polynucleotide genetically maintained in the plant cell, and the method may include a step of verifying stable integration of the construct. The method may also comprise the additional step of collecting seeds from selected progeny plants.

Aspects of the invention also extend to products derived, preferably directly derived, from harvestable parts of the plant, such as dry granules or powders, oils, fats and fatty acids, starches or proteins. The invention also relates to foods and food supplements comprising the plants of the invention or parts thereof.

According to various aspects of the invention, nitrogen metabolism is increased in transgenic plants compared to control plants. A control plant according to all aspects of the invention as used herein is a plant which has not been modified according to the methods of the invention. Therefore, control plants were not genetically modified to express the nucleic acid encoding HY5. In one embodiment, the control plant is a wild type plant. The control plant is usually the same plant species, preferably of the same genetic background as the modified plant.

Nitrogen metabolism according to the invention includes increased NUE and nitrogen uptake (NO3 ⁻ uptake) according to the invention. The terms "increase", "improvement" or "enhancement" as used in accordance with various aspects of the present invention are used interchangeably. NUE can be defined as the grain yield per unit of N available in soil (including residual N present in soil and fertilizer). The overall N use efficiency of a plant includes both uptake and use efficiency and can be calculated as UpE. In one embodiment, NUE is increased by more than 5%-50%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In another embodiment, nitrogen uptake is increased by more than 5%-50%, such as at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, compared to control plants % or 50%.

Increased nitrogen metabolism leads to increased yield. Thus, the method of the invention can be used for increased yields. The term "yield" includes one or more of the following characteristics in a non-limiting list: early flowering time, biomass (plant biomass (root and/or shoot biomass) or seed/grain biomass), Seed/grain yield, seed/grain viability and germination efficiency, seed/grain size, grain starch content, early vigor, greenness value, increased growth rate, delayed senescence of green tissue. The term "yield" generally means the measurable production of economic value typically associated with a given crop, area and time period. Individual plant parts directly contribute to yield based on their number, size and/or weight. Actual yield is the yield per square meter per year of the crop year, determined by dividing the total yield (including harvested and assessed yield) by the square meter planted.

Thus, according to the present invention, yield comprises one or more of, and can be measured by evaluating one or more of: increased seed yield per plant, increased seed filling, Increased number of filled seeds, increased harvest index, increased viability/germination efficiency, increased number or size of seeds/capsules/pods/grains, increased growth or increased branching, e.g. with more Multi-branched inflorescences for increased biomass or grain filling. Preferably, increased yield comprises increased number of grains/seeds/capsules/pods, increased biomass, increased growth, increased floral organs and/or increased number of branched flowers. Increased yield relative to control plants. For example, an increase in yield of more than 2%, 3%, 4%, 5%-50%, e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, compared to control plants , 45% or 50%.

The method for increasing nitrogen metabolism in plants as described above (comprising introducing and expressing in plants a nucleic acid construct comprising a plant HY5 nucleic acid sequence) may comprise further steps comprising evaluating the phenotype of the transgenic plant, measuring One or more of NUE and/or NO3 ^- uptake, comparing NUE and/or NO3 ^- uptake to control plants, measuring yield and comparing yield to control plants.

Transformation methods for producing transgenic plants of the invention are known in the art. Thus, according to various aspects of the invention, a nucleic acid comprising a HY5 nucleic acid, such as SEQ D NO. 1 , functional variants or homologues thereof, is introduced into plants and expressed as a transgene. The nucleic acid sequence is introduced into the plant by a process called transformation. The term "introduction" or "transformation" as referred to herein includes the transfer of an exogenous polynucleotide into a host cell, regardless of the method used for the transfer. Plant tissues capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with the genetic constructs of the invention and whole plants produced therefrom. The particular tissue chosen will vary according to the clonal propagation system available for, and best adapted to, the particular species being transformed. Exemplary tissue targets include leaf discs, pollen, embryos, cotyledons, hypocotyls, female gametophytes, callus tissue, existing meristems (e.g., apical meristems, axillary buds, and root meristems), and Induced meristems (eg, cotyledon meristem and hypocotyl meristem). A polynucleotide can be introduced into a host cell transiently or stably, and can remain unintegrated, eg, as a plasmid. Alternatively, it can integrate into the host genome. The resulting transformed plant cells can then be used to regenerate transformed plants in a manner known to those skilled in the art.

The transfer of a foreign gene into the genome of a plant is called transformation. Transformation of plants is now a routine technique in many species. Advantageously, any of a variety of transformation methods can be used to introduce the gene of interest into suitable progenitor cells. Methods described for transformation and regeneration of plants from plant tissues or plant cells can be used for transient or stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase the uptake of free DNA, direct injection of DNA into plants, particle gun bombardment, transformation using viruses or pollen, and microprojection. Methods can be selected from the calcium/polyethylene glycol method for protoplasts, electroporation of protoplasts, microinjection into plant material, bombardment of DNA or RNA-coated particles, infection with (non-integrating) virus, etc. Transgenic plants, including transgenic crop plants, are preferably produced by Agrobacterium tumefaciens-mediated transformation.

In order to select transformed plants, the plant material obtained in the transformation is subjected to selective conditions so that transformed plants can be distinguished from non-transformed plants. For example, the seeds obtained in the above manner can be planted and, after the initial growth period, suitable selections can be carried out by spraying. Another possibility is, if appropriate, to grow the seeds after sterilization on agar plates with a suitable selection agent so that only transformed seeds can grow into plants. Alternatively, transformed plants are screened for the presence of a selectable marker. Following DNA transfer and regeneration, putative transformed plants can also be assessed for the presence, copy number and/or genomic composition of the gene of interest, eg, using DNA analysis. Alternatively or additionally, expression levels of newly introduced DNA can be monitored using Northern blot and/or Western blot analysis, both techniques are well known to those of ordinary skill in the art.

The resulting transformed plants can be propagated in a variety of ways, such as by clonal propagation or classical breeding techniques. For example, first generation (or T1) transformed plants can be selfed and homozygous second generation (or T2) transformants selected, and the T2 plants can then be further propagated by classical breeding techniques. The resulting transformed organism can take a variety of forms. For example, they can be chimeras of transformed and untransformed cells; clonal transformants (e.g., transforming all cells to contain the expression cassette); grafts of transformed and non-transformed tissues (e.g., in plants, Transformed rhizomes grafted onto untransformed scions).

The method of the present invention for increasing nitrogen metabolism in plants comprises introducing and expressing a nucleic acid construct comprising a plant HY5 nucleic acid sequence. The nucleic acid construct preferably comprises a regulatory element operably linked to a plant HY5 nucleic acid sequence.

According to all aspects of the invention, including the methods described above and including plants, methods and uses as described herein, the term "regulatory element" is used herein interchangeably with "control sequence" and "promoter", and all terms are used in a broad sense Considered in the context of , regulatory nucleic acid sequences capable of affecting the expression of sequences to which they are linked.

The term "promoter" generally refers to a nucleic acid control sequence located upstream of the initiation of transcription of a gene, which is involved in the binding of RNA polymerase and other proteins to direct the transcription of an operably linked nucleic acid. Included in the above terms are transcriptions derived from classical eukaryotic genomic genes (including the TATA box required for precise transcription initiation, with or without CCAAT box sequences) and additional regulatory elements (i.e., upstream activating sequences, enhancers and silencers) Regulatory sequences, which alter gene expression in response to developmental and/or external stimuli or in a tissue-specific manner. Also included within the term are the transcriptional regulatory sequences of classical prokaryotic genes, which in this case may include the -35 box sequence and/or the -10 box transcriptional regulatory sequence.

The term "regulatory element" also includes synthetic fusion molecules or derivatives that confer, activate or enhance the expression of a nucleic acid molecule in a cell, tissue or organ.

A "plant promoter" comprises regulatory elements that regulate the expression of a coding sequence segment in plant cells. Thus, a plant promoter need not be of plant origin, but may be derived from viruses or microorganisms, for example from viruses that attack plant cells. A "plant promoter" may also be derived from a plant cell, eg from a plant transformed with a nucleic acid sequence to be expressed in the methods of the invention and described herein. This also applies to other "plant" regulatory signals, such as "plant" terminators. The promoter upstream of the nucleotide sequence used in the method of the invention may be modified by one or more nucleotide substitutions, insertions and/or deletions without interfering with the promoter, open reading frame (ORF) or 3'-regulation The function or activity of any of the regions such as the terminator or other 3' regulatory regions remote from the ORF. It is also possible that the activity of promoters is increased by modifying their sequences, or that they are completely replaced by more active promoters, even by promoters from heterologous organisms. For expression in plants, a nucleic acid molecule as described above is preferably operably linked to or comprises a suitable promoter which expresses the Gene.

To identify functionally equivalent promoters, the promoter strength and/or expression pattern of a candidate promoter can be analyzed, for example, by operably linking the promoter to a reporter gene and determining the expression level and pattern of the reporter gene in different tissues of the plant . Suitable well known reporter genes are known to the skilled person and include for example beta-glucuronidase or beta-galactosidase.

The term "operably linked" as used herein refers to a functional linkage between a promoter sequence and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

For example, a nucleic acid sequence can be expressed using a promoter that drives overexpression. Overexpression according to the invention means that the transgene is expressed at a level higher than that of its endogenous counterpart driven by its endogenous promoter. For example, overexpression can be performed using strong promoters, such as constitutive promoters. A "constitutive promoter" refers to a promoter that is transcriptionally active in at least one cell, tissue or organ during most, but not necessarily all, stages of growth and development and under most environmental conditions. Examples of constitutive promoters include cauliflower mosaic virus promoter (CaMV35S or 19S), rice actin promoter, maize ubiquitin promoter, ribulose bisphosphate hydroxylase (rubisco) small subunit, maize or alfalfa H3 histone, OCS, SAD1 or 2, GOS2 or any promoter that produces enhanced expression. Alternatively, enhanced or increased expression can be achieved through the use of transcriptional or translational enhancers or activators, and enhancers can be incorporated into genes to further increase expression.

