WO2010132988A1

WO2010132988A1 - Increased seed oil and abiotic stress tolerance mediated by plant chd3 protein

Info

Publication number: WO2010132988A1
Application number: PCT/CA2010/000755
Authority: WO
Inventors: Nirmala Sharma; Pierre R. J. A. Fobert
Original assignee: National Research Council Of Canada
Priority date: 2009-05-19
Filing date: 2010-05-19
Publication date: 2010-11-25

Abstract

Increased seed oil content, decreased abscisic acid sensitivity and/or increased drought resistance in a plant may be accomplished by introducing into the plant means for encoding a PICKLE-RELATED1 (PKR1 ) protein to thereby increase expression of PKR1 in the plant compared to a plant grown under similar conditions in which the means for encoding the PKR1 protein has not been introduced.

Description

INCREASED SEED OIL AND ABIOTIC STRESS TOLERANCE MEDIATED BY PLANT

CHD3 PROTEIN

Cross-reference to Related Applications

This application claims the benefit of United States Provisional Patent Application USSN 61/213,238 filed May 19, 2009, the entire contents of which is herein incorporated by reference.

Field of the Invention

This invention is related to genetic manipulation of plants to alter plant phenotype. In particular, the present invention is related to altering expression of a PICKLE- RELATED1 (PKR1) protein in a plant to alter seed oil content and abiotic stress responses.

Background of the Invention

In most years, canola is the top Canadian cash crop, generating some $11 B of economic activity. Canola is valued for its superior oil quality and seed oil represents an estimated 80% of the worth of the crop. Recent changes to the registration standards for Canadian canola focus upon an increase in oil content for new varieties and the Canadian industry is targeting a 2.5% increase of seed oil levels to 45% by 2015. Economically, it has been estimated that a 1 % increase in seed oil yield translates to an annual value of $80 M CAD. Not surprisingly, increasing seed oil content has been identified by the industry as an important research objective. Achieving this goal is a considerable challenge when one considers that the general trend in the past has been towards a slow upward drift in oil content. According to information from the Canadian Grain Commission, harvest surveys dating back to 1956 show a linear rise (nonsignificant) of only 0.05% in oil.

Several strategies can be used to increase seed oil content, including conventional breeding, marker assisted breeding and transgenic modifications. Previous and on-going studies using transgenics have focused on manipulation of lipid biosynthetic genes such as diacylglycerol acyltransferase (DGAT) or genes encoding regulatory elements including kinases such as pyruvate dehydrogenase complex kinase (PDCK) and transcription factors such as WRINKLED1 (WRH). Increases in seed oil modification using the existing approaches are often modest, necessitating the use of multiple genes in combination. Stacking genes with different modes of action may be advantageous, resulting in additive or synergist effects.

CHD3 is a subfamily of the SWI/SNF DNA-dependent ATPases that use energy from ATP to remodel chromatin without covalent modifications. The family is highly conserved throughout eukaryotes and has been studied intensely in yeast and animals where CHD3 proteins are found in complexes with histone deacetylase activity that repress transcription and embryonic programs during developmental transitions.

There remains a need in the art for approaches to modifying seed oil content and/or abiotic stress responses in a plant.

Summary of the Invention

It has now been found that increasing levels of the transcriptional regulator

PICKLE-RELATED1 (PKR1), a putative member of the CHD3 family of chromatin remodelling ATPases, is an effective means of increasing seed oil content. In addition, expression or overexpression of plant PKR 1 increases tolerance of plants to various abiotic stresses.

Thus, there is provided a method of increasing seed oil content, decreasing abscisic acid sensitivity and/or increasing drought resistance in a plant comprising: introducing into the plant means for encoding a PICKLE-RELATED1 (PKR1) protein to thereby increase expression of PKR1 protein in the plant to thereby increase seed oil content, decrease abscisic acid sensitivity and/or increase drought resistance in the plant compared to a plant grown under similar conditions in which the means for encoding the PKR1 protein has not been introduced.

There is also provided a nucleic acid construct comprising means for encoding a PICKLE-RELATED1 (PKR1) protein operably linked to one or more nucleic acid sequences required for transforming the construct into a cell and/or for expressing or overexpressing the PKR1 protein encoding means in the cell.

There is also provided a cell, seed or plant comprising the nucleic acid construct of the present invention.

Further features of the invention will be described or will become apparent in the course of the following detailed description. Brief Description of the Drawings

In order that the invention may be more clearly understood, embodiments thereof will now be described in detail by way of example, with reference to the accompanying drawings, in which:

Fig. 1 depicts a vector map for a binary T-DNA PKR1 transformation vector.

Fig. 2A depicts a graph of seed oil content in different Arabidopsis PKR1 transgenic lines, the Arabidopsis knockout mutant line (p/σ7-ko) and corresponding Arabidopsis wild types. PKR1 3, PKR1 6, PKR1 12 and PKR1 15 represent independent transgenic events. Transgenic lines compare with the CoI-O wild type and the knockout mutant line compares with the Col-2 wild type.

Fig. 2B depicts a graph of seed oil content comparing Arabidopsis wild type (Col-2) to Arabidopsis pkr knockout (pkr1 ko) lines in two independent experiments.

Fig. 2C depicts a graph of seed oil content comparing Arabidopsis wild type (CoI-O) to Arabidopsis PKR1 over-expression (PKR1 3-7-7) lines.

Fig. 3A depicts graphs showing rate of germination of PKR1 transgenic lines and wild type seeds on half-strength MS medium supplemented with 0.25 μM ABA.

Fig. 3B depicts a graph showing germination of pkr1 knockout mutant line and wild type seeds on half-strength MS medium supplemented with various concentrations of ABA, 48 hrs after stratification.

