WO2025016551A1

WO2025016551A1 - Stay green cucumber plant

Info

Publication number: WO2025016551A1
Application number: PCT/EP2023/085399
Authority: WO
Inventors: Mattheus Nicolaas GROOT; Miquel Angel CABALLERO PEREZ; Bart KAIJ; Karoline Adriana Christine BOOGERT
Original assignee: Enza Zaden Beheer BV
Current assignee: Enza Zaden Beheer BV
Priority date: 2023-07-14
Filing date: 2023-12-12
Publication date: 2025-01-23
Anticipated expiration: 2026-01-14
Also published as: WO2025016901A1

Abstract

The present invention relates to a Cucumber plant (Cucumis sativus) which provides fruits having an improved shelf life, wherein said plant comprises a mutated stay green gene providing the improved fruit shelf life, wherein the expression of said stay green gene is reduced as compared to the expression of said stay green gene in a cucumber plant not providing fruits having an improved shelf life or the enzymatic activity of said protein is reduced as compared to the enzymatic activity of said protein in a cucumber plant not providing fruits having an improved shelf life.

Description

STAY GREEN CUCUMBER PLANT

Description

The present invention relates to a cucumber plant (Cucumis sativus) which provides fruits having an improved shelf life. Further, the present invention relates to seeds, plant tissue, fruits or plants parts of a cucumber plant (Cucumis sativus). A further aspect of the present invention relates to a method for providing a cucumber plant (Cucumis sativus which provides fruits with an improved shelf life. According to yet another aspect the present invention relates to nucleic acids and amino acids related to the present improved shelf life trait.

Cucumber plants have already been cultivated for at least 3.000 years and several different cultivars have emerged, which are grown and market on the global market. The cucumber fruits are mainly eaten in the unripe green form since the ripe yellow form normally becomes bitter and sour. Accordingly, cucumber fruits are commonly harvested while still green and are generally used for both the pickling industry and the fresh market, which latter usage has the greatest added value for the farmers. Due to its relatively short shelf life, storage and shipping of fresh cucumber fruits is, however, difficult, and expensive. Cooling, which is generally used to extend the shelf life of fresh products, cannot be applied for cucumber fruits as these fruits are not suitable for storage at low temperatures due to chill injury.

It is known to extend the shelf life of cucumber fruits by for example wrapping them in sealing foil or storing them under controlled conditions (>10° C). However, additional processing steps or specific storage measures thus have to be taken. In addition, in this way the shelf life is extended only in the specific cucumbers that have been wrapped in foil, or have been stored under said specific conditions. Further, although the shelf life is extended by wrapping cucumbers in foil, these wrapped cucumbers turn yellow within a time period of about two weeks.

Furthermore, under stress conditions (i.e., drought, disease), Cucumber plant senescence start very early, and leaves become yellow which negatively affects plants health and crop yield. Therefore, it would be of importance to find a solution to inhibit this natural process given the advantage in the field in terms of plant continuation, crop yield and harvest period extension. Given the above, there is a need in the art for Cucumber plants to provide for fruits having an extended shelf life and wherein the process of yellowing of the leaves of the plants is inhibited or reduced. Furthermore, there is a need to provide for cucumber plants that have increased shelf life in relation to increased disease tolerance and their maintained or increased agronomical performance.

Considering the above, it is an object of the invention, amongst other objects, to provide Cucumber plants providing fruits having an extend shelf life and wherein the plant and fruits are more resistant to yellowing, while maintaining their agronomical properties. It is an object of the present invention, amongst other objects, to address the above need in the art. The object of present invention, amongst other objects, is met by the present invention as outlined in the appended claims.

Specifically, this object, amongst other objects, is met by providing a cucumber plant (Cucumis sativus) which provides fruits having an improved shelf life, wherein said plant comprises a genomic sequence encoding a stay green (SGR) gene providing the improved fruit shelf life, wherein said stay green gene encodes a SGR protein, wherein said plant comprises a G1410T mutation in the genomic sequence of SEQ ID No. 1 or in the genomic sequence having at least 98% sequence identity, preferably at least 99% sequence identity with SEQ ID No. 1, resulting in a truncated or non-functional SGR protein. The expression of said SGR gene is reduced as compared to the expression of said SGR gene in a cucumber plant not providing fruits having an improved shelf life or the enzymatic activity of said protein is reduced as compared to the enzymatic activity of said protein in a cucumber plant not providing fruits having an improved shelflife. In the research that led to the present invention, it was surprisingly found that a reduced expression of the present gene or a reduced enzymatic activity of the present protein provided fruits having an extended shelf life for a time period up to five weeks when stored under standard storing conditions for cucumbers.