In one embodiment, the promoter is a constitutive or strong promoter. In preferred embodiments, the regulatory sequence is an inducible promoter or a stress-inducible promoter. The stress-inducible promoter is selected from the following non-limiting list: HaHB1 promoter, RD29A (which drives the drought-inducible expression of DREB1A), maize rabl7 drought-inducible promoter, P5CS1 (which drives the expression of the proline biosynthesis enzyme P5CS1 Drought-inducible expression), the ABA- and drought-inducible promoters of Arabidopsis clade A PP2C (ABI1, ABI2, HAB1, PP2CA, HAI1, HAI2 and HAI3) or their corresponding crop homologues.

Promoters can also be tissue specific. These types of promoters are described in the prior art, but non-limiting examples are listed below. Other suitable promoters are also known to the skilled person.

Tissue-specific promoters are transcriptional control elements that are active only in specific cells or tissues, such as in vegetative or reproductive tissues, at specific times during plant development. Examples of tissue-specific promoters under developmental control include promoters that are expressed only (or predominantly only) in specific tissues (such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruits, ovules, seeds, pollen, pistols ), flower) or any promoter that initiates transcription in embryonic tissue or epidermis or mesophyll. Reproductive tissue-specific promoters can be, for example, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and testa-specific, pollen-specific, petal-specific, Sepals - specific, or some combination thereof. In some embodiments, the promoter is cell type specific, eg, guard cell-specific.

In one embodiment, a green tissue-specific promoter can be used. Green tissues include leaves and shoots. For example, the green tissue-specific promoter can be selected from the group consisting of maize orthophosphokinase promoter, maize phosphoenolpyruvate carboxylase promoter, rice phosphoenolpyruvate carboxylase promoter, rice small subunit nuclear Ketose bisphosphate hydroxylase (rubisco) promoter, rice β-expandin EXBP9 promoter, pigeon pea small subunit ribulose bisphosphate hydroxylase (rubisco) promoter, mesophyll-specific promoter CAB3 (Warnasooriya, S.N. , and Montgomery, B.L. (2009), Plant Physiol 149, 424-433) or the pea RBS3A promoter.

Examples of leaf-specific promoters also include the ribulose diphosphate carboxylase (RBCS) promoter. For example, the tomato RBCS1, RBCS2, and RBCS3A genes are expressed in leaves and light-grown seedlings, and only RBCS1 and RBCS2 are expressed in developing tomato fruit (Meier FEBS Lett. 415:91-95, 1997). The ribulose diphosphate carboxylase promoter described by Matsuoka Plant J. 6: 311-319, 1194, which is expressed at a high level almost only in mesophyll cells in leaves and leaf sheaths, can be used. Another leaf-specific promoter is the light-harvesting chlorophyll a/b binding protein gene promoter, see, eg, Shiina Plant Physiol. 115:477-483, 1997; Casal Plant Physiol. 116: 1533-1538, 1998. The Arabidopsis myb-related gene promoter (Atmyb5) described by Li FEBS Lett. 379:1 17-121, 1996 is also leaf-specific. The Atmyb5 promoter is expressed in developing leaf trichomes on young rosettes and stem leaf margins, stipules and epidermal cells, and immature seeds. Atmyb5mRA appears at the 16-cell stage of fertilization and embryonic development and persists beyond the heart stage.

Another type of useful vegetative tissue-specific promoter is a meristem promoter, which drives expression in shoot tips. For example, the "SHOOTMERISTEMLESS" and "SCARECROW" promoters, which are active in developing shoot or root apical meristems, can be used by Di Laurenzio, Cell 86:423-433, 1996; and Long, Nature 379: 66-69, 1996 description. Another useful promoter is one that controls the expression of the 3-hydroxy-3-methylglutaryl-CoA reductase HMG2 gene, the expression of which is restricted to the meristems and flowers (secretory region of the stigma, mature pollen pistil group vascular tissue and fertilized ovule) tissue (see, eg, Enjuto Plant Cell. 7:517-527, 1995). Also available are knl-related genes from maize and other species that exhibit meristem-specific expression, see, e.g., Granger Plant Mol. Biol. 31:373-378, 1996; Kerstetter Plant Cell 6:1877- 1887, 1994; Hake Philos. Trans. R. Soc. Lond. B. Biol. Sci. 350:45-51, 1995.

The invention also relates to genetically altered plants comprising a HY5 nucleic acid sequence and tissue-specific regulatory sequences.

In one aspect, the present invention relates to a transgenic plant comprising a nucleic acid construct comprising a HY5 nucleic acid sequence and a tissue-specific regulatory sequence.

The terms HY5 nucleic acid sequence and tissue-specific regulatory sequence are described elsewhere herein. Preferably, the tissue-specific regulatory sequence is a leaf or shoot-specific promoter. In one embodiment, the plant is not Arabidopsis.

The invention also extends to transgenic plants comprising a nucleic acid construct comprising a HY5 nucleic acid sequence, wherein said plant is not Arabidopsis.

The present invention also relates to a nucleic acid construct comprising a plant HY5 nucleic acid sequence operably linked to a tissue-specific promoter. Tissue-specific promoters may be selected from green tissue-specific promoters as described above. The present invention also relates to an expression vector comprising a nucleic acid construct comprising a plant HY5 nucleic acid sequence operably linked to a tissue-specific promoter.

In another aspect, the invention relates to an isolated host cell transformed with a nucleic acid construct or vector as described above. The host cell can be a bacterial cell, such as Agrobacterium tumefaciens, or an isolated plant cell. The invention also relates to a culture medium or a kit comprising a culture medium and an isolated host cell as described above.

The nucleic acid constructs or vectors described above can be used to generate transgenic plants using transformation methods known in the art. Thus, in a further aspect, the present invention relates to the use of the above-mentioned nucleic acid construct or vector for increasing nitrogen metabolism in plants. In another aspect, the present invention relates to the use of a nucleic acid construct or vector comprising a HY5 nucleic acid sequence and a tissue-specific promoter for increasing the level of HY5 in plant roots.

The present invention also relates to a method for producing plants with increased nitrogen metabolism, comprising introducing and expressing in plants a nucleic acid construct comprising a HY5 nucleic acid sequence operably linked to a green tissue-specific promoter.

In another aspect, the present invention relates to a method for increasing the presence of HY5 protein in plant roots, which comprises introducing and expressing in a plant a nucleic acid construct comprising a HY5 nucleic acid sequence operably linked to a green tissue-specific promoter.

The inventors also demonstrated that HY5 regulates the balance between C and N metabolism. The relative abundance of transcripts encoding proteins associated with the availability of fixed carbon in the form of sucrose (e.g. TPS1) or encoding sucrose efflux transporters (e.g. SWEET11 and 12) was strongly reduced in hy5 seedlings (lacking HY5), suggesting HY5 regulates both sucrose synthesis and migratory aspects of C metabolism. They also found that sucrose or glucose substantially enhanced AtNRT2.1 transcript abundance and NO3 ^- uptake in WT plants, but these effects were substantially reduced in hy5 mutants. High-affinity nitrate transporter expression and HY5-dependent sucrose induction of N-uptake promote a coordinated homeostatic balance of C and N metabolism. Therefore, the present invention also relates to a method for regulating the balance between C and N metabolism in plants, which comprises introducing and expressing a nucleic acid construct comprising a HY5 nucleic acid sequence in a plant. In one embodiment, the HY5 nucleic acid sequence is operably linked to a green tissue-specific promoter.

The present invention also relates to a method for increasing HY5 levels in plant roots, which comprises introducing and expressing in plants a nucleic acid construct comprising a HY5 nucleic acid sequence operably linked to a green tissue-specific promoter.

The invention also extends to plants obtained or obtainable by a method as described herein, for example a method of increasing nitrate metabolism.

In another aspect, the present invention relates to mutant plants, i.e. plants having increased expression of an endogenous HY5 nucleic acid, wherein the endogenous HY5 promoter carries a mutation introduced by mutagenesis or targeted genome editing, and said mutation Resulting in increased expression of endogenous HY5 nucleic acid. In this embodiment, the increase in expression is relative to the level in a control or wild-type plant, as described elsewhere herein. In one embodiment, targeted genome editing is used to modify (eg, insert or alter) at least one nucleic acid in the nucleic acid sequence of the HY5 promoter, or a functional homologue or variant thereof. In preferred embodiments, the mutation increases the activity of the endogenous promoter. For example, targeted genomic modification can be used to insert at least one enhancer or promoter site, such as a TATA box (TATAAA), GC box (GGGCGG) or CAAT (GGCCAATCT) box, or functional variants thereof. In another aspect, the present invention relates to plants having increased expression of an endogenous HY5 nucleic acid, wherein the endogenous HY5 nucleic acid carries a mutation introduced by mutagenesis or targeted genome editing, which results in increased expression of said nucleic acid. In another aspect, the present invention relates to plants having increased expression of a HY5 nucleic acid, wherein HY5 expression is increased by incorporation of one or more additional copies of the HY5 nucleic acid into the plant genome. In preferred embodiments, said combined genome editing is achieved.

In one embodiment, the HY5 nucleic acid is SEQ ID NO: 1 or 2 or a functional homologue or variant thereof. In one embodiment, the endogenous promoter is SEQ ID NO: 35 or a functional homolog or variant thereof.

In another aspect of the invention, there is provided a method of increasing nitrogen metabolism in a plant, the method comprising introducing at least one mutation into an endogenous HY5 sequence or a regulatory sequence thereof to enhance expression of an endogenous HY5 nucleic acid. Also provided are methods of producing plants with increased nitrogen uptake, methods of increasing the presence of HY5 protein in plant roots, methods of modulating the balance between C and N metabolism in plants and methods of increasing the presence of HY5 protein in plant roots, Wherein the method comprises introducing at least one mutation into an endogenous (ie native or native) HY5 sequence or its regulatory sequence to enhance expression of an endogenous HY5 nucleic acid.

In one embodiment, said at least one mutation is introduced using targeted genome editing. In one embodiment, targeted genome editing is used to modify (eg, insert or alter) at least one nucleic acid in the nucleic acid sequence of the HY5 promoter, or a functional homologue or variant thereof. In preferred embodiments, the mutation increases the activity of the endogenous promoter. For example, targeted genomic modification can be used to insert at least one enhancer or promoter site, such as a TATA box (TATAAA), GC box (GGGCGG) or CAAT (GGCCAATCT) box, or functional variants thereof. In another embodiment, targeted genome editing is used to incorporate one or more additional copies of the HY5 nucleic acid into the plant genome.