Fig. 3C depicts a graph showing ABI3 gene expression 96 hr and 120 hr post- stratification in 0.3 μM ABA-treated Arabidopsis seedlings of pkr1 knockout mutant line (pkr1-6) compared to the wild type (Col-2).

Fig. 3D depicts a graph showing ABI5 gene expression 96 hr and 120 hr post- stratification in 0.3 μM ABA-treated Arabidopsis seedlings of pkr1 knockout mutant line (pkr1-6) compared to the wild type (Col-2).

Fig. 4 depicts a graph showing drought sensitivity of pkr1 knockout mutant line (pkr1-ko) compared to the wild type (Col-2).

Fig. 5 depicts a graph showing expression of embryogenic (oil related) gene LEC1 in Arabidopsis wild type (Wild-type), pkr1 knockout mutant (pkr1-1) and pkl1 knockout mutant (pkl1-1) lines. Description of Preferred Embodiments

An example of one means for encoding a PICKLE-RELATED1 (PKR1) protein is an Arabidopsis nucleic acid molecule (pkr1) (SEQ ID NO 1) This nucleic acid molecule encodes a putative CHD3 chromatin remodeling protein (SEQ ID NO 2) and has been characterized using forward and reverse genetics approaches This nucleic acid molecule is a gene involved in regulating oil disposition in seeds and in mediating ABA responses in germinating seeds and drought responses in juvenile plants

Other means for encoding a PICKLE-RELATED1 (PKR1) protein include, for example, nucleic acid molecules that encode proteins having at least 80% sequence identity to SEQ ID NO 2

Seed oil content in several independent transgenic lines was elevated compared to the wild type, whereas opposite phenotype was observed for a T-DNA insertion mutant in this gene We have shown that increasing levels of PKR 1 increases the level of seed oil in Arabidopsis thaliana from about 30% to up to about 42% (seed DW) There appears to be no overt developmental effects of PKR1 overexpression in Arabidopsis PKRI's closest characterized relative, PICKLE1 (PKL1 ) having about 40% ammo acid sequence identity with PKR1 , has been shown to repress embryogenic programs upon seed germination However, PKL1 knockout mutants have not been reported to affect seed oil accumulation, nor has it been reported that overexpression of PKL1 increases seed oil levels Since PKR1 may be involved in regulating chromatin structure, PKR1 is likely to regulate seed oil accumulation by mechanisms that are distinct from those of previously reported genes

In addition to producing more seed oil, transgenic lines also germinated faster and better in presence of ABA than the wild type, whereas the knockout mutant line was hypersensitive to ABA and hence displayed delayed or poor germination compared to its wild type Thus, Arabidopsis plants overexpressing PKR1 display greater tolerance to the plant hormone ABA, which is involved in regulating many physiological processes, including seed maturation and tolerance to abiotic stresses Conversely, loss of PKR1 function results in reduced tolerance to ABA and reduced drought tolerance

Terms

In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terms are provided Complementary nucleotide sequence: "Complementary nucleotide sequence" of a sequence is understood as meaning any DNA whose nucleotides are complementary to those of sequence of the disclosure, and whose orientation is reversed (antiparallel sequence).

Degree or percentage of sequence homology: The term "degree or percentage of sequence homology" refers to degree or percentage of sequence identity between two sequences after optimal alignment. Percentage of sequence identity (or degree or identity) is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical amino-acid residue or nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

Isolated: As will be appreciated by one of skill in the art, "isolated" refers to polypeptides or nucleic acids that have been "isolated" from their native environment.

Nucleotide, polynucleotide, or nucleic acid sequence: "Nucleotide, polynucleotide, or nucleic acid sequence" will be understood as meaning both a double-stranded or single-stranded DNA in the monomeric and dimeric (so-called in tandem) forms and the transcription products of said DNAs.

Sequence identity: Two amino-acid or nucleotide sequences are said to be "identical" if the sequence of amino-acids or nucleotidic residues in the two sequences is the same when aligned for maximum correspondence as described below. Sequence comparisons between two (or more) peptides or polynucleotides are typically performed by comparing sequences of two optimally aligned sequences over a segment or "comparison window" to identify and compare local regions of sequence similarity. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Smith 1981), by the homology alignment algorithm of Neddleman and Wunsch (Neddleman 1970), by the search for similarity method of Pearson and Lipman (Pearson 1988), by computerized implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science Dr., Madison, Wis.), or by visual inspection. Isolated and/or purified sequences of the present invention may have a percentage identity with the bases of a nucleotide sequence, or the amino acids of a polypeptide sequence, of at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, or 99.7%. This percentage is purely statistical, and it is possible to distribute the differences between the two nucleotide sequences at random and over the whole of their length.

The definition of sequence identity given above is the definition that would be used by one of skill in the art. The definition by itself does not need the help of any algorithm, said algorithms being helpful only to achieve the optimal alignments of sequences, rather than the calculation of sequence identity. From the definition given above, it follows that there is a well defined and only one value for the sequence identity between two compared sequences which value corresponds to the value obtained for the best or optimal alignment. In the BLAST N or BLAST P "BLAST 2 sequence", software which is available in the web site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, and habitually used by the inventors and in general by the skilled man for comparing and determining the identity between two sequences, gap cost which depends on the sequence length to be compared is directly selected by the software (i.e. 11.2 for substitution matrix BLOSUM-62 for length>85).

It will be appreciated that this disclosure embraces the degeneracy of codon usage as would be understood by one of ordinary skill in the art and as illustrated in Table 1. Furthermore, it will be understood by one skilled in the art that conservative substitutions may be made in the amino acid sequence of a polypeptide without disrupting the structure or function of the polypeptide. Conservative substitutions are accomplished by the skilled artisan by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Additionally, by comparing aligned sequences of homologous proteins from different species, conservative substitutions may be identified by locating amino acid residues that have been mutated between species without altering the basic functions of the encoded proteins. Table 2 provides an exemplary list of conservative substitutions. Table 1

Codon Degeneracies

Table 2 Conservative Substitutions

Expression

PKR1 nucleotide sequences can be expressed in alternate plant hosts to impart characteristics of improved agronomic performance via recombinant means. The methods to construct DNA expression vector and to transform and express foreign genes in plant and plant cells are well known in the art.