Improved shelf life, as used in the present context, is meant an improved storability of harvested unripe green cucumbers, which cucumbers stay green for longer periods of time than comparable standard harvested unripe green cucumbers. Preferably, the present cucumbers stay green for a time period up to 4 or 5 weeks. Plants of the invention can be distinguished from prior art plants by measuring the time period under standard storage conditions that the cucumber fruits stay green. Conventional cucumber fruits turn yellow within 1 or 2 weeks; whereas cucumber fruits according to the present invention turn yellow after a time period of at least 4 or at least 5 weeks. Further, expression levels in the present plants and the reference plants can be determined using any suitable and generally known Molecular Biology technique such as a quantitative Polymerase Chain Reaction (PCR) or mRNA hybridization.

As disclosed herein, the percentage (%) sequence identity is known to the person skilled in the art. Preferably it is to be understood in relation to a query sequence having at least 98% of the sequence length of the gene or protein sequence as claimed herein, preferably at least 99%, more preferably 100%, most preferably 100% sequence length. Alternatively, or additionally, the gene or protein sequence alignment is performed from start(codon) to stop(codon) of the coding sequence or protein sequence. For example, Geneious Prime (Clustal Omega algorithm) can be used to align sequences and calculate the % sequence identity.

According to the present invention, an enzymatic activity is reduced in comparison with the activity of the present protein in a cucumber plant which not provides cucumber fruits having an extended shelf life. The term ‘not providing cucumber plants having an extended shelf life’ indicates a shelf life, determined in an appropriate storability test and appropriate reference plant, such as a parent plant, being less than the shelf life than observed for cucumber fruits of the present invention. Suitable reference plants can, besides parent plants, also be plants generally designated as providing marketable or agronomical cucumber fruits.

The present protein has a chlorophyll decarboxylase function wherein, amongst other reactions, a carboxyl group is removed, and carbon dioxide is released. Accordingly, the present reduced activity can be determined using an assay measuring compounds being either the starting compounds or the resulting compounds of the enzymatic reaction. As a suitable alternative, protein levels, being inherently indicative of a reduced activity, of the present proteins can be determined by, for example, ELISA or protein hybridization both being techniques commonly known to the skilled person.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein the genomic sequence encoding said SGR gene is mutated resulting in a pre-mature stop codon. According to another preferred embodiment of this first aspect of the present invention, the present improved shelf life is provided by one or more mutations in the genomic sequence encoding the SGR gene resulting in a truncated or non-functional SGR protein. Truncated proteins can be readily determined by analyzing gene transcripts at the mRNA or cDNA level and nonfunctional proteins can be determined in enzyme assays or using conformation-dependent antibodies. Mutations which can be assayed at the transcript level are, for example, amino acid substitutions, frame-shifts or pre-mature stop codons.

According to another preferred embodiment, the present invention relates to the cucumber plant, wherein expression of the SGR gene represented by SEQ ID No.2 is reduced as compared to the expression of said SGR gene in a cucumber plant not providing fruits having an improved shelf life. SEQ ID No.2 represents the coding sequence of the non-mutated SGR gene encoding a functional SGR protein represented by SEQ ID No. 3.

According to yet another preferred embodiment, the present invention relates to the cucumber plant, wherein the enzymatic activity of the SGR protein represented by SEQ ID No.3 is reduced as compared to the enzymatic activity of said SGR protein in a cucumber plant not providing fruits having an improved shelf life.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein said plant comprises the genomic sequence of SEQ ID No. 4.