In one embodiment, the HY5 nucleic acid is SEQ ID NO: 1 or 2 or a functional homologue or variant thereof. In one embodiment, the endogenous promoter is SEQ ID NO: 35 or a functional homolog or variant thereof.

In the above embodiments, an 'endogenous' nucleic acid may refer to a sequence that is native or native to the genome of a plant.

Targeted genome modification or targeted genome editing is a genome engineering technique that uses targeted DNA double-strand breaks (DSBs) to stimulate genome editing through homologous recombination (HR)-mediated recombination events. To achieve efficient genome editing by introducing site-specific DNADSBs, four main classes of customizable DNA-binding proteins are available: meganucleases derived from mobile genetic elements of microorganisms, ZFs based on eukaryotic transcription factors Ribozymes, transcription activator-like effectors (TALEs) from Xanthomonas bacteria, and CRISPR (clustered regularly interspaced short palindromic repeats) RNA-guided DNA endonuclease Cas9. Meganucleases, ZFs, and TALE proteins all recognize specific DNA sequences through protein-DNA interactions. Whereas meganucleases integrate nuclease and DNA-binding domains, ZF and TALE proteins consist of individual modules of 3 or 1 nucleotide (nt), respectively, that target DNA. ZFs and TALEs can be assembled in desired combinations and linked to the nuclease domain of Fokl to direct nucleolytic activity against specific genomic loci.

Upon delivery into a host cell via the bacterial type III secretion system, TAL effectors enter the nucleus, bind effector-specific sequences in host gene promoters and activate transcription. Their targeting specificity is determined by a central domain of tandem 33-35 amino acid repeats. This is followed by a single truncated 20 amino acid repeat. Most naturally occurring TAL effectors examined have between 12 and 27 complete repeats.

These repeats differ from each other only by two adjacent amino acids, their repeat-variable diresidues (RVD). Decide which single nucleotide RVD the TAL effector will recognize: one RVD corresponds to one nucleotide, and the four most common RVDs are each preferably associated with one of the four bases. The naturally occurring recognition site is consistently followed by a T required for TAL effector activity. TAL effectors can be fused to the catalytic domain of FokI nuclease to generate TAL effector nucleases (TALENs), which generate targeted DNA double-strand breaks (DSBs) for genome editing in vivo. The use of this technique in genome editing is well described in the art, for example in US 8,440,431 , US 8,440,432 and US 8,450,471 . Reference 38 describes a set of custom-made plasmids that can be used with the Golden Gate cloning method to assemble multiple DNA fragments. As described therein, the Golden Gate method uses a type IIS restriction endonuclease that cleaves outside its recognition site to generate a unique 4 bp overhang. Cloning is accelerated by digestion and ligation in the same reaction mixture, since enzyme recognition sites are eliminated for proper assembly. Assembly of custom TALEN or TAL effector constructs involves two steps: (i) assembly of repeat modules into intermediate arrays of 1-10 repeats, and (ii) joining of intermediate arrays into a backbone to make the final construct.

Another genome editing method that can be used in accordance with various aspects of the invention is CRISPR. The use of this technique in genome editing is well described in the art, for example in US 8,697,359 and references cited therein. In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain combinations of CRISPR-associated (Cas) genes and specific non-coding RNA elements capable of programming CRISPR-mediated nucleic acid cleavage (sgRNA). Three types of (I-III) CRISPR systems have been identified in a wide range of bacterial hosts. A key feature of each CRISPR locus is the presence of an array of repeat sequences (direct repeats) interspersed by short non-repetitive sequence segments (spacers). The non-coding CRISPR array is transcribed and cleaved within the direct repeat into short crRNAs containing individual spacer sequences that guide the Cas nuclease to the target site (protospacer). Type II CRISPR is one of the best characterized systems and carries targeted DNA double-strand breaks in four sequential steps. First, two noncoding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the repeat region of the pre-crRNA and mediates the processing of the pre-crRNA into a mature crRNA containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 through Watson-Crick base-pairing between the spacer arm on the crRNA and the protospacer on the target DNA adjacent to the protospacer adjacent motif (PAM) to the target DNA, which is additionally required for target recognition. Finally, Cas9 regulates cleavage of target DNA to generate double-strand breaks within the protospacer.

Thus, Cas9 is the hallmark protein of type II CRISPR-Cas systems and is a large monomeric DNA nuclease guided by a complex of two noncoding RNAs, CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA) To a DNA target sequence adjacent to a PAM (protospacer adjacent motif) sequence motif. The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand and thereby introduces a blunt cleavage in the target DNA. Heterologous expression of Cas9 together with sgRNA can introduce site-specific double-strand breaks (DSBs) into the genomic DNA of living cells from various organisms. For eukaryotic application, a codon-optimized version of Cas9, originally from the bacterium Streptococcus pyogenes, was used.

A single guide RNA (sgRNA), the second component of the CRISPR/Cas system, forms a complex with the Cas9 nuclease. sgRNAs are synthetic RNA chimeras produced by fusing crRNA to tracrRNA. The sgRNA guide sequence at its 5' end confers DNA target specificity. Therefore, by modifying the guide sequence, it is possible to generate sgRNAs with different target specificities. A typical length of the leader sequence is 20bp. In plants, plant RNA polymerase III promoters such as U6 and U3 have been used to express sgRNAs.

Cas9 expression plasmids used in the methods of the invention can be constructed as described in the art.

Thus, aspects of the invention relate to targeted mutagenesis methods, in particular genome editing, and in preferred embodiments exclude embodiments based solely on the generation of plants by traditional breeding methods.

In another aspect of the present invention, there is provided a screening method for detecting plant species with increased or high expression levels of HY5 and/or increased or high levels of nitrogen metabolism, said method comprising detecting HY5 expression levels in at least one plant , and selecting the one or more plants with the highest or higher expression level of HY5. In preferred embodiments, selected plants are also propagated by various means, such as those described above.

The term HY5 and plant is defined above.

The foregoing disclosure provides a general description of subject matter encompassed within the scope of the invention, including methods of making and using the invention, and the best mode thereof, the following examples are provided to further enable those skilled in the art to practice the invention, and a complete written description of . However, those skilled in the art will appreciate that the specifics of these examples should not be construed as limiting the invention, the scope of which should be understood from the appended claims of this disclosure and their equivalents. Various other aspects and embodiments of the invention will be apparent to those skilled in the art from the present disclosure.

"And/or" as used herein is to be considered as a specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be understood as specific disclosure of each of (i) A, (ii) B, and (iii) A and B, as if each were individually listed herein.

Unless the context dictates otherwise, the described definitions of the above features are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments described.

The foregoing applications, and all documents and sequence accession numbers cited therein or during their prosecution (“Application Cited Documents”) and all documents cited or referenced in Application Cited Documents, and all documents cited or referenced herein ("documents cited herein"), and all documents cited or referenced in documents cited herein, together with any manufacturer's instructions, instructions for any product described herein or in any document incorporated by reference herein , Product Specification and Product Brochure, which are hereby incorporated by reference and may be used in the practice of the present invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.

Example

Materials and methods

Plant material and growing conditions. Arabidopsis seeds were imbibed at 4 °C for three days and then placed on 1/2 MS medium. Exposed seedlings were exposed to a 16h photoperiod at 22°C. Experiments involving grafting of seedlings were performed as previously described (17).

Plasmid constructs. HY5 cDNA was amplified and subcloned into pCaMV35S:nos vector (32). The sequences of the HY5, SUC2 and CAB3 promoters were amplified and subcloned into the pCaMV35S:nos vector, as were both the HY5 promoter and the GFP coding sequence to generate the pHY5:GFP expression cassette. The HY5 coding sequence was cloned into the pSK-N-Tagged-myc vector (33) and then subcloned into the pCaMV35S:nos vector. To prepare the pCAB3:2×GUS-TEVrs-HY5-GFP fusion construct, a TEV recognition site (TEV ^rs ) was fused to the 3′-end of the 2×GUS coding sequence by PCR, and the PCR product was introduced into pCAB3:HY5- GFP vector. The TEV protease was amplified and cloned into pCaMV35S:nos vector (32). To construct the pHY5:HY5-GFP transgene, the HY5 coding sequence was cloned into the pHY5:GFP construct. The primer sequences used for PCR amplification are given in Table 2.

Transcript analysis. Total RNA was extracted using TRIzol reagent (Invitrogen, New York, USA) and reverse transcribed using M-MLV reverse transcriptase kit (Promega, Wisconsin, USA). qRT-PCR analysis was performed as previously described (34). Each experiment was represented by three biological replicates with at least three technical replicates per biological replicate. Arabidopsis actin 2 was used as a reference gene. The relevant primers are given in Table 3.

Western blot analysis. Crude protein was prepared by extraction in 50 mM Tris-HCl (pH 7.5), 150 mM KCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 0.1% NP-40, 1X complete protease inhibitor (Roche). Aliquots were electrophoresed on 10% (w/v) SDS-polyacrylamide gels and transferred to Hybond ECL nitrocellulose membranes. The membrane was then operated according to the established protocol (35). Anti-myc antibody (Santa Cruz Biotechnology, Santa Cruz, USA) and anti-GFP antibody (Roche Diagnostics GmbH, Germany) were used to detect myc-HY5 and HY5-GFP fusion proteins, respectively, and uperSignal West Pico chemiluminescent substrate (Thermo Fisher Scientific, Waltham, USA) to visualize the signal.

Measurement of total carbon and nitrogen content. The dehydrated plant tissue was ground and the total carbon and nitrogen content of the powdered material was determined using a high temperature combustion process (36) using an Elementar Vario PYROCube analyzer (Elementar Analysensysteme GmbH, Frankfurt, Germany).

¹⁵ NO3-uptake activity assay. Root ¹⁵ NO3 ^-influx was determined as described elsewhere (3). The roots were dried overnight at 80°C and ^the15N content was measured using the ANCA-MS system (PDZ Europa Ltd).

ChIP-PCR assay. hy5 and pHY5:myc-HY5hy5 seedlings were grown on ½ MS plates for 14 days. A 2 g aliquot of the plant tissue was then fixed by formaldehyde crosslinking. ChIP assays used anti-myc antibodies (Santa Cruz Biotechnology, Santa Cruz, USA) as previously described (37). Enrichment of DNA fragments was determined by qRT-PCR analysis. Three independent biological replicates were performed. The relevant primer sequences are given in Table 4.

