Patent Description:
Cucumber plants are already cultivated for at least <NUM> 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, the 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 (><NUM>). 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.

Zhou et al. (<NUM>) disclose a functional analysis of a STAY-GREEN gene in Medicago truncatula and an effective use thereof for Alfalfa improvement.

<CIT> discloses methods and compositions for enhancing agronomic properties in plants through modification of senescence. Nucleic acid constructs therefore are described and transgenic plants are provided that exhibit enhanced agronomic properties.

Given the above, there is a need in the art for cucumber plants providing fruits having an extended shelf life.

Therefore, it is an object of the invention, amongst other objects, to provide cucumber plants providing fruits having an extend shelf life.

This object, amongst other objects, is met by providing a cucumber plant according to the appended claim <NUM>.

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 gene, or stay green gene, providing the improved fruit shelf life, wherein said gene, or stay green gene, encodes a protein comprising the amino acid sequence as shown in SEQ ID NO. <NUM>, or amino acid sequences with more than <NUM>% identity, such as more than <NUM>%, <NUM>% or <NUM>%, preferably more than <NUM>% identity such as more than <NUM>%, more preferably more than <NUM>% identity such as more than <NUM>%, even more preferably more than <NUM>% identity or more than <NUM>% identity; and wherein 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.

In the research that led to the present invention, it was surprisingly found that 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.

With 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 <NUM> or <NUM> 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 <NUM> or <NUM> weeks; whereas cucumber fruits according to the present invention turn yellow after a time period of at least <NUM> or at least <NUM> weeks.

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 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.

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 (<NPL>, and <NPL>). 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 (<NPL>) individual plants having a mutation in the present genes are identified.

The present improved shelf life is provided by one or more mutations in the coding sequence of the present gene resulting in a truncated or non-functional protein. Truncated proteins can be readily determined by analyzing gene transcripts at the mRNA or cDNA level and non-functional 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.

In a preferred embodiment of the present invention the present mutation results in an amino acid substitution comprising a substitution of leucine, or designated as 'L' (Leu), on position <NUM> of SEQ ID NO. More preferably the leucine on position <NUM> of SEQ ID NO. <NUM> is substituted by an phenyl alanine, or 'F' (Phe). Accordingly, in a further preferred embodiment, the present stay green gene encodes a protein comprising an amino acid sequence as shown in SEQ ID NO. This protein comprising the present amino acid sequence as shown in SEQ ID NO. <NUM> is highly correlated with the improved shelf life trait of the present invention, since an improved shelf life of at least <NUM> weeks is observed for fruits from a cucumber plant comprising a gene encoding the present protein.

The present stay green gene is present in homozygous form, thereby providing an improved shelf life of the cucumber fruits.

According to a second aspect, the present invention relates to seeds, plant tissue, fruits or plants parts of a cucumber plant (Cucumis sativus) comprising a stay green gene providing an improved fruit shelf life, or alternatively obtainable or derived from the present cucumber plant, wherein the stay green gene encodes an amino acid sequence as shown in SEQ ID NO. <NUM>, or amino acid sequences with more than <NUM>% identity, preferably more than <NUM>% identity, more preferably more than <NUM>% identity, even more preferably more than <NUM>% identity; and wherein 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.

Disclosed is 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 stay green gene encoding a protein comprising the amino acid sequence as shown in SEQ ID No. <NUM>, or a gene with more than <NUM>% sequence identity, preferably more than <NUM>% sequence identity, more preferably more than <NUM>% sequence identity with identity SEQ ID No. <NUM>; and wherein 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.

Disclosed is the use of a gene, or the cDNA sequence thereof, encoding a protein comprising the amino acid sequence as shown in SEQ ID No. <NUM> or a gene encoding a protein with more than <NUM>% sequence identity, preferably more than <NUM>% sequence identity, more preferably more than <NUM>% sequence identity with identity SEQ ID No. <NUM> for providing cucumber plants (Cucumis sativus) which provides fruits with an improved shelf life, or preferably fruits which stay green during storage for a time period of at least <NUM> or <NUM> weeks.

The present invention relates to proteins comprising an amino acid sequence as shown in SEQ ID NO <NUM>.

The present invention relates to nucleic acids, preferably isolated nucleic acids, comprising SEQ ID NO <NUM>.

The present invention relates to a gene, or stay green gene, encoding a protein having an amino acid sequence comprising SEQ ID No. <NUM>.

The present invention relates to a gene, or stay green gene, comprising a nucleic acid sequence comprising SEQ ID NO.