According to another preferred embodiment, the present invention relates to the cucumber plant, wherein the truncated or non-functional SGR protein comprises the sequence of SEQ ID No. 6. This truncated protein comprising the present amino acid sequence as shown in SEQ ID NO. 6 is highly correlated with the improved shelf life trait of the present invention, since an improved shelf life of at least 4 weeks is observed for fruits from a cucumber plant comprising a mutated SGR gene encoding the present protein.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein said genomic sequence stay encoding the SGR gene is present in heterozygous or homozygous form, preferably homozygous form in said plant. The mutated SGR genomic sequence encoding the mutated SGR gene is present in homozygous form, thereby providing an improved shelf life of the cucumber fruits. Alternatively, the present stay green gene may be present in heterozygous form, thereby providing an intermediary phenotype.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein the G1410T mutation is a natural or a non-natural mutation, preferably a natural mutation as found in or obtainable from deposit NCIMB 44206.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein the G1410T mutation is provided by gene editing techniques, preferably by mutagenesis or CRISPR/Cas. The present cucumber plants can be obtained by mutagenesis of cucumber plants. For example, mutations, either at the expression level or the protein level, can be introduced in these plants by using mutagenic chemicals such as ethyl methane sulfonate (EMS) or by irradiation of plant material with gamma rays or fast neutrons. The resulting mutations can be directed or random. In the latter case, mutagenized plants carrying mutations in the present gene can be readily identified by using the TILLING (Targeting Induced Local Lesions IN Genomes) method (McCallum et al. (2000) Targeted screening for induced mutations. Nat. Biotechnol. 18, 455-457, and Henikoff et al. (2004) TILLING. Traditional mutagenesis meets functional genomics. Plant Physiol. 135, 630-636). Briefly, this method is based on the PCR amplification of a gene of interest from genomic DNA of a large collection of mutagenized plants in the M2 generation. By DNA sequencing or by scanning for point mutations using a single-strand specific nuclease, such as the CEL-I nuclease (Till et al. (2004) Mismatch cleavage by single-strand specific nucleases. Nucleic Acids Res. 32, 2632-2641) individual plants having a mutation in the present genes are identified.

According to a preferred embodiment, the present invention relates to the cucumber plant, wherein the mutated genomic sequence encoding the SGR gene is obtainable from deposit number NCIMB 44206. Seeds comprising the mutated SGR gene are deposited at NCIMB Ltd. Wellheads Place, Aberdeen, Dyce, AB21 7GB Scotland on 9 August 2023 under the number NCIMB 44206.

According to a second aspect, the present invention relates to Seeds, plant tissue, plant cell, fruits or plants parts of a cucumber plant (Cucumis sativus) as disclosed herein comprising a stay green gene providing an improved fruit shelf life. Preferably the stay green gene providing an improved fruit shelf life is comprised in homozygous form in said seeds, plant tissue, plant cell, fruits or plants parts. According to a third aspect the present invention relates to a method for providing or producing a cucumber plant (Cucumis sativus) which provides fruits with an improved shelf life, said method comprises the step of introducing in a cucumber plant (Cucumis sativus) a mutation in a genomic sequence encoding a stay green (SGR) gene, wherein said mutation is a G1410T mutation in the genomic sequence of SEQ ID No. 1 or in the genomic sequence having at least 98% sequence identity, preferably at least 99% sequence identity with SEQ ID No. 1, resulting in a truncated or non-functional SGR protein. The expression of said gene is reduced as compared to the expression of said gene in a cucumber plant not providing fruits having an improved shelf life or the enzymatic activity of said protein is reduced as compared to the enzymatic activity of said protein in a cucumber plant not providing fruits having an improved shelf life. Preferably said G1410T mutation is provided by gene editing techniques, preferably by mutagenesis or CRISPR/Cas.

According to a fourth aspect, the present invention relates to a SGR protein comprising an amino acid sequence as shown in SEQ ID No. 6 for providing a cucumber plant (Cucumis sativus) which provides fruits with an improved shelf life.

According to a further aspect, the present invention relates to a nucleic acid sequence comprising SEQ ID No. 4 or SEQ ID No. 5 for providing a cucumber plant (Cucumis sativus which provides fruits with an improved shelf life.