EMSA assay. HY5 cDNA was amplified and cloned into pMAL ^™ -c2X vector (New England Biolabs, Ipswich, USA). MBP and MBP-HY5 fusion proteins were purified according to the manufacturer's instructions. The DNA probe was amplified and labeled at its 3-terminus with biotin using a biotin labeling kit (Invitrogen, New York, USA). DNA gel shift assays were performed using the LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Waltham, USA). The primer sequences used are given in Table 5.

result

Although plant shoots and roots follow different developmental trajectories, their biology is coordinated to optimize overall plant performance in fluctuating environments. Coordination of metabolic uptake is crucial: shoots fix atmospheric carbon (C; CO ₂ ), and roots acquire soil ionized nitrogen (N; mainly nitrate (NO3 ⁻ )) (1). Because N is essential, soil NO3 ^- levels often limit productivity in natural and agricultural environments (2, 3). The regulation of N and C uptake rates is known to be closely linked (4, 5), and light is used to regulate these two processes (6). However, the underlying molecular mechanisms regulating long-distance shoot-root communication are unknown.

As an apparent reporter of regulation of shoot-root communication, and because roots are not normally exposed to light, we therefore determined the effect of separate shoot or root irradiation on root growth (Fig. 1A). We found that primary roots of wild-type (WT) Arabidopsis (Col-0 experimental strain) seedlings whose shoots were (only) exposed to light (S(L)/R(D)) were significantly different from those fully exposed to light (S(L)/R(D)). Primary roots of WT seedlings of (L)/R(L) had similar lengths (Fig. 1A-C). In contrast, roots of WT S(D)/R(L) seedlings were more D) The seedlings are short and have similar length to S(D)/R(D) seedlings (Fig. 1B, C). In addition, the lateral root proliferation of S(L)/R(D) seedlings is similar to that of S(L)/ R(L), while S(D)/R(L) seedlings had much less lateral root proliferation, similar to that of S(D)/R(D) (Fig. 7). Therefore, shoot irradiation most likely Promotes root growth through shoot-to-root signaling.

We next screened Arabidopsis mutants specifically disrupted in shoot-irradiation-promoted root growth and found that one of them contained a novel HY5 loss-of-function allele (hy5-526; Fig. 1B; Fig. 8) . Further analysis showed that the hy5 null allele (7; Figure 8) also abolished shoot-irradiation-promoted root growth (Figure 1C). HY5 encodes the photosensitive morphogenetic bZIP transcription factor HY5 (8). HY5 is regulated by the COP1 ubiquitin ligase, which targets HY5 for protein degradation in the dark (9, 10). Thus, we found that loss-of-function copl-4 mutants mimic shoot-irradiation promotion of root growth in dark-grown seedlings (Figure 1C). These results suggest that HY5 regulates shoot-irradiation promotion of root growth.

We next found that WT root NO3 ^- uptake was promoted by irradiation of shoots, but not roots (Fig. 1D). NO3 ^- Uptake by plants via dual low-affinity/high-affinity CHL1/NRT1.1/NPF6.3 and high-affinity NRT2.1 transporters (11-15). Shoot-irradiation-promoted NO3 ^- uptake was substantially reduced in the nrt2.1-2 mutant lacking NRT2.1, but was relatively unaffected in the chl1-5 mutant lacking CHL1/NRT1.1/NPF6.3 ( Figure 1D), showing that shoot-irradiation promotion of root NO3 ^- uptake is significantly NRT2.1-dependent. Furthermore, shoot-irradiation-promoted root NO3 ^- uptake was largely abolished in both hy5 and hy5-526 (Fig. 1D), suggesting that HY5 regulates NRT2.1-dependent shoot-irradiation promotion of root NO3 ^- uptake .

Subsequent experiments used hypocotyl grafted chimeras and found that HY5 scions allowed shoot-irradiated enhancement of root growth and NO3 ^- uptake (relative to hy5-526 scions; Fig. 1E,F), suggesting a HY5-dependent shoot-irradiation enhancement. Shoot-derived signals regulate root NO3 ^- uptake. To determine whether HY5 is (part of) this signal, we next used non-tissue-specific pHY5 (8), photosynthetic tissue-specific pCAB3 (16, 17) or phloem companion cell-specific pSUC2 promoters (18) in hy5 Transgenes encoding HY5-GFP or myc-HY5 fusion proteins were expressed in , and both HY5-GFP and myc-HY5 were found to complement the hy5 phenotype ( FIG. 9 ), and thus retain HY5 activity. We also found that pHY5:HY5-GFP, pCAB3:HY5-GFP and pSUC2:HY5-GFP all restored shoot irradiation regulation of primary root growth of hy5 (Fig. 2A). Because pCAB3-driven expression is photosynthetic tissue-specific, these observations suggest that HY5 transcripts, HY5, or HY5-dependent signals move from shoots to roots.

We next found that HY5-GFP was detectable in the whole root of S(L)/R(D)pHY5:HY5-GFP hy5 seedlings but not in S(D)/R(D) (Fig. 2B) , which suggested that HY5 was relatively stable in dark rooting, and especially root HY5 abundance was regulated by shoot irradiation. We also detected HY5-GFP in the roots of S(L)/R(D)pCAB3:HY5-GFP hy5 seedlings, but not in S(D)/R(D) controls (Fig. 2C), suggesting that It is suggested that HY5 transcripts, or HY5 (or both) move from shoots to roots. We next compared the distribution of myc-HY5 transcripts and myc-HY5 in pCAB3:myc-HY5hy5 seedlings (Fig. 2D, E). Although myc-HY3 transcripts could only be detected in S(L)/R(D) shoots (Fig. 2D), myc-HY5 was detected in S(L)/R(D) shoots and roots (Fig. 2E). This result was confirmed by detection of HY5-GFP in roots of S(L)/R(D)-grown pCAB3:HY5-GFP hy5/hy5 graft chimeras (Fig. 2F; Fig. 10). Further experiments with pHY3:HY5-GFP hy5 seedlings detected HY5-GFP in S(L)/R(D) rootstock roots of grafted plants (Fig. 11), suggesting that HY5 migrates from shoots to root.

We then expressed the fused HY5-GFP, TEVrs (TEV protease recognition site) and double β-glucuronidase (2×GUS) (2×GUS-TEVrs-HY5-GFP) proteins from pCAB3. The protein was detected in shoots but not in roots of S(L)/R(D) plants, probably because its relatively large size prevents shoot-root movement (Fig. 2G; Fig. 12). However, co-expression of 35S:TEVP (expressing TEV protease) enabled cleavage at TEV ^rs (19), which resulted in the detection of HY5-GFP in shoots and roots (Fig. 2G; Fig. 12). These changes in HY5-GFP mobility mirrored parallel effects on shoot-irradiation-promoted primary root growth (Fig. 2H) and NO3 ^- uptake (Fig. 2I). These results suggest that shoot-root movement of HY5 is required for shoot-irradiation-promoted root growth and NO3 ^- uptake. Interestingly, HYH, a transcriptional regulator closely related to HY5 (20), is not shoot-root mobile (Fig. 13). Thus, HY5 (but not HY5 mRNA) is a light-regulated shoot-root movement signal that regulates root growth and NO3 ^- uptake.

The distribution of HY5-GFP to hy5 roots was different from pHY5:HY5-GFP (Fig. 2B), pCAB3:HY5-GFP (Fig. 2C), and pHY5:HY5-GFP hy5/hy5 graft chimera (Fig. 11). pHY5:HY5-GFP resulted in HY5-GFP being detected throughout the root, whereas pCAB3:HY5-GFP and pHY5:HY5-GFP hy5/hy5 seedlings conferred HY5-GFP positions more closely associated with root vasculature. This difference may be due to HY5 activation of root HY5 expression. First, shoot-irradiation induction of root expression of the pHY5:GFP transgene (reporting pHY5 activity) was reduced in hy5 (Fig. 2J), suggesting that shoot-derived HY5 normally activates root pHY5. Second, HY5-GFP was detectable in the roots of shoot-irradiated pCAB3:myc-HY5hy5/pHY5:HY5-GFP hy5 grafts, but not in shoot-irradiated hy5/pHY5:HY5-GFP hy5 grafts could not be detected in the roots of (Fig. 2K). Third, the binding of HY5 to pHY5 was confirmed using EMSA assay and ChIP analysis of roots of pHY5:myc-HY5hy5 seedlings (21; Figure 14). These observations suggest that shoot-root transferred HY5 activates root HY5 through a self-regulatory feedback loop.

We next found that shoot-irradiation promotion of root NRT2.1 transcript levels was largely eliminated in hy5 (Fig. 3A). Further experiments showed that HY5 scion/pHY5:HY5-GFP hy5 rootstock or pCAB3:HY5-GFP hy5 scion/HY5 rootstock allowed a shoot-irradiation-dependent increase in root NR72.1 transcript levels comparable to that observed in HY5 plants To a similar extent, pCAB3:HY5-GFP hy5 scion/hy5 rootstock levels were reduced relative to that observed in HY5 plants (Fig. 3A). These differential effects paralleled effects on shoot-irradiation and NRT2.1-dependent NO3 ^- uptake (Fig. 3B), and involved the requirement for functional HY5 in roots. ChIP and EMSA assays demonstrated the in vivo and in vitro binding of HY5 to the NRT2.1 promoter (Fig. 3C,D), suggesting that HY5 directly regulates NRT2.1 expression. Taken together, these results suggest that shoot-root mobility of HY5 activates root NRT2.1 through a mechanism amplified by self-activation of root HY5, thereby increasing NO3 ^- uptake.