Disclosed is a gene, or stay green gene, as defined above, i.e. wherein said gene, or stay green gene, encodes a protein comprising the amino acid sequence as shown in SEQ ID NO. <NUM>, or amino acid sequences with more than <NUM>% identity, such as more than <NUM>%, <NUM>% or <NUM>%, preferably more than <NUM>% identity such as more than <NUM>%, more preferably more than <NUM>% identity such as more than <NUM>%, even more preferably more than <NUM>% identity or more than <NUM>% identity; wherein said gene comprises a mutation in the coding sequence resulting in one or more amino acid substitutions in comparison with the unmutated gene encoding a protein comprising the amino acid sequence as shown in SEQ ID NO.

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

Two thousand seeds of the Cucumber (Cucumis sativus) hybrid Kurios were treated with <NUM> % (W/V) Ethyl Methane Sulfonate (EMS) for <NUM> hours. After rinsing the seeds several times with water, seeds were sown in peet blocks. After 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 <CIT>.

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

The cucumbers according to the invention stayed green during the time period of <NUM> weeks, with a continuous score (<NUM>). The conventional cucumbers show a normal yellowish colour due to chlorophyll degradation during the <NUM> week test period, reaching score <NUM> after <NUM> weeks. <FIG> shows a picture with on the left side <NUM> weeks old conventional cucumbers having a yellowish colour, and on the right side <NUM> weeks old cucumbers according to the present invention having a full green colour. Further, the presence of SEQ ID NO. <NUM> also affected the flesh colour of cucumber fruits. <NUM> week old conventional cucumbers show a normal lighter colour (see <FIG>, left side) compared to cucumbers harvested from a plant of the present invention, (see <FIG>, right side).

Plants derived from the EMS treatment comprising SEQ ID NO. <NUM> were grown in the greenhouse together with plants derived from untreated seeds (Wild type/WT) and the reference variety Kurios. From the plants carrying the present stay green gene, both heterozygous (Het) and homozygous (Hom) forms were planted.

Fruits of the <NUM> different plant types were harvested on day <NUM> and stored for <NUM> weeks in a crate at <NUM>. 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 <NUM>, day <NUM>, day <NUM>, and day <NUM> 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. The scores, using the scale <NUM>-<NUM>, are shown in <FIG>. It can be concluded that there is little difference between the taste of the <NUM> different types (Kurios, WT, Het, Hom) at the <NUM> moments (day <NUM>, day <NUM>, day <NUM>, day <NUM>) of tasting.

After <NUM> weeks of storage, pictures were taken from the fruits. They are shown in <FIG> and <FIG>.

It is clear that the exterior color of the fruit of the homozygous SGR type after <NUM> 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 can be concluded from <FIG> and <FIG>, that the other aspects of shelf life (like blisters, soft neck) are not altered by the presence of the stay green gene.

Seeds of plants with either the present stay green gene in homozygous form or in heterozygous form 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 <NUM> weeks of storage, the picture as shown in <FIG> was visible.

Cotyledons from plants of seeds that were harvested from the plant with the stay green in the homozygous form were placed on the grid A1-C1 <NUM>. Cotyledons from plants of seeds that were harvested from the plant with the stay green gene in heterozygous form were placed on the grid D1-F11. In the grid D <NUM>-F11 there are <NUM> cotyledons expressing the stay green gene in a homozygous form (as in the grid A1-C11), and <NUM> cotyledons lacking this expression. These cotyledons may either lack the stay green gene or have it in a heterozygous form. The ratio <NUM>/<NUM> confirms the expected ratio for a single recessive gene, meaning that the present stay green gene is present in homozygous form.

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 being 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 <NUM> weeks) lower leaves were picked and placed next to each other. The result is shown in <FIG>.

Claim 1:
Cucumber plant (Cucumis sativus) which provides fruits having an improved shelf life, wherein said plant comprises a stay green gene, said stay green gene is present in homozygous form, providing the improved fruit shelf life, wherein said stay green gene encodes a protein comprising the amino acid sequence as shown in SEQ ID NO. <NUM>, or amino acid sequences with more than <NUM>% identity, preferably more than <NUM>% identity, more preferably more than <NUM>% identity, even more preferably more than <NUM>% identity; and wherein 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, said reduced enzymatic activity is provided by one or more mutations in the coding sequence of said stay green gene resulting in one or more amino acid substitutions, frame-shifts or pre-mature stop codons resulting in a truncated or non-functional protein.