According to a further aspect, the present invention relates to use of a genomic sequence, gene sequence, or the cDNA sequence thereof, encoding a protein comprising the amino acid sequence as shown in SEQ ID No. 6 for providing cucumber plants (Cucumis sativus) which provides fruits with an improved shelf life. Preferably fruits stay green during storage for a time period of at least 4 or 5 weeks.

According to a further aspect, the present invention relates to a method for identifying or screening of (i) a cucumber plant (Cucumis sativus) as disclosed herein or (ii) a seed of said plant, the method comprises the step of establishing, in the genome of the plant or seed the presence of a nucleic acid sequence of comprising SEQ ID No. 4 or SEQ ID No. 5, encoding an SGR protein of SEQ ID No. 6.

The invention is further elucidated in the illustrative examples below. In the examples, reference is made to figures wherein:

Figure 1: Shows cucumber plants according to present invention comprising the mutated

SGR gene homozygous providing the “stay green” effect and reduced leaf yellowing, and masking Downey mildew infection, see upper panels. The right upper panel shows aspects of fruit of the plant of present invention, which is unaffected showing total absence of yellowing. Lower panels show a control plant (Marumba variety, which does not comprise the mutated SGR gene) that is affected by Downy Mildew as observed by the yellowing and curled leaves. The right lower panel shows the fruit from the control plant heavily affected showing yellowing.

Example 1

Two thousand seeds of the Cucumber (Cucumis sativus) hybrid Kurios were treated with 0.75 % (W/V) Ethyl Methane Sulfonate (EMS) for 16 hours. After rinsing the seeds several times with water, seeds were sown in peet blocks. After the appearance of the primary leaf, the apical meristem was removed to induce the development of the lateral meristem. This procedure was repeated to induce outgrowth of the lateral meristem of the first side shoot. Samples were taken from the top of this developing shoot for further molecular analysis i.e. the detection of mutations in candidate genes as described by Van Eijk and Van Tunen in EP 1929039.

A plant derived from the EMS treatment comprising SEQ ID NO. 4 was grown in a greenhouse and cucumber fruits were harvested. The shelf life of the fruits was measured during 4 weeks in a storability test using the following scale: (1) Good colour (green); (2) Acceptable colour (somewhat lighter green); (3) Unacceptable colour (first occurrence of yellowing); (4) Unacceptable colour (yellowing); (5) Unacceptable colour (extreme yellowing). A conventional cucumber fruit derived from a plant not comprising SEQ ID NO. 4 was also measured for 4 weeks in the same storability test.

The cucumbers according to the invention stayed green during the time period of 4 weeks, with a continuous score (1). The conventional cucumbers show a normal yellowish colour due to chlorophyll degradation during the 4- week test period, reaching score 4 after 4 weeks. Further, the presence of SEQ ID NO. 4 also affected the flesh colour of cucumber fruits. 4-week-old conventional cucumbers show a normal lighter colour compared to cucumbers harvested from a plant of the present invention.

Example 2

Cucumber plants, including plants comprising SEQ ID NO. 4, as deposited as NCIMB 44206, were grown in the greenhouse together with plants derived from non-mutated wild type seeds (i.e. lacking the mutated stay green (SGR) gene) and the reference variety Kurios. From the plants carrying the stay green gene, both heterozygous (Het) and homozygous (Hom) forms were planted.

Fruits of the 4 different plant types were harvested on day 0 and stored for 4 weeks in a crate at 17 °C. A taste panel has been evaluating the taste of the fruits, giving attention to Freshness, Moisture, Sour/sweet, Aftertaste, Color, Smell, and General impression. Evaluations took place at day 0, day 6, day 13, and day 20 after the fruits were harvested. As a control, at a day of tasting, a fresh fruit of the reference variety Kurios was also harvested and evaluated. It can be concluded that there is little difference between the taste of the 4 different types (Kurios, WT, Het, Hom) at the 4 moments (day 0, day 6, day 13, day 20) of tasting.

It is clear that the exterior color of the fruit of the homozygous SGR type after 28 days is at least as dark as the fresh picked Kurios fruit. Also, the interior color of the fruit of the homozygous SGR type is darker than the fresh picked Kurios fruit, the wild type and the heterozygous type. Also, it can be concluded that the other aspects of shelf life (like blisters, soft neck) are not altered by the presence of the stay green gene.