Recent studies have shown that HY5 regulates photosynthetic capacity in part by controlling the expression of genes related to chlorophyll biosynthesis and photosynthesis, such as phytoene synthase (PHYTOENE SYNTHASE) (PSY, Fig. 3E) (22, 23). Immobilized C is mainly transported via the phloem in the form of sucrose to sink tissues (e.g., roots) (24), and the trehalose precursor trehalose-6-phosphate (T6P) is used as the carbohydrate state for photosynthesis alternatives (25). We also found that HY5 affects sucrose metabolism and shoot growth by promoting the expression levels of TPS1, a gene encoding trehalose-6-phosphate synthase (25), and SWEET11 and SWEET12, genes encoding sucrose efflux transporters (24) - Root transport (Fig. 3E). Further ChIP experiments confirmed the binding of HY5 to the TPS1, SWEET11 and SWEET12 promoters (Fig. 3F). Thus, HY5 regulates C fixation and import into phloem cells for shoot-root migration.

Because C-states regulate N-states (4, 5), we next determined whether sugar levels affect root NRT2.1 transcript levels and light-regulated NRT2.1-dependent NO3 ^- uptake. We found that sugar (sucrose or glucose) enhanced NRT2.1 expression and NO3 ^- shoot-irradiation promotion of uptake (effect largely eliminated in hy5) (Fig. 3G,H; Fig. 15). Furthermore, ChIP assays showed that sucrose promoted the in vivo binding of light-activated myc-HY5 to the NRT2.1 promoter, and this effect was abolished in the sugar-insensitive mutant sis4 (Fig. 3I). Since neither HY5 promoter activity nor HY5-GFP abundance was increased by sucrose (Figure 16), it is unlikely that increased HY5 levels contributed to the sucrose-induced increase in the binding affinity of myc-HY5 to the NRT2.1 promoter. Regardless of the mechanism, sucrose induction of NRT2.1 expression and NO3 ^- uptake was dependent on HY5 (Fig. 17). In summary, HY5 protein movement promotes a coordinated homeostatic balance of C and N metabolism.

Because natural fluctuations in incident light fluence affect both C-fixation (26) and HY5 abundance (27), we therefore determined that increasing fluence (5-100 μmol.s ⁻¹ .m ⁻² ) has a significant effect on Effects on root growth and NO3 ^- uptake. We found that increasing energy density promoted WT primary root extension length (Fig. 4A) and NO3 ^- uptake (Fig. 4B), which caused an energy density-dependent increase in biomass accumulation (Fig. 4C). However, these energy density-dependent effects were largely abolished in hy5 (Fig. 4A-C). We next determined whether HY5 continues to coordinate growth, C and N metabolism in more mature plants. First, we found that lack of HY5 reduced the biomass of 21-day-old soil-grown plants, an effect that increased with increasing energy density (Fig. 4D, Fig. 18). Next, we found that the C content of 21-day-old WT and hy5 intact plants was relatively unchanged with increasing energy density (Fig. 4E). In contrast, while the N content of WT intact plants remained relatively unchanged, the N content of hy5 decreased significantly with increasing energy density (Fig. 4F). As a result, absence of HY5 caused an energy density-dependent increase in the C/N content ratio (which remained relatively unchanged in WT; FIG. 4G ). These results suggest that mobile HY5 regulates the coordination of shoot and root growth, C and N acquisition. Notably, HY5 maintains a homeostatic balance of C and N metabolism under altered light energy density.

Although previous studies have shown that phloem-mobilized sucrose acts as a cotyledon-derived signal to control primary root extension during early seedling development in Arabidopsis (28), shoot-root long-distance signaling that regulates lateral root growth and N uptake The molecular mechanism remains unclear (29). Here, we show that HY5 is a shoot-root mobility signal that regulates shoot growth and C uptake linked to photoregulation of root growth and N uptake. This association is achieved by HY5-regulation of C fixation in shoots and by sucrose-enhanced promotion of HY5-dependent N-uptake in roots. As a result, HY5 regulates the homeostatic regulation of intact plant C relative to intact plant N status. HY5 is already known to integrate various phytohormone (eg, abscisic acid) and environmental (eg, low temperature) signaling inputs in the control of plant growth and development (30, 31).

Our finding that HY5 is a mobile signal adds another dimension to this knowledge. Our findings that HY5 mobilization regulates homeostatic coordination of C and N metabolism enhance understanding of how plant C and N nutrient balance is maintained in fluctuating environments and suggest new strategies to improve nutrient use efficiency in crop species.

Identification of HY5 homologues

The percent identity between two protein sequences is obtained by dividing the number of matches by the total positions (including gap positions).

Table 1: HY5 Homologue Identity (Based on Protein Sequence)

At: Arabidopsis thaliana; Bn: Brassica napus; Gb: Gossypium barbadense; Gm: soybean (Glycine max); Hv: barley (Hordeum vulgare); Ta: common wheat (Triticum astivum); Os: rice (Oryza sativa); So: sugarcane (Saccharum officinarum); Zm: corn (Zea mays); Sm: Selaginella moellendo rffii; Pp: Physcomitrella patens.

Expression of PpHY5 in Arabidopsis and expression of OsHY5 in Arabidopsis (Figure 5)

To investigate whether there is evolutionary conservation of HY5 function, we next generated transgenic plants expressing OsHY5-GFP and PpHY5-GFP fusion proteins in hy5 using the promoter of the Arabidopsis HY5 gene. We found that pAtHY5:OsHY5-GFP and pAtHY5:PpHY5-GFP complement the hy5 hypocotyl elongation phenotype (Fig. 5a). We also found that both pAtHY3:OsHY5-GFP and pAtHY5:PpHY5-GFP restored root meristem size of hy5 and shoot-irradiation regulation of NRT2.1 expression (Fig. 5b,c). Subsequent experiments used hypocotyl grafted chimeras and found that the distribution of OsHY5-GFP and PpHY5-GFP could be detected in rootstock roots of grafted plants, suggesting that both PpHY5-GFP and OsHY5-GFP fusion proteins are mobile of (Fig. 5d).

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sequence information

Arabidopsis

SEQ ID NO: 1 AtHY5CDS used in the construct:

atgcaggaacaagcgactagctctttagctgcaagctctttaccatcaagcagcgagaggtcatcaagctctgctccacatttggagatcaaagaaggaattgaaagcgatgaggagatacggcgagtgccggagtttggaggagaagctgtcggaaaagaaacttccggtagagaatctggatcggcgaccggtcaggagcggacacaggcgactgtcggagaaagtcaaaggaagcgagggaggacaccggcggagaaagagaacaagcggctgaagaggttgttgaggaacagagtttcagctcagcaagcaagagagaggaaaaaggcttacttgagcgagttggaaaacagagtgaaagacttggagaacaaaaactctgaacttgaagagcgactctctactcttcagaacgagaaccagatgcttagacatattctgaagaacacaacaggaaacaagagaggaggtggtggtggttctaatgctgatgcaagcctttga

SEQ ID NO: 2AtHY5 mRNA (NM_121164)

cagagatctgacggcggtagccagagtaatctattccttcccaaaatgtctcgcaattagattctttccaagttcttctgtaaatcccaagtcccgctcttttcctctttatccttttcaccagcttcgctactaagacaacaaatctttccctctctctctcgcctgatcgatcttcaaagagtaagaaaaatgcaggaacaagcgactagctctttagctgcaagctctttaccatcaagcagcgagaggtcatcaagctctgctccacatttggagatcaaagaaggaattgaaagcgatgaggagatacggcgagtgccggagtttggaggagaagctgtcggaaaagaaacttccggtagagaatctggatcggcgaccggtcaggagcggacacaggcgactgtcggagaaagtcaaaggaagcgagggaggacaccggcggagaaagagaacaagcggctgaagaggttgttgaggaacagagtttcagctcagcaagcaagagagaggaaaaaggcttacttgagcgagttggaaaacagagtgaaagacttggagaacaaaaactctgaacttgaagagcgactctctactcttcagaacgagaaccagatgcttagacatattctgaagaacacaacaggaaacaagagaggaggtggtggtggttctaatgctgatgcaagcctttgatctccttcttcttcttgtgttatatttttgtggataaaatttacagagaattgtatcaataattatcatgttaaaattatatgggatgtgagagctaatattgcaattgtagaccaagttctcttattgtagtcttagatttctcttaattgaaacataatgttgttttataacaaaaataagctaatttttgttctatgata

SEQ ID No: 3AtHY5 protein

mqeqatsslaasslpsssserssssaphleikegiesdeeirrvpefggeavgketsgresgsatgqertqatvgesqrkrgrtpaekenkrlkrllrnrvsaqqarerkkaylselenrvkdlenknseleerlstlqnenqmlrhilknttgnkrgggggsnadasl

Rice (Oryza sativa)

SEQ ID NO: 4 OsHY5 CDS used in the construct:

atgcaggagcaggcgacgagctcgcggccgtccagctccgagaggtcgtccagctccggcggccaccacatggagatcaaggaaggaatggagagcgacgaggagatagggagagtgccggagctggggctggagccgggcggcgcttcgacgtcggggagggcggccggcggcggcggcggcggggcggagcgcgcgcagtcgtcgacggcgcaggccagcgcgcgccgccgcgggcgcagccccgcggataaggagcacaagcgcctcaaaaggttgctgaggaaccgggtatcagcgcagcaggcaagggagagaaagaaggcatacttgaatgatcttgaggtgaaggtgaaggacttggagaagaagaactcagagttggaagaaagattctccaccctacagaatgagaaccagatgctcagacagatactgaagaatacaactgtgagcagaagagggccaggtagcactgctagtggagagggtcaatag

SEQ ID NO: 5OsHY5 mRNA

atgcaggagcaggcgacgagctcgcggccgtccagctccgagaggtcgtccagctccggcggccaccacatggagatcaaggaaggaatggagagcgacgaggagatagggagagtgccggagctggggctggagccgggcggcgcttcgacgtcggggagggcggccggcggcggcggcggcggggcggagcgcgcgcagtcgtcgacggcgcaggccagcgcgcgccgccgcgggcgcagccccgcggataaggagcacaagcgcctcaaaaggttgctgaggaaccgggtatcagcgcagcaggcaagggagagaaagaaggcatacttgaatgatcttgaggtgaaggtgaaggacttggagaagaagaactcagagttggaagaaagattctccaccctacagaatgagaaccagatgctcagacagatactgaagaatacaactgtgagcagaagagggccaggtagcactgctagtggagagggtcaatag

SEQ ID NO: 6OsHY5 protein

mqeqatssrpsssserssssgghhmeikegmesdeeigrvpelglepggastsgraaggggggaeraqsstaqasarrrgrspadkehkrlkrllrnrvsaqqarerkkaylndlevkvkdlekknseleerfstlqnenqmlrqilknttvsrrgpgstasgegq