Additionally, it is evident that the sequences can be used in the construction of a construct or an expression vector. It is well known that nucleotide sequences encoding PKR1 can be inserted within an expression vector for heterologous expression in diverse host cells and organisms, for example plant cells and plant, by conventional techniques. These methods, which can be used in the invention, have been described elsewhere (Potrykus 1991 ; Vasil 1994; Walden 1995; Songstad 1995), and are well known to persons skilled in the art. As known in the art, there are a number of ways by which genes and gene constructs can be introduced into plants and a combination of transformation and tissue culture techniques have been successfully integrated into effective strategies for creating transgenic plants. For example, one skilled in the art will certainly be aware that, in addition to Agrobacteήum-meό^'iated transformation of Arabidopsis by vacuum infiltration (Bechtold 1993) or wound inoculation (Katavic 1994), it is equally possible to transform other plant species, using Agrobacterium Ti-plasmid mediated transformation (e.g., hypocotyl (DeBlock 1989) or cotyledonary petiole (Moloney 1989) wound infection), particle bombardment/biolistic methods (Sanford 1987; Nehra 1994; Becker 1994) or polyethylene glycol-assisted, protoplast transformation (Rhodes 1988; Shimamoto 1989) methods.

As will also be apparent to persons skilled in the art, and as described elsewhere (Meyer 1995; Datla 1997), it is possible to utilize plant promoters to direct any intended regulation of transgene expression using constitutive promoters (e.g., those based on CaMV35S), or by using promoters which can target gene expression to particular cells, tissues (e.g., napin promoter for expression of transgenes in developing seed cotyledons), organs (e.g., roots), to a particular developmental stage, or in response to a particular external stimulus (e.g., heat shock). Promoters for use herein may be inducible, constitutive, or tissue-specific or cell specific or have various combinations of such characteristics. Useful promoters include, but are not limited to constitutive promoters such as carnation etched ring virus (CERV), cauliflower mosaic virus (CaMV) 35S promoter, or more particularly the double enhanced cauliflower mosaic virus promoter, comprising two CaMV 35S promoters in tandem (referred to as a "Double 35S" promoter). Meristem specific promoters include, for example, STM, BP, WUS, CLV gene promoters. Seed specific promoters include, for example, the napin promoter. Other cell and tissue specific promoters are well known in the art.

Promoter and termination regulatory regions that will be functional in the host plant cell may be heterologous (that is, not naturally occurring) or homologous (derived from the plant host species) to the plant cell and the gene. Suitable promoters which may be used are described above. The termination regulatory region may be derived from the 3' region of the gene from which the promoter was obtained or from another gene. Suitable termination regions which may be used are well known in the art and include Agrobacterium tumefaciens nopaline synthase terminator (Tnos), A. tumefaciens mannopine synthase terminator (Tmas) and the CaMV 35S terminator (T35S). Particularly preferred termination regions for use herein include the pea ribulose bisphosphate carboxylase small subunit termination region (TrbcS) or the Tnos termination region. Such gene constructs may suitably be screened for activity by transformation into a host plant via Agrobacterium and screening for the desired activity using known techniques.

Preferably, a nucleic acid molecule construct for use herein is comprised within a vector, most suitably an expression vector adapted for expression in an appropriate plant cell. It will be appreciated that any vector which is capable of producing a plant comprising the introduced nucleic acid sequence will be sufficient. Suitable vectors are well known to those skilled in the art and are described in general technical references. Particularly suitable vectors include the Ti plasmid vectors. After transformation of the plant cells or plant, those plant cells or plants into which the desired nucleic acid molecule has been incorporated may be selected by such methods as antibiotic resistance, herbicide resistance, tolerance to amino-acid analogues or using phenotypic markers. Various assays may be used to determine whether the plant cell shows an increase in gene expression, for example, Northern blotting or quantitative reverse transcriptase PCR (RT-PCR). Whole transgenic plants may be regenerated from the transformed cell by conventional methods. Such plants produce seeds containing the genes for the introduced trait and can be grown to produce plants that will produce the selected phenotype.

Expression or overexpression of genes encoding PKR1 may done in combination with overexpression or expression of one or more other genes involved in seed oil production and/or abiotic stress tolerance, for example, lipid biosynthetic genes such as diacylglycerol acyltransferase (DGAT) or genes encoding regulatory elements including kinases such as pyruvate dehydrogenase complex kinase (PDCK) and transcription factors such as WRINKLED1 (WRM)

Preferred plants in which PKR1 activity may be expressed or overexpressed include crop species, especially oilseed plant species Some examples include Brassicaceae spp (e g rapeseed and Canola), Borago spp (borage), Ricinus spp (e g Ricinus communis (castor)), Theobroma spp (e g Theobroma cacao (cocoa bean)), Gossypium spp (cotton), Crambe spp , Cuphea spp , Linum spp (flax), Lesquerella spp , Limnanthes spp , Linola, Tropaeolum spp (nasturtium), Olea spp (olive), Elaeis spp (palm), Arachis spp (peanut), Carthamus spp (saff lower), Glycine spp (soybean), Soja spp (soybean), Helianthus spp (sunflower), Vernonia spp Plants of particular note are from the family Brassicaceae, especially Arabidopsis thaliana, Brassica napus, Brassica rapa, Brassica cannata, Brassica juncea, and Camelina sativa Arabidopsis thaliana, Brassica spp and Glycine spp are of particular note