Example 3

It is common knowledge that leaves of a cucumber plant which are in the lower part of the plant, become old and yellow, and start to disintegrate when they are covered by upper leaves. The disintegration of the leaf is, amongst others, caused by the inactivity of the chlorophyll. The chlorophyll is giving the green color to the leaf. Plants of the wild type (lacking the present stay green gene), plants with the present stay green gene in heterozygous form and plants with the present stay green gene in homozygous form have been planted in the greenhouse. At mature stage of the plants (i.e. after 10 weeks) lower leaves were picked and placed next to each other.

It is clear the plant with the present stay green gene in homozygous form is able to maintain its chlorophyll for a longer period. Likewise, it is expected that the plant containing this gene in homozygous form is able to be photosynthetic active for a longer period.

Example 4

It was observed that the generated EMS plant of present invention has superior shelf life and disease tolerance characteristics in view of downy mildew infection. Molecular marker analysis revealed that a genetic factor influencing the value of the observed traits is located on Chr5 of the cucumber genome, by using Illumina short read sequencing technology (2x125 bp) on whole genome DNA processed for sequencing with Illumina TruSeq sample preparation. Sequencing results were processed according to standard procedure and aligned to public reference genome sequence 9930 v2 (http://cucurbitgenomics.org/organismZ2), along with larger set of 284 other diverse cucumber germplasm samples. A single position was identified that is unique and linked to the stay green and masking phenotype observed. The unique position results in the loss of a splice site. The possible splice site abolishing effect of plant of present invention was determined as a G>T mutation on position 1410 in the genomic sequence of SEQ ID No.l (referred to herein as G1410T mutation). The mutation was further investigated by producing cDNA sequence results. The splicing acceptor sequence “AG”, typically present at the 3’ end of an intron, is found as “AT” in the plant. This AT will not function as acceptor and the next “AG” site downstream of the original will gain the function of splicing acceptor. This next possible splice acceptor site is located 25 bp downstream of the original, resulting in an additional 25 bp deletion from the cDNA sequence (represented by SEQ ID No. 5), compared to the wildtype sequence (represented by SEQ ID No. 2). The 25 bp deletion in the coding sequence for SGR effectively results in a frameshift mutation, causing the translational frame to jump from frame 1 to frame 2. In effect, this frameshift results in a premature stop located 29 amino acids downstream and this premature stop results in a non-functional truncated SGR protein (represented by SEQ ID No. 6).

Seeds of the cucumber plants with a mutated SGR gene (i.e. comprising the G1410T mutation) in homozygous form and wild type plants (i.e. not comprising the mutated SGR gene) were grown till the cotyledon stage. The cotyledons were removed from the plantlets and placed in a plastic container covered with a glass plate. It can be considered that there was no air exchange possible between the content of the container and the environment. After 2 weeks of storage the leaves of the plant comprising the mutated SGR gene remained green, whereas the wild type plant yellowing of the leaves is observed.

Example 5

Leaves of the cucumber plant which are in the lower part of the plant, become old and yellow, start to disintegrate when they are covered by upper leaves. The disintegration of the leaf is, amongst others, caused by the inactivity and degradation of the chlorophyll. Chlorophyll is giving the green color to the leaf. Downy mildew (caused by Pseudoperonospora cubensis) in cucumber causes light green to yellow angular spots on the upper surfaces of leaves. With time these lesions turn brown and dry up.

Plants of the wild type (lacking the present mutant stay green gene), plants with the present stay green gene in heterozygous form, and plants with the present stay green gene comprising the mutation in homozygous form have been planted in the greenhouse and subjected to Downy Mildew infection. At mature stage of the plants (i.e. after 10 weeks) were placed next to each other and infected by Downey Mildew. The result is shown in Figure 1. It is clear that the plant (Upper panels, a plant according to present invention) with the mutated SGR gene in homozygous form can maintain its chlorophyll for a longer period as compared to the control plant (Marumba variety) not comprising the mutated SGR gene. Likewise, it is expected that the plant containing this mutated SGR gene in homozygous form can be photosynthetic active for a longer period. The plant of present invention provides a “stay green” effect by masking Downey mildew infection showing total absence of yellowing, see Figure 1 upper panels. Figure 1 lower panels shows the control that is affected by Downy Mildew as observed by the yellowing and curled leaves.