Physcomitrella patens

SEQ ID NO: 7 PpHY5 CDS for construct:

atggcagacgcacaaaatggtaagggcttttcgcagttgacttcagttattggaaacatcaatagtgttgcaaatagtagcaggaggggtcccagagaagcggttgatattggccggaactggaaacctgtcaattttggtgagtaccaaggtttgggcgaaatcttgcccatgcaggcctctactgtgggtcccgcttcttctccgccctcttcgaagcagcagacgggcactgatatatcagtatcacctcctttggcgactgcagctgttgacaaactcatgaaagacggcaatgaaagcgactctgatgttaggagggttccagagctatctgcgaagactagtggaggggtttctgggtcgcatacacaagataaaggtctcactggatcttctcatcggaaaagagggggagcttctgctgataaagaacacaagcgtctgaagaggctgcttaggaaccgtgtttcagctcagcaagccagagaacgaaaaaaagcatatctcggtgaattggaagttagatcaaaggagttggagcatcgaaatgcagaattagaagaaagagtgtccaccctacaaagggagaaccagatgttgcgtcaaatcgtcaagaacactgctctgaagaagacttatagtggtggtaacgctgaggatggtgcgcaatga

SEQ ID NO: 8: PpHY5 mRNA

cagttttgttccattgtagtatgtactttgagccagtaatgcgttgtagttaaactgagagtcctgcactattatttagctaggtctgttcatgtttttcccattgcagtagggattgaagagtgttggaaacaacgacagtaaaactaaacgttcatatgggatggcagacgcacaaaatggtaagggcttttcgcagttgacttcagttattggaaacatcaatagtgttgcaaatagtagcaggaggggtcccagagaagcggtttgtgttggccggaactggaaacctgtcaattttggtgagtaccaaggtttgggcgaaatcttgcccatgcaggcctctactgtgggtcccgcgtcttctccgccctcttcgaagcagcagacgggcactgatatatcagtatcacctcctttggcgactgcagctgttgacaaactcatgaaagacggcaatgaaagcgactctgatgttaggagggttccagagctatctgcgaagactagtggaggggtttctgggtcgcatacacaagataaaggtctcactggatcttctcatcggaaaagagggggagcttctgctgataaagaacacaagcgtctgaagaggctgcttaggaaccgtgtttcagctcagcaagccagagaacgaaaaaaagcatatctcggtgaattggaagttagatcaaaggagttggagcatcgaaatgcagaattagaagaaagagtgtccaccctacaaagggagaaccagatgttgcgtcaaatcgtcaagaacactgctctgaagaagacttatagtggtggtaacgctgaggatggtgcgcaatgatggaatattcgagagatgtgtggcctaccgttttttgttattcataatcaaccatttagtatgtaatgcaggaagtttttatttcaagtatgccccgcttcacattaagttcaaaataagtttggtaatcgaaggtcagaattctctcagcgttgtccttca ttccatcaacttccgttgttttatggaatgaccgttggatgctttgttgctgttacaatcgcaagaactatatttccattgaagtcatatttttgaagcaattctcaataactgtatgaaggccatggttcattataaccaagcgattcttgatagcat

SEQ ID NO: 9PpHY5 protein

Madaqngkgfsqltsvigninsvanssrrgpreavcvgrnwkpvnfgeyqglgeilpmqastvgpassppsskqqtgtdisvsppplataavdklmkdgnesdsdvrrvpelsaktsggvsgshtqdkgltgsshrkrggasadkehkrlkrllrnrvsaqqarerkkaylgelevrskelehrnaeleervsggntalkedrkgq

Selaginella moellendorffii

SEQ ID NO: 10SmHY5A mRNA (XM_002970018)

atagtcacgtgaaatatctttctctcttggcggagaagctgaaagcggtccaagagctttcggtgtccggatcgagctagtgcttgggggaggcggtgaattttggttccatcgcaatgcggtgactcgggggtgtgaggagtatctattcagtcatgcgatcggcgtccgcggtgaagaggccggttgcgagtttcaatcccggcagggtggagaatttgtcgcggaagaaaggctcgattggcggtgaagcagcggagaatgccgaggtcgtggatgatttgagcagcctcaaagctgagtgtgatatcgagtctgttggatacgagagccaggcggcggccagcattgcgaatttcggctgcaagtcgtttgcaggggcttggaagcccgtcatcgacggaaaaatccaacctttggagccaccaaacagcgccaccaacagcatcacctggaattggacgagcgagtccaacaggacgaattcgtcatcgaggcatggatcagacaggccctctgtttcgaacatctccggctcatcggatatgaagaaagacgacgaaggcaacgatagcgactcggacatcagacgagtgcccgagctgccagagaagagcagcaaaggtcgctctcagaagcttgtcggtggaagctcgtcgtcgagaaggcgatccggaggctcttccaatgacaaggaaggcaagcgactaaagaggttgctgaggaatcgcgtgtcggcacaacaggcccgagagcgcaaaaaggcttacttggtcgagctggagcaaaaggccaaggatctggaaactagaaacgctgagctggaggagaaaaacgcgacgcttcaaagagaaaactacatgctccgacagattgtcaagaacactaccatccggggcggtggcgattgataatccaaatcttccaagacgaaagaaacaggagtaaacaagatctttggccattttacaacatacattggctatataacaagagaagt tatggtgttacaacaaaaataatttagctatgactaagat

SEQ ID NO: 11SmHY5A protein

Mrsasavkrpvasfnpgrvenlsrkkgsiggeaaenaevvddlsslkaecdiesvgyesqaaasianfgcksfagawkpvidgkiqpleppnsatnsitwnwtsesnrtnsssrhgsdrpsvsnisgssdmkkddegndsdsdirrvpelpeksskgrsqklvggssssrrrsggssndkegkrlkrllrnrvsaqqarerkkaylveleqkakdletrnaeleeknatlqrenymlrqivknttirgggd

SEQ ID NO: 12SmHY5B mRNA (XM_002973152)

Ggagttttcttggtgctggtgtttctcgcttccacagctcggcattgccccaatcgcgacgagaggacgcatccatagcaatcgacgcgatgattctgaggaacgattgctagaagttcctctctctctctctctgcctctccagccagctccagatttgttccaggatgcaagcagcggcatcagcgtcgccgacaatgcacaagcaattcagcgatttgatggctcttcccaacgccgagatgaaagtggacgaggattgggcaggaaatgagagtgactcggaagtaagaaaggtccccgacttacctggaggtaaaatcgtgactgcgttgccagagcaagacacggcagcatccaattctcgcaagaggggtgctgttcctgctgacaaagaacacaagcgattgaaaagacttttacgcaatcgagtctcggcacaacaagccagggagagaaaaaaggcttacgttgttgagctcgaagcaaaagcccgggatttggagctcaggaatgcggagctagaggaacgggtaaacacgttacaaaaggaaacattcatgctgcgacagattttgaagaacataaagaacaatggctccactgctggactagaacaagcacagtaaaaaaccaaaaactttacttgagggggccaacgtagcaacgaaagtcggccaccactgtaagattgtttctccacaatcagttttggtaaccatcttctcggc

SEQ ID NO: 13SmHY5B protein

mqaaasasptmhkqfsdlmalpnaemkvdedwagnesdsevrkvpdlpggkivtalpeqdtaasnsrkrgavpadkehkrlkrllrnrvsaqqarerkkayvveleakardlelrnaeleervntlqketfmlrqilkniknngstagleqaq

Barley (Hordeum vulgare)

SEQ ID NO: 14HvHY5 mRNA (AK365526)

Ggttttcctcgggattgggagggacgagagaggcggggagagaatgcaggagcagggggagagctcgcggccttcgagcagcgagaggtcgtccagctccggcaaccacatggagcacaaggaagggatggagagcgacgacgaaatagggacggtgccggagctaggcctggggccaagcggcgcgtccacgtccggcaggagggaagccgacgggccggagcgtgcccagtcctccaacgcgcaaggcagcgcgcgccgccgcggacgcaccccggctgacaaggagcacaagcgcctcaagaggttgctgaggaaccgggtatcagcccagcaggcaagggagaggaagaaagcttatttgggcgatctggaggtgaaggtgaaggacctagagaagaagaattcggagctggaagagaggcattccaccctacagaatgagaaccagatgctccgacagatcctgaagaacaccactgtgagcagaagagggccaagtgagggtcaatagcacagaagttgtaagggtcgattcgcagaattttcacagcagaatcaaagaagccctaggatcgaatatagctgcgttgattgatcccaaaatacaccatgtctcgaacttaaaatggttggaagctttctgaccaatggataacctcaaaaactggggtcaaaaacctgtgtagatcttcagagatgtccccatcatactctatgaagttcagcactacgtgttgcacttcagtaataatttcagaaaattagttttgggtggtttaacatatgatctgtactatcatttttatgtatctacaagtacaatccaaacattttattgttggataatttactttctactattggaaaatgcgc

SEQ ID NO: 15HvHY5 protein (AK365526)

mqeqgessrpsssserssssgnhmehkegmesddeigtvpelglgpsgastsgrreadgperaqssnaqgsarrrgrtpadkehkrlkrllrnrvsaqqarerkkaylgdlevkvkdlekknseleerhstlqnenqmlrqilknttvsrrgpsegq

Common Wheat (Triticum astivum)