Examples

Example 1 Transformation of plant material

Genomic protein coding region of At5g44800, lacking the first 14 codons, was cloned from the bacterial artificial chromosome K23L20 into cloning vector pJM1 by recombination as described by Liu et al (Liu 2003) and subsequently into the binary T- DNA vector pDMC32 At2S3 using Gateway technology (Invitrogen) Agrobacterium strain GV3101 (MP90) harboring the T-DNA vector was used to transform the At2S3 (napin) PKR1 gene into Arabidopsis thaliana (Columbιa-0 ecotype) by floral dipping The PKR1 T-DNA vector map is shown in Fig 1

A potential pkr1 T-DNA insertion line (WISCDSLOX377-380E20) was identified in Arabidopsis thaliana Columbιa-2 background from the SaIk Institute Genomic Laboratory Genomic database (http //signal salk edu) and seeds were obtained from the Arabidopsis Biological Resource Centre (ABRC) A homozygote T-DNA line was identified using PCR genotyping Several independent T4 or T5 transgenic lines, and the T-DNA insertion line, were analyzed for seed oil content, fatty acid profile, and ABA responses during germination The pkr1 T-DNA insertion mutant line was also evaluated for drought response Example 2: Seed oil analysis

Seeds of PKR1 transgenic lines, T-DNA insertion lines and their corresponding wild types were stratified in the dark for 2-3 days at 4⁰C and sown on germination medium

Sunshine™ Mix#4 (Sun Gro Horticulture, Canada). Plants were germinated and grown in chambers (Conviron) under a 16 hr photoperiod, 21/18⁰C day/night temperature cycle and about 250 μE irradiance. Secondary shoots were trimmed away and siliques on primary shoots were allowed to dry on plants before moving to a finishing chamber for two weeks to ensure complete after-ripening of seeds. Seeds were harvested only from main shoots and allowed to dry at room temperature for another 2-3 weeks before analyzing for seed oil content and fatty acid profiles.

Total lipids were extracted by grinding seeds in chloroform :isopropanol (2:1). The solvent was evaporated off at room temperature under a stream of nitrogen gas and total lipids were transmethylated by heating samples with 3 N methanolic HCI at 80⁰C for 3 hrs. Fatty acids methyl esters (FAMES) were then extracted with GC grade hexane in presence of 0.9% NaCI. The solvent (hexane) was evaporated under a stream of nitrogen gas and FAMES were re-dissolved in 500 μl of methyl esters standards (17:0 M. E. and 23:0 M. E.) in hexane and analyzed by GC. Seed oil content and fatty acid profiles were calculated as a percentage (%) of dry seed weight.

Oil content was measured on four independent PKR1 transgenic lines and the pkr1 T-DNA insertion mutant grown at three separate occasions, and consistently show increases (transgenics) or decreases (T-DNA insertion mutant) in seed oil content (Fig.

2A and Table 3). However, profiles of fatty acids were not altered in transgenic lines or the mutant line.

In two independent experiments, comparison of seed oil content in wild type line (Col-2) to pkr1 mutant line (pkr1 ko) consistently showed a decrease in seed oil content in the mutant line (Fig. 2B), demonstrating that PKR1 is active in seed oil production. Further, a comparison of seed oil content in the PKR1 3-7-7 over-expression transgenic line to the wild type line (CoI-O) shows an increase in seed oil content of over 4% based on the total weight of the seed (Fig. 2C). Table 3

Example 3 ABA responses

Seeds were surface-sterilized with 30% bleach (0 01% Tween™-20), rinsed several times with sterilize water, and sown on 1 5% agar plates containing half-strength

Murashige and Skoog (MS) salt solution supplemented or not with 0 μM, 0 25 μM or 0 3 μM ABA (± ABA, Toray batch, PBI 58, NRC/PBI Saskatoon) The plates were transferred to a tissue culture room after a cold treatment of 2 days at 4°C in the dark, and incubated at 20°C/16°C day/night temperatures under a 16 hrs/8 hrs light/dark regime and 80-100 μE irradiance

Germination (defined as endosperm rupture and radical emergence) was scored starting 24 hrs after seed stratification Germination events are expressed as a percentage of the total number of seeds per plate Germination experiments were repeated at least three times using three different seed batches of wild type, PKR1 transgenic lines or the pkri mutant grown in parallel

PKR1 transgenic lines showed a decreased sensitivity to ABA during germination as indicated by faster and higher overall germination than in the wild type in presence of ABA in the germination medium (Fig 3A) On the other hand, the T-DNA insertion mutant line showed an increased sensitivity to ABA, and hence a slower or reduced germination in the presence of ABA compared to its wild type (Fig 3B) Further, ABI3 and ABI5 are two genes known to inhibit seed germination by arresting embryo growth The pkr1 knockout mutant expresses these genes at higher level than the wild type during seed germination 96 hr and 120 hr after stratification (Fig. 3C and Fig. 3D). This could explain poor germination of this mutant in presence of ABA.

Example 4: Drought responses

Wild type and the mutant seeds were germinated directly on Sunshine™ Mix4 (Sun Gro Horticulture, Canada) and grown under regular watering and fertilization regime until the plants were three-weeks-old. At that point, plants were subjected to drought stress by withholding water and wilting symptoms were monitored daily thereafter until more than 80% plants displayed some degree of wilting. Drought response was evaluated in three independent batches of plants. Results are presented as a percentage of wilted or dead plants 10 days (10 d) after imposing drought stress by withholding water. The pkr1 T-DNA insertion mutant showed an increased sensitivity to drought as indicated by higher percentage wilting or death of the whole plant compared to its wild type at a certain time point after imposing drought stress (Fig. 4).