Claims

1. Cucumber plant (Cucumis sativus) which provides fruits having an improved shelf life, wherein said plant comprises a genomic sequence encoding a stay green (SGR) gene providing the improved fruit shelf life, wherein said stay green gene encodes a SGR protein, wherein said plant comprises a G1410T mutation in the genomic sequence of SEQ ID No. 1 or in the genomic sequence having at least 98% sequence identity, preferably at least 99% sequence identity with SEQ ID No. 1, resulting in a truncated or non-functional SGR protein.

2. Cucumber plant (Cucumis sativus) plant according to claim 1, wherein the genomic sequence encoding said SGR gene is mutated resulting in a premature stop codon.

3. Cucumber plant (Cucumis sativus) according to claim 1 or 2, wherein expression of the SGR gene represented by SEQ ID No.2 is reduced as compared to the expression of said SGR gene in a cucumber plant not providing fruits having an improved shelf life.

4. Cucumber plant (Cucumis sativus) according to any one of the claims 1 to 3, wherein the enzymatic activity of the SGR protein represented by SEQ ID No.3 is reduced as compared to the enzymatic activity of said SGR protein in a cucumber plant not providing fruits having an improved shelf life.

5. Cucumber plant (Cucumis sativus) according to any one of the claim 1 to 4, wherein said plant comprises the genomic sequence of SEQ ID No. 4.

6. Cucumber plant (Cucumis sativus) according to any one of the claims 1 to 5, wherein the truncated or non-functional SGR protein comprises the sequence of SEQ ID No. 6.

7. Cucumber plant (Cucumis sativus) plant according to any of the claims 1 to 6, wherein said genomic sequence stay encoding the SGR gene is present in heterozygous or homozygous form, preferably homozygous form in said plant.

8. Cucumber plant (Cucumis sativus) according to any one of the claims 1 to 7, wherein the G1410T mutation is a non-natural mutation.

9. Cucumber plant (Cucumis sativus) according to any one of the claims 1 to 8, wherein the G1410T mutation is provided by gene editing techniques, preferably by mutagenesis or CRISPR/Cas.

10. Cucumber plant (Cucumis sativus) according to any one of the claims 1 to 9, wherein the mutated genomic sequence encoding the SGR gene is obtainable from deposit number NCIMB 44206.

11. Seeds, plant tissue, plant cell, fruits or plants parts of a cucumber plant (Cucumis sativus) of any one of the claims 1 to 10.

12. Method for providing a cucumber plant (Cucumis sativus) which provides fruits with an improved shelf life, said method comprises the step of introducing in a cucumber plant (Cucumis sativus) a mutation in a genomic sequence encoding a stay green (SGR) gene, wherein said mutation is a G1410T mutation in the genomic sequence of SEQ ID No. 1 or in the genomic sequence having at least 98% sequence identity, preferably at least 99% sequence identity with SEQ ID No. 1, resulting in a truncated or non-functional SGR protein.

13. Method according to claim 12 wherein the G1410T mutation is provided by gene editing techniques, preferably by mutagenesis or CRISPR/Cas.

14. A protein comprising an amino acid sequence as shown in SEQ ID No. 6 for providing a cucumber plant (Cucumis sativus) which provides fruits with an improved shelf life.

15. A nucleic acid sequence comprising SEQ ID No. 4 or SEQ ID No. 5 for providing a cucumber plant (Cucumis sativus) which provides fruits with an improved shelf life.

16. Use of a genomic sequence, gene sequence, or the cDNA sequence thereof, encoding a protein comprising the amino acid sequence as shown in SEQ ID No. 6 for providing cucumber plants (Cucumis sativus) which provides fruits with an improved shelf life.

17. Method for identifying or screening of (i) a cucumber plant (Cucumis sativus) according to any one of the claims 1 to 10 or (ii) a seed of said plant, the method comprises the step of establishing, in the genome of the plant or seed the presence of a nucleic acid sequence of claim 15 encoding an SGR protein as defined in claim 14.