SEQ ID NO: 16TaHY5 mRNA

Gagtaagtagcagctgggaggaggagccgaggaagaggagcagaagataggaggagaggagcagcggtagcctcgtcttcctcgggattgggagggacgagagaggcggggggagaatgcaggagcagggggagagctcgcggccttcgagcagcgacaggtcgtccagctccggcaaccacatggggcacaaggaagggatggagagcgacgacgagatagggacggtgccggagctgggcctggggccaagcggcgcgtccacgtccggcaggagggaagccgacgggccggagcgtgcccagtcctccaccgcgcaaggcagcgcgcgccgccgcggacgcaccccggccgacaaggagcacaagcgcctcaagaggttgctgaggaaccgggtatcagcccagcaggcaagggagaggaagaaagcttatttgggcgatctggaggtgaaggtgaaggacctagagaagaagaattcggagctggaagagaggcattccaccctacagaatgagaaccagatgctccgacagatcctgaagaacaccactgtgagcagaagagggccaagtgagggtcaatagcacagaggttgtcagggttgattcacagaattttcacagcagacagttcaattccagtgaatcaaagaggccctaggatcgaatatagccgcgttgatcgatcccaaaacacaccatgtctggaacttaaactggttggaagctttctggccaaaggataacctcaaaaaccgggggcctaaacctgtgtagatcttcacagatgtccccatcatactctatgaagttcagcaccacgtgctgtgcttcgttaataatttcacaaaattagttttgggtgatttaacatatgatctgtatcattttttatgtatctacaagcacaatccaaacattttattgttgggcaatttactttgtactattggaaaatgcgcacgtccggtgtcggcaatgcagccctgtcatgttgg ctcttgcagaccagactatttgttttgttgaacttgcttcaataaagtggccgcatgcatctttccgatgcagaggccggggaagtcctccttttcaaaaaaaaaaaaaaaacga

SEQ ID NO: 17TaHY5 protein

mqeqgessrpsssdrssssgnhmghkegmesddeigtvpelglgpsgastsgrreadgperaqsstaqgsarrrgrtpadkehkrlkrllrnrvsaqqarerkkaylgdlevkvkdlekknseleerhstlqnenqmlrqilknttvsrrgpsegq

Corn (Zea mays)

SEQ ID NO: 18ZmHY5/ZmbZIP61 mRNA (KJ726945)

Atgcaggagcaggcggcgagctcgcggccttccagcagcgagaggtcgtccagctccgggcaccacgtggacatggaggtcaaggaagggatggagagcgacgatgagataaggagagtgccggagctgggcctggagttgccgggagcctccacgtcgggcagggaggctggccctggcgctgcgggcgcagaccgcgcccttgcccagtcgtccacggcgcaggccagcgcgcgccgccgcgtccgcagccacgccgacaaggagcacaagcgcctcaaaaggttactgaggaacagggtgtcagctcaacaggctagagagaggaagaaggcttatttaactgatctggaggtgaaggtgagagatctggagaagaagaactcggagatggaagagaggctctccaccctccagaacgagaaccagatgctccgacagatactgaagaacaccgctgtaaacagaagaggttcaggaagcactgctagtggagagggccacggccaa

SEQ ID NO: 19ZmHY5/ZmbZIP61 protein

mqeqaassrpsssserssssghhvdmevkegmesddeirrvpelglelpgastsgreagpgaagadralaqsstaqasarrrvrshadkehkrlkrllrnrvsaqqarerkkayltdlevkvrdlekknsemeerlstlqnenqmlrqilkntavnrrgsgstasgeghgq

Sugarcane (Saccharum officinarum L.)

SEQ ID NO: 20 SoHY5 mRNA (CA121289)

Agacaggaaggatcgcaggggaggaggagatagggaaggagaagcggagtgcgcgcgggcgactctgcagggcctcagtcggaggcggaggtggagagcgagccagaatgcaggagcaggcgacgagctcgcggccttccagcagcgagaggtcgtccagctccgcgcaccacatggacatggaggtcaaggaagggatggagagcgacgaggagataaggagagtgccggagctgggcctggagctgccgggcgcttccacgtcgggcagggaggctggcccgggcgccgccggcgcagaccgcgccttggcccagtcgtccacggcgcaggccagcgagcgccgccgcgtccgcagccccgccgacaaggagcacaagcgcctcaaaagattactgaggaaccgggtgtcagctcaacaggcaagagagaggaagaaggcttatttgactgatctggaggtgaaggtgaaacaccctgaagaagaagaacttcgaggttcgaagaagaggctctctacccttaaaaacgaagaaccagatgctccgggcagatacctgaagaatacccactgtaagcagaaagaggtttacggaagcactggttagtggaaaaaggccaattagttcaaaatgacaggaaaaatggtaatggcctatgcttaaatatatgtttatgggga

SEQ ID NO: 21 SoHY5 protein

mqeqatssrpssssersssssahhmdmevkegmesdeeirrvpelglelpgastsgreagpgaagadralaqsstaqaserrrvrspadkehkrlkrllrnrvsaqqarerkkayltdlevkvkhpeeeelrgskkrlstlkneepdapgrylknthckqkevygstg

Brassica napus

SEQ ID NO: 22BnHY5 mRNA (EV071015)

gaaagtcccgctcttttccatctctatcttcatcaccagcttctgtaaatcccaatccatcttcaaaggagattcaaagagtaaggaaaaaaaaatgcaggagcaaacgactagctctttacctgcaagctctctaccatcaagcagcgagagatcctcaagctctgctcctcatttggagatcaaagaaggaattgaaagcgatgaagagatacggcgagttccggagtttggaggagaagctaccggaaaggaaatctctggatcggcgaccggtcaggaccagacacaagcaacggtcggaggagagggtcaaaggaagagagggaggactccggctgagaaagagaccaagcggcttaagaggttgttgaggaacagagtttcagcacagcaagcaagagagaggaagaaagcttacttgggtgagttggaaaacagagtgaaagacttggagaacagaaactctgaacttgaagagagactctctaccttgcagaacgagaaccagatgcttagacagattctgaagaacacaacaggaaacaagaggggaagcggtggttctaacgctgatgcaagcctatgatctccttgttcttgtattattatttacctggataaactttacaaggaattgtattaaataaatattttt

SEQ ID NO: 23BnHY5 protein

mqeqttsslpasslpsssserssssaphleikegiesdeeirrvpefggeatgkeisgsatgqdqtqatvggegqrkrgrtpaeketkrlkrllrnrvsaqqarerkkaylgelenrvkdlenrnseleerlstlqnenqmlrqilknttgnkrgsggsnadasl

Sea Island cotton (Gossypium barbadense)

SEQ ID NO: 24GbHY5 mRNA (JK803801)

Ccaaaaaattaattttattttgttttttctataacgaagaaatgcaagaacaaggaacgagttcaatagcagctagttccttaccttcaagcagtgaaagatcttcaagctctgctcttcaagttgaagtcaaggaaggcatggaaagtgatgaagagatccggagagtgcctgagataggaggtgaagcatcagcagctccggccgccggtcgtgaacccggttcactgacccgactggaccggcctcaaccatcgggtgaaggcggtcagagaaagagagggagaagcccaacggataaagaaaacaagcgcttaaagaggttgttgaggaacagagtatcagcgcaacaagcaagggaaaggaaaaaggcgtacttgaatgaaccggaaaccagagttagagacttggagaagaagaactctgaactagaagagaggctatccacgttgcacaatgagaatcagatgcttcgacaaatagtcaagaacacaactgctagcaggagaggtggaaatggcagttcaaatgcagctgatggaaccctttaaaggaaatgcatgggtataaaaaattataaagggattttttttaaaaaaatattaattttatgctttagggaaaaaaaaaccgctattagcaatgcaatgaagtagcaaaacatgacaattggaactttggttctctctaatcttaatcataaaagtaaattatc

SEQ ID NO: 25GbHY5 protein

mqeqgtssiaasslpsssserssssalqvevkegmesdeeirrvpeiggeasaapaagrepgsltrldrpqpsgeggqrkrgrsptdkenkrlkrllrnrvsaqqarerkkaylnepetrvrdlekknseleerlstlhnenqmlrqivknttasrrggngssnaadgtl

soybeans

SEQ ID NO: 26GmHY5L mRNA (BT093548)

gggaagaaagagagagagagagagagagagaggtgtgaagttggtgaaggtttttgagaagaaagatggaacgaagtggcggaatggtaactgggtcgcatgaaaggaacgaacttgttagagttagacacggctctgatagtaggtctaaacccttgaagaatttgaatggtcagagttgtcaaatatgtggtgataccattggattaacggctactggtgatgtctttgtcgcttgtcatgagtgtggcttcccactttgtcattcttgttacgagtatgaactgaaacatatgagccagtcttgtccccagtgcaagactgcattcacaagtcaccaagagggtgctgaagtggagggagatgatgatgatgaagacgatgctgatgatctagataatgagatcaactatggccaaggaaacagttccaaggcggggatgctatgggaagaagatgctgacctctcttcatcttctggacatgatttctcaaataccaaacccccatctagcaaacgggcaaccgatgtctggtgagtttccatgtgctacttctgatgctcaatctatgcaaactacatctataggtcaatccgaaaaggttcactcactttcatatgctgatccaaagcaaccaggtcctgagagtgatgaagagataagaagagtgccagagattggaggtgaaagtgccggaacttcggcctctcagccagatgccggttcaaatgctggtacagagcgtgttcaggggacaggggagggtcagaagaagagagggagaagcccagctgataaagaaagtaaacggctaaagaggctactgaggaaccgagtttcagctcagcaagcaagggagaggaagaaggcatacttgattgatttggaaacaagagtcaaagacttagagaagaagaactcagagctcaaagaaagactttccactttgcagaatgagaaccaaatgcttagacaaatattgaagaacacaacagcaa gcaggagagggagcaataatggtaccaataatgctgagtgaacataatgtcaaaagatggcagagaaaacttatagatggaatagatttagaaagagagaatacattagccagaaagagaaaaaaaaaattggacattagttgatgattctttctaggtgtgcgtttggaatacaatgaagtaaaggatgaaccttaagacatgctttatcctaaaatagtgtgatctgatattccattgttaatgagtaatgtaattatcatacaaacaatttgtagtctcattttaattaataattattaaactacttgattaaaaaaaaaaaaaaaaa

SEQ ID NO: 27GmHY5L protein

mrstmaketvprrgcygkkmltslhlldmisqipnphlangqpmsgefpcatsdaqsmqttsigqsekvhslsyadpkqpgpesdeeirrvpeiggesagtsasqpdagsnagtervqgtgegqkkrgrspadkeskrlkrllrnrvsaqqarerkkaylidletrvkdlekknselkerlstlqnentmlqnengq