Example 5: Comparison of PKR1 to PKL 1

Phenotypes of the PICKLE-RELATED1 φkr1) knockout mutant were compared to the PICKLE1 {pkl1) mutant. First, the appearance of pkl-like roots was examined in seedling of both the pkr1 mutant and the pkl1 mutant, before and after administration of the fungicide uniconazole-P (Uni-P). As evidenced in Table 4, pkr doesn't mimic pkl in terms of production of pkl-like roots. Second, expression of the embryogenic LEC1 gene, an oil-related gene expressed in the roots of a plant, was examined. As evidenced by Fig. 5, the pkr1 mutant, like the wild type, does not express the LEC1 gene, whereas pkl1 does express the LEC1 gene. Collectively, these data show that PKR1 and PKL1 have different functions.

Table 4

Free Listing of Sequences

PKR1 - nucleotide sequence (SEQ ID NO: 1) - Arabidopsis thaliana (6729 bp)

ATGAAAGATTCAGGCAGTGAAATGATTAAAAGAGATTGGGTCATGAAGCAGAAACGAAGAAAACTT CCGTCTATACTAGATATATTAGACCAAAAAGTGGATAGTTCTATGGCTTTTGATTCCCCGGAATAC ACTTCTTCCTCTAAACCAAGTAAGCAGCGGCTTAAGACTGATTCGACTCCTGAAAGGAACTCCTCT AAGAGGAAAGGAAATGATGGGAATTATTTTGAATGTGTGATCTGTGACCTTGGTGGTGATTTATTG TGTTGTGATAGTTGTCCTCGGACCTATCATACCGCATGCCTCAATCCACCTCTTAAGCGGATTCCA AATGGTAAGTGGATCTGCCCAAAATGTTCCCCAAACAGTGAAGCACTCAAGCCTGTCAATCGTTTA GATGCCATTGCTAAGCGAGCAAGAACAAAAACCAAGAAAAGAAATTCAAAAGCCGGACCAAAGTGC GAAAGAGCTTCTCAGATTTATTGCAGTTCTATAATTTCTGGAGAACAATCTTCAGAGAAAGGGAAA TCTATATCGGCCGAAGAGAGCAAATCCACAGGAAAGGAAGTTTATTCTTCCCCGATGGATGGCTGT TCAACTGCTGAGCTTGGTCATGCATCTGCGGATGACCGACCTGATTCATCGTCTCATGGAGAAGAT GATTTGGGGAAACCCGTCATACCCACTGCAGATTTACCATCTGATGCAGGATTAACGTTGCTGTCC TGTGAAGATCTCTCCGAATCTAAACTATCAGATACGGAGAAAACTCATGAAGCACCCGTGGAGAAG TTGGAACATGCTTCCAGTGAGATCGTGGAGAACAAGACAGTTGCTGAAATGGAGACTGGAAAAGGT AAAAGGAAAAAACGGAAGCGTGAACTAAATGATGGGGAAAGTCTTGAAAGGTGCAAGACTGATAAG AAACGCGCGAAGAAAAGTTTGTCCAAAGTGGGTTCCAGTTCTCAGACTACCAAATCACCGGAGTCT TCGAAAAAAAAGAAAAAGAAAAATCGTGTGACTTTAAAATCCTTGTCCAAACCTCAGTCCAAGACA GAAACACCAGAAAAAGTGAAGAAGCTTCCCAAGGAGGAACGTCGTGCAGTACGTGCCACTAATAAA TCTTCTAGTTGTTTGGAAGATACAAACTCTCTTCCGGTTGGAAACCTCCAGGTTCATCGTGTTTTA GGATGCCGAATCCAAGGTCTGACTAAAACCTCGCTGTGTAGTGCTCTTTCAGATGACTTGTGTTCG GATAATTTACAAGCTACTGACCAACGGGATAGCTTAGTACAAGATACGAATGCTGAATTAGTAGTT GCTGAGGACAGAATAGATTCTTCTTCTGAGACAGGTAAAAGTTCGAGGGATTCACGACTGAGGGAT AAAGATATGGATGATTCTGCTTTAGGTACCGAGGGTATGGTTGAGGTGAAAGAAGAGATGCTTTCT GAAGACATTTCCAATGCCACATTGAGTAGACATGTGGATGATGAAGATATGAAAGTTAGTGAAACG CATGTATCTGTTGAGAGGGAGTTACTTGAAGAAGCACATCAGGAAACAGGGGAAAAAAGCACTGTG GCTGATGAAGAAATTGAGGAGCCTGTTGCTGCTAAAACTTCAGATCTTATTGGTGAGACTGTATCA TATGAGTTTCTTGTTAAATGGGTGGATAAATCTAATATTCATAATACTTGGATTTCTGAGGCGGAG CTGAAAGGTCTAGCTAAAAGAAAACTAGAGAACTACAAAGCAAAGTACGGAACAGCTGTAATAAAC ATCTGTGAAGATAAATGGAAACAGCCTCAGCGAATAGTTGCTCTCCGGGTTTCAAAAGAAGGTAAC CAAGAAGCTTACGTAAAGTGGACAGGCTTAGCTTATGATGAATGCACGTGGGAGAGCTTGGAGGAG CCTATTCTTAAACATTCATCCCATTTAATAGATCTTTTTCATCAGTATGAGCAGAAAACATTGGAA AGGAATAGTAAGGGTAATCCCACAAGGGAAAGGGGTGAAGTCGTTACCCTCACAGAACAACCTCAA GAGCTCAGAGGAGGTGCCTTGTTTGCCCATCAGCTTGAGGCTTTGAATTGGCTGCGTAGATGCTGG CATAAATCAAAAAATGTAATACTTGCTGATGAGATGGGGCTTGGAAAAACTGTGTCTGCTAGTGCA TTCCTCTCCTCCCTTTATTTTGAATTTGGAGTTGCAAGACCTTGTTTAGTCCTGGTTCCACTTTCA ACAATGCCAAACTGGCTATCAGAGTTTTCTCTTTGGGCTCCACTCCTTAATGTTGTGGAGTATCAT GGAAGTGCAAAGGGACGAGCCATAATTCGAGACTATGAGTGGCATGCTAAGAATTCTACTGGGACG ACCAAGAAGCCGACATCCTACAAATTTAATGTCCTTTTAACTACTTATGAAATGGTTCTGGCTGAC TCATCTCATCTACGTGGGGTTCCATGGGAAGTTCTTGTGGTTGATGAAGGGCATCGTCTAAAGAAT TCAGAAAGTAAGCTGTTTAGCTTGCTCAACACATTCTCTTTTCAACACCGTGTGCTCTTGACTGGC ACCCCTCTTCAGAATAACATTGGTGAGATGTATAATCTGCTCAACTTCTTGCAACCATCTTCATTC CCTTCTTTGTCTTCTTTTGAGGAGAGGTTCCATGATTTGACAAGTGCTGAGAAAGTAGAAGAACTG AAGAAACTTGTTGCTCCTCATATGCTTCGCCGGCTTAAAAAAGATGCGATGCAGAATATTCCTCCA AAGACAGAGAGGATGGTCCCTGTCGAGTTGACATCGATCCAGGCGGAATATTATCGTGCAATGCTA ACTAAGAACTATCAGATACTACGAAATATCGGAAAAGGGGTAGCGCAACAATCAATGCTTAACATA GTGATGCAGTTGAGAAAGGTTTGCAATCACCCATATCTCATACCAGGTACTGAGCCAGAGTCTGGG TCATTGGAGTTTCTTCACGATATGAGAATAAAAGCGTCAGCCAAGTTGACTCTGTTGCACTCTATG CTTAAGGTGCTACATAAGGAAGGCCATAGAGTTCTGATATTTTCACAGATGACAAAGCTTCTAGAC ATTCTGGAGGACTACCTGAACATAGAATTTGGGCCTAAAACATTTGAAAGGGTAGATGGTTCTGTT GCTGTAGCTGATCGTCAGGCAGCTATAGCACGTTTCAACCAAGACAAAAATCGGTTCGTTTTTCTG TTATCAACTCGTGCCTGTGGTCTTGGTATCAATCTGGCAACAGCTGATACTGTTATTATCTATGAC TCTGATTTCAACCCTCACGCTGATATCCAAGCCATGAATAGAGCTCATCGAATTGGACAGTCCAAA CGACTTTTGGTATACAGACTTGTTGTCCGTGCCAGCGTTGAAGAGCGCATTTTGCAGCTGGCCAAG