SEQ ID NO: 28GmbZIP36/STF1 mRNA (NM_001250343.1)

actgaagtaagaaagagagagagagagagaaagagaagtgtgtagttggtgaagtttttgagaagaatatggaacgaagtggcggaatggtaacggggtcgcatgaaaggaacgaacttgttagagttagacacggttctgacagtgggatttgtcaaatatgtggtgacaccattggattaacggctactggtgacctctttgttgcttgtcatgagtgtggcttcccactttgtcattcttgttacgagtatgagctgaaaaatgtgagccaatcttgtccccagtgcaagactacattcacaagtcgccaagagggtgctgaagtggagggagatgatgatgacgaagacgatgctgatgatctagataatgggatcaactatggccaaggaaacaattccaagtcggggatgctgtgggaagaagatgctgacctctcttcatcttctggacatgattctcatataccaaacccccatctagtaaacgggcaaccgatgtctggtgagtttccatgtgctacttctgatgctcaatctatgcaaactacatcagatcctatgggtcaatccgaaaaggttcactcacttccatatgctgatccaaagcaaccaggtcctgagagtgatgaagagataagaagagtgccggagattggaggtgaaagcgctggaacttcagcctctcggccagatgccggttcaaatgctggtacagaacgtgctcaggggacaggggacagccagaagaagagagggagaagcccagctgataaagaaagcaagcggctaaagaggctactgaggaatagagtttcggctcagcaagcaagggagaggaagaaggcatatttgattgatttggaaacaagagtcaaagacttagagaagaagaactcagagctcaaagaaagactttccactttgcagaatgaaaaccaaatgcttagacaaatattgaagaacacaacagcaagcaggagagggagcaatagtgg taccaataatgctgagtaaacttatagatggagtagatatagagagagagaaagagaaaaaaattaaacattagttgatgattctttctaggtgtgcgtttggaatacaatgaagtaaaggatgaaccttaagacatgctttgtcctaaaatagtgtgatctgatgtaccattgttgatgagtaatgtaattatcatacacagttttttacagtctcattttaattaataattatcaaactacttgattacttatggttaa

SEQ ID NO: 29GmbZIP36/STF1 protein

Mersggmvtgshernelvrvrhgsdsgskplknlngqicqicgdtigltatgdlfvachecgfplchscyeyelknvsqscpqckttftsrqegaevegddddeddaddldnginygqgnnsksgmlweedadlssssghdshipnphlvngqpmsgefpcatsdaqsmqttsdpmgqsekvhslpyadpkqpgpesdeeirrvpeiggesagtsasrpdagsnagteraqgtgdsqkkrgrspadkeskrlkrllrnrvsaqqarerkkaylidletrvkdlekknselkerlstlqnenqmlrqilknttasrrgsnsgtnnav

SEQ ID NO: 35: HY5 promoter (2.2.kb)

cttcgtcgtcaggattatccgtcaacagagttctgtttcggaatcggaacgctagtccttgtggtctatctcttccaattctcaatccgtctcggtctgtacttgtttttgctcgtggcaagaatcgaaaaggattcgtgtcttcgtcttcttcgtctccgaagaaaaacaaaaaggtaagaaaatcagaatgagaatgatttatgttcgcaattgctcttacctgctgctttcgccgaatttgactagaatttggatacactaactaagtgagctattatcggttagaattggtgaatcctcgaattagactttggctctttttgattttgtatcagcctttgatttcatggtttgtggcagaaaagtttggatggagctgataatggaggaggtgaggaggaggaggatccatttgaggcattgtttaatcttttagaagaagatttgaagaatgataactcggatgatgaggagataagtgaggaagagttagaggcattagccgatgagttagctcgagcattgggtgttggtgacgatgttgatgacatcgacttgtttggttcagtcactggtgatgttgatgttgatgttgataatgatgatgatgataatgatgatgatgataatgatgatgatgatgatgacagcgaagaagatgaaagaccgactaagctgaagaattggcagcttaaaagactggcttatgcattgaaagctgggcgtcgtaaaactagtgtaagttttagttcagtgttgaagtgatgttgaatacattgtgtaaagcatagtgcttgagttagtgtctgctttggagattttgcttacgaattgtttaagagttgacaaagaacaagtagttctctggttttcaaatgcataattgataacggatggttttgttgtgatctgtagattaaaaatctggcagctgaggtttgtcttgacagggcttatgttcttgaattgcttcgtgacccgcctccaaagctgctaa tgctaagtgctacactaccagatgaaaaaccgccagtagcagcacctgagaactcctcacctgatccaagtcctgtggaatcattatcagctgaagatgttgtggtcgaacctaaggaaaaagtaaaagatgaagcagttcatgtgatgcaacagagatggtctgctcagaaaagagtgaagaaagctcacattgaaacgctagagaaagtttacagaagatcaaaacgacccactgtaaggattctccttttacatttgaatcaatttctatgttacttgaatgctctatctcacatatgatcatgtttgatgatgctgtgaatagaatgctgtggttagcagcattgttcaagtgaccaatcttccaaggaagcgagttttgaagtggttcgaagataaaagagcagaagacggagttccagataagcgagctccatatcaagctccggtttgatctaatgttaacgttgagatggcaatgatttgtatacttgattctcagaaactcatcaacattgtcgtcaaggacaagtttttttggtgatacgaggagtgtttatagtagtagattctgtccaatggtgtggctggatatgttggactatgaaattttaggatatcttgtattcagtttttagttatttccttgctgagattgtgtcttgtagaaaaccgtttcaactttgtttggtttatggcggctataaagtttaattttaatgcatgacaaaaacaaatcaccaaaaataaaataaattactttcacgacacttttgaaagcactgccctaggcgtgggccatgtgacagaatgaaagaactcagaccaaacttttctgtccaaggacaggaatggggcccacccaattagctcccctatccattattcaccgtaagatgctaaccagatctaacggctaaaatccacccacgttccaatctcaattgcctttggatccttgtatttcctcaaggctcacctttctccacgattcactc tcgatatccgttcgattcttcagagatctgacggcggtagccagagtaatctattccttcccaaaatgtctcgcaattagattctttccaagttcttctgtaaatcccaagtcccgctcttttcctctttatccttttcaccagcttcgctactaagacaacaaatctttccctctctctctcgcctgatcgattagtcaa

Table 2: Primer sequences used to generate DNA constructs

Table 3: Primer sequences used for qRT-PCR analysis

Primer name Primer sequence (5' to 3') SEQ ID NO: qactin 2-F CTGGATCGGTGGTTCCATTC 57 qactin 2-R CCTGGACCTGCCTCATCATAC 58 qHY5-F GAACGAGAACCAGATGCTTAGAC 59 QHY5-R TGCAATATTAGCTCTCCACATCCC 60 OCS-R CATAGGCGTCTCGCATATCTC 61 qHY5-GFP-F CAGAACGAGAACCAGATGCTTAG 62 qHY5-GFP-R CAGATGAACTTCAGGGTCAGC 63 qNRT1.1-F TCTAAGACCGCTTCAACGGATCG 64 qNRT1.1-R ACTGTTGGACCATGAGCGTGTG 65 qNRT2.1-F AACAAGGGCTAACGTGGATG 66 qNRT2.1-R CTGCTTCTCCTGCTCATTCC 67 qTPS1-F GGTCATTTCTTGGGGAAGGA 68 qTPS1-R TCTCCTGATGATGACTTGGC 69 qSWEET11-F GCGAACAAGTGTACCTGCGG 70 qSWEET11-R GGGTACACGTGGTGGTTGGT 71 qSWEET12-F TCGTCCGATCGGTGAACACA 72 qSWEET12-R ACTAGTACACGTGGACAATGGTGA 73

Table 4: Primer sequences for ChIP-PCR

Table 5: DNA probes assayed with dry EMSA

Claims (17)

1. for increasing the method for nitrogen metabolism in plant, methods described, which is included in plant, to be introduced and expresses comprising HY5 nucleic acid sequences The nucleic acid construct of row.

2. according to the method described in claim 1, wherein the nucleic acid construct includes coding SEQ ID NO：3 are limited The SEQ ID NO of AtHY5 albumen or its functional variety：1 or 2 or coding SEQ ID NO：The nucleotide sequence of 3 homologue.

3. method according to claim 2, wherein the homologue is selected from SEQ ID NOs：4 to 29.

4. according in the method described in previous claims, wherein the plant is selected from corn, paddy rice, wheat, colea/plus taken Big rape, sorghum, soybean, sunflower, clover, potato, tomato, tobacco, grape, barley, pea, beans, broad bean, lettuce, cotton Flower, sugarcane, sugar beet, broccoli or other brassica vegetables or willow.

5. according in the method described in previous claims, wherein the nucleic acid construct includes regulatory sequence.

6. method according to claim 5, wherein the regulatory sequence is selected from constitutive promoter or tissue specificity is opened Mover.

7. method according to claim 6, wherein the tissue-specific promoter is chlorenchyma specificity promoter.

8. nucleic acid construct, it includes HY5 nucleotide sequences and tissue-specific promoter.

9. nucleic acid construct according to claim 8, wherein the tissue-specific promoter is chlorenchyma specificity Promoter.

10. nucleic acid construct according to claim 8 or claim 9, wherein the nucleic acid construct includes coding SEQ ID NO：3 The SEQ ID NO of the AtHY5 albumen limited：1 or 2 or coding SEQ ID NO：The nucleotide sequence of 3 homologue.

11. nucleic acid construct according to claim 10, wherein the homologue is selected from SEQ ID NOs：4 to 29.

12. recombinant vector, it includes the nucleic acid construct according to any one of claim 8 to 11.

13. host cell, it is comprising the nucleic acid construct according to any one of claim 8 to 11 or according to claim Recombinant vector described in 12.

14. the method for producing the plant absorbed with increased nitrogen, it includes introducing and expressing into plant comprising HY5 cores Acid sequence and the nucleic acid construct of tissue-specific promoter.

15. for increasing the method for the presence of HY5 albumen in plant roots, it, which is included in plant to introduce and express, includes and green The nucleic acid construct for the HY5 nucleotide sequences that tissue-specific promoter is operatively connected.

16. the method for adjusting the balance in plant between C and N metabolism, it, which is included in plant, introduces and expresses comprising HY5 The nucleic acid construct of nucleotide sequence.

17. for increasing the method for the presence of HY5 albumen in plant roots, it, which is included in the chlorenchyma of plant, introduces and expresses Include the nucleic acid construct of HY5 nucleotide sequences.