AAGAAGTTGATGCTCGATCAGCTTTTTGTAAACAAGTCGGGATCCCAGAAGGAATTTGAAGATATT CTACGCTGGGGTACTGAAGAACTTTTCAACGACTCCGCTGGTGAGAACAAGAAAGATACAGCTGAA AGTAATGGAAACTTAGATGTAATCATGGATTTAGAAAGCAAGAGTAGGAAAAAAGGTGGTGGCCTC GGAGATGTTTATCAAGACAAATGTACAGAAGGAAATGGGAAGATTGTTTGGGATGATATTGCAATT ATGAAGTTGCTTGATCGGTCAAATCTTCAATCTGCCTCCACTGATGCCGCTGATACTGAGTTGGAT AATGATATGCTCGGCTCCGTGAAGCCTGTGGAATGGAATGAGGAAACAGCTGAAGAACAAGTTGGA GCTGAATCACCTGCACTGGTGACTGATGATACTGGTGAACCGAGTTCAGAGAGGAAAGATGATGAT GTCGTTAATTTTACTGAAGAAAATGAATGGGACAGGCTTCTGCGTATGAGGTTGGAGTTCCCTCTT TCTCTGAGTTCAGCGTCTTGGCTTTGGTCTTGGCAGCATATATGGGAGAAATATCAGAGCGAGGAA GAAGCAGCGCTTGGCAGAGGGAAGCGTTTGAGAAAGGCTGTTTCGTATAGGGAAGCATATGCCCCA CATACCAGTGGACCTGTAAATGAGAGTGGTGGTGAAGATGAGAAAGAACCAGAACCAGAACTTAAG AAGGAATATACACCGGCAGGGCGAGCCCTAAAAGAAAAGTTTACCAAACTGCGAGAGAGGCAAAAG AACCTGATTGCGAGAAGGAATTCTGTTGAAGAGTCTCTTCCTAGCGGCAATGTGGATCAGGTAACT GAAGTAGCTAATCAGGACGAAGAAAGCCCTACATCAATGGACTTGGACGATAGCAAAGCTAGCCAG CAATGTGATGCACAGAAAAGAAAAGCCAGTTCTTCAGATCCTAAACCAGATCTTCTAAGCCAACAT CATCATGGCGCAGAATGTCTGCCATCTTTACCCCCAAACAACCTGCCAGTCCTTGGACTGTGTGCT CCTAATTTTACTCAGTCAGAATCCTCCCGGAGAAATTATTCTCGTCCAGGTAGTAGACAGAACAGA CCCATAACAGGACCCCATTTTCCCTTCAATCTACCCCAAACATCGAACTTGGTTGAGAGGGAAGCA AATGACCAGGAACCTCCTATGGGTAAACTAAAACCACAGAACATAAAGGAAGAACCTTTTCAGCAG CCTCTTAGTAATATGGATGGTTGGCTTCCACATCGTCAGTTTCCTCCGTCAGGGGATTTTGAGCGT CCTCGAAGTTCTGGTGCTGCTTTTGCTGATTTCCAGGAGAAGTTTCCGTTGCTTAACCTTCCATTT GATGATAAGCTGCTTCCTCGATTTCCATTTCAGCCGAGAACAATGGGAACTTCGCATCAAGACATA ATGGCCAATCTTTCGATGAGGAAAAGATTTGAAGGTACTGGTCATTCTATGCAAGACCTATTTGGC GGAACACCAATGCCGTTTCTACCCAATATGAAAATCCCTCCTATGGATCCACCTGTCTTCAACCAA CAAGAGAAGGACTTACCGCCTTTGGGTTTGGATCAGTTTCCATCAGCTCTTTCATCTATCCCAGAG AACCATCGAAAGGTGCTGGAGAATATAATGCTAAGAACTGGCTCTGGAATTGGGCACGTACAGAAA AAGAAAACAAGAGTAGATGCATGGTCCGAGGATGAACTAGATTCTCTCTGGATTGGGATTCGCAGA CATGGGTATGGAAACTGGGAGACAATTCTCAGAGATCCAAGGCTCAAATTTTCGAAATTTAAAACA CCAGAGTACTTGGCAGCTAGGTGGGAAGAAGAGCAACGTAAATTCTTGGATAGTCTTTCATCTCTG CCATCTAAATCAAGCAGGACTGATAAGTCCACCAAATCTTCCTTGTTTCCTGGTCTTCCCCAAGGA ATAATGAATCGGGCCTTACATGGTAAATATGCCACTCCTCCAAGGTTCCAATCCCATCTCACAGAC ATAAAACTCGGATTCGGCGATCTAGCATCTCCCCTTCCGTTATTTGAACCATCTGATCACCTGGGA TTTCGAAGTGAGCATTTTCCTCCCATGGCAAATCTGTGCACTGACAATCTTCCCGGGGAGCCTTCT GCTGGACCATCTGAACGAGCAGGGACATCGACAAATATTCCCAACGAGAAGCCTTTTCCACTCAAC TCTCTTGGAATGGGCAACTTAGGTTCATTGGGTTTGGATAGTTTAAGTTCCTTAAACACACTGAGA GCAGAGGAAAAACGGGATGCTATTAAGCGCGGGAAACTACCCTTGTTTTTAGATATGCCGTTACCT CAGATGCTTGATTCAAGCAACAACGTATTCTTGGGAAGATCAGCCAATCCATCTTTCCTTCACCCA AATCGAGGGTTGAATCCCTCCAATCCCATGGGGAGAGACATAATGGGAATTAGCTCTTCAGAGAAC AAGCTACCTCATTGGTTACGGAATGTTGTGACTGTTCCTACCGTGAAGTCACCTGAACCACCCACT CTACCTCCAACTGTGTCAGCTATAGCTCAATCAGTCCGCGTTTTΆTATGGTGAAGACTCTACAACC ATTCCACCGTTTGTGATACCAGAGCCGCCACCTCCTGCTCCCAGAGATCCAAGACACAGTCTGCGT AAGAAAAGGAAACGTAAATTGCATTCATCGAGTCAAAAGACTACAGACATTGGTAGTAGCAGCCAC AATGCTGTAGAAAGCAGCTCTCAAGGCAATCCACAAACATCTGCAACTCCTCCTTTGCCTCCACCG TCTCTGGCGGGTGAAACTTCAGGGTCTTCTCAACCCAAATTACCTCCTCACAACCTAAATAGCACA GAACCACTGTCCTCTGAAGCAATCATAATTCCACCACCTGAAGAAGATTCTGTGATAGCAGCAGCG CCATCTGAAGCACCAGGGCCTAGTCTAGAGGGAATCACTGGTACAACAAAGTCAATCTCGCTAGAG AGCCAAAGCTCTGAACCAGAAACTATTAATCAAGATGGAGACTTAGATCCAGAAACTGATGAGAAA GTTGAGTCTGAACGAACCCCGCTTCATTCAGATGAGAAACAAGAGGAGCAAGAATCTGAAAATGCA TTGAACAAGCAGTGTGAGCCCATAGAGGCTGAAAGTCAAAACACCAATGCAGAAGAAGAAGCAGAG GCACAAGAAGAAGATGAAGAATCCATGAAGATGGTGACTGGTAATTCTCTTAGTGACGACTGA

PKR 1 - amino acid sequence (SEQ ID NO: 2) - Arabidopsis thaliana (2242 aa) MKDSGSEMIKRDWVMKQKRRKLPSILDILDQKVDSSMAFDSPEYTSSSKPSKQRLKTDSTPERNSS KRKGNDGNYFECVICDLGGDLLCCDSCPRTYHTACLNPPLKRIPNGKWICPKCSPNSEALKPVNRL DAIAKRARTKTKKRNSKAGPKCERASQIYCSSIISGEQSSEKGKSISAEESKSTGKEVYSSPMDGC STAELGHASADDRPDSSSHGEDDLGKPVIPTADLPSDAGLTLLSCEDLSESKLSDTEKTHEAPVEK LEHASSEIVENKTVAEMETGKGKRKKRKRELNDGESLERCKTDKKRAKKSLSKVGSSSQTTKSPES SKKKKKKNRVTLKSLSKPQSKTETPEKVKKLPKEERRAVRATNKSSSCLEDTNSLPVGNLQVHRVL GCRIQGLTKTSLCSALSDDLCSDNLQATDQRDSLVQDTNAELVVAEDRIDSSSETGKSSRDSRLRD KDMDDSALGTEGMVEVKEEMLSEDISNATLSRHVDDEDMKVSETHVSVERELLEEAHQETGEKSTV ADEEIEEPVAAKTSDLIGETVSYEFLVKWVDKSNIHNTWISEAELKGLAKRKLENYKAKYGTAVIN ICEDKWKQPQRIVALRVSKEGNQEAYVKWTGLAYDECTWESLEEPILKHSSHLIDLFHQYEQKTLE RNSKGNPTRERGEVVTLTEQPQELRGGALFAHQLEALNWLRRCWHKSKNVILADEMGLGKTVSASA FLSSLYFEFGVARPCLVLVPLSTMPNWLSEFSLWAPLLNVVEYHGSAKGRAIIRDYEWHAKNSTGT TKKPTSYKFNVLLTTYEMVLADSSHLRGVPWEVLVVDEGHRLKNSESKLFSLLNTFSFQHRVLLTG TPLQNNIGEMYNLLNFLQPSSFPSLSSFEERFHDLTSAEKVEELKKLVAPHMLRRLKKDAMQNIPP KTERMVPVELTSIQAEYYRAMLTKNYQILRNIGKGVAQQSMLNIVMQLRKVCNHPYLIPGTEPESG SLEFLHDMRIKASAKLTLLHSMLKVLHKEGHRVLIFSQMTKLLDILEDYLNIEFGPKTFERVDGSV AVADRQAAIARFNQDKNRFVFLLSTRACGLGINLATADTVIIYDSDFNPHADIQAMNRAHRIGQSK RLLVYRLVVRASVEERILQLAKKKLMLDQLFVNKSGSQKEFEDILRWGTEELFNDSAGENKKDTAE SNGNLDVIMDLESKSRKKGGGLGDVYQDKCTEGNGKIWDDIAIMKLLDRSNLQSASTDAADTELD NDMLGSVKPVEWNEETAEEQVGAESPALVTDDTGEPSSERKDDDVVNFTEΞNEWDRLLRMRLEFPL SLSSASWLWSWQHIWEKYQSEEEAALGRGKRLRKAVSYREAYAPHTSGPVNESGGEDEKEPEPELK KEYTPAGRALKEKFTKLRERQKNLIARRNSVEESLPSGNVDQVTEVANQDEESPTSMDLDDSKASQ QCDAQKRKASSSDPKPDLLSQHHHGAECLPSLPPNNLPVLGLCAPNFTQSESSRRNYSRPGSRQNR PITGPHFPFNLPQTSNLVEREANDQEPPMGKLKPQNIKEEPFQQPLSNMDGWLPHRQFPPSGDFER PRSSGAAFADFQEKFPLLNLPFDDKLLPRFPFQPRTMGTSHQDIMANLSMRKRFEGTGHSMQDLFG GTPMPFLPNMKIPPMDPPVFNQQEKDLPPLGLDQFPSALSSIPENHRKVLENIMLRTGSGIGHVQK KKTRVDAWSEDELDSLWIGIRRHGYGNWETILRDPRLKFSKFKTPEYLAARWEEEQRKFLDSLSSL PSKSSRTDKSTKSSLFPGLPQGIMNRALHGKYATPPRFQSHLTDIKLGFGDLASPLPLFEPSDHLG FRSEHFPPMANLCTDNLPGEPSAGPSERAGTSTNIPNEKPFPLNSLGMGNLGSLGLDSLSSLNTLR AEEKRDAIKRGKLPLFLDMPLPQMLDSSNNVFLGRSANPSFLHPNRGLNPSNPMGRDIMGISSSEN KLPHWLRNVVTVPTVKSPEPPTLPPTVSAIAQSVRVLYGEDSTTIPPFVIPEPPPPAPRDPRHSLR KKRKRKLHSSSQKTTDIGSSSHNAVESSSQGNPQTSATPPLPPPSLAGETSGSSQPKLPPHNLNST EPLSSEAIIIPPPEEDSVIAAAPSEAPGPSLEGITGTTKSISLESQSSEPETINQDGDLDPETDEK VESERTPLHSDEKQEEQESENALNKQCEPIEAESQNTNAEEEAEAQEEDEESMKMVTGNSLSDD References: The contents of the entirety of each of which are incorporated by this reference.

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Other advantages that are inherent to the structure are obvious to one skilled in the art. The embodiments are described herein illustratively and are not meant to limit the scope of the invention as claimed. Variations of the foregoing embodiments will be evident to a person of ordinary skill and are intended by the inventor to be encompassed by the following claims.

Claims

Claims:

1. A method of increasing seed oil content, decreasing abscisic acid sensitivity and/or increasing drought resistance in a plant comprising: introducing into the plant means for encoding a PICKLE-RELATED1 (PKR1) protein to thereby increase expression of PKR 1 protein in the plant to thereby increase seed oil content, decrease abscisic acid sensitivity and/or increase drought resistance in the plant compared to a plant grown under similar conditions in which the means for encoding the PKR1 protein has not been introduced.

2. The method according to claim 1 , wherein the PKR1 protein has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a conservatively substituted amino acid sequence thereof.

3. The method according to claim 1 , wherein the PKR1 protein has an amino acid sequence as set forth in SEQ ID NO: 2 or a conservatively substituted amino acid sequence thereof.

4. The method according to any one of claims 1 to 3, wherein the means for encoding comprises a nucleic acid molecule having a nucleotide sequence having at least 80% sequence identity to SEQ ID NO: 1 or a codon degenerate nucleotide sequence thereof.

5. The method according to claim 4, wherein the nucleic acid molecule has a nucleotide sequence as set forth in SEQ ID NO: 1 or a codon degenerate nucleotide sequence thereof.

6. The method according to any one of claims 1 to 5, wherein the plant is from family Brassicaceae.

7. A nucleic acid construct comprising means for encoding a PICKLE-RELATED1 (PKR1) protein operably linked to one or more nucleic acid sequences required for transforming the construct into a cell and/or for expressing or overexpressing the PKR1 protein encoding means in the cell.

8. The construct according to claim 7, wherein the PKR1 protein has an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 2 or a conservatively substituted amino acid sequence thereof.

9. The construct according to claim 7, wherein the PKR1 protein has an amino acid sequence as set forth in SEQ ID NO: 2 or a conservatively substituted amino acid sequence thereof.

10. A cell, seed or plant comprising the nucleic acid construct as defined in claim 7.