WO2013190322A1

WO2013190322A1 - Regulation of seed dormancy

Info

Publication number: WO2013190322A1
Application number: PCT/GB2013/051637
Authority: WO
Inventors: Steven Penfield; Sarah KENDALL; Dana MACGREGOR
Original assignee: The University Of Exeter
Priority date: 2012-06-22
Filing date: 2013-06-21
Publication date: 2013-12-27
Also published as: GB201211079D0

Abstract

The disclosure relates to means for increasing seed vigour by providing a plant that expresses variants of ubiquitin E3 ligases and optionally variants of an ICE1 transcription factor.

Description

Regulation of Seed Dormancy

Field of the Invention

The disclosure relates to means for increasing seed vigour by providing a plant that expresses variants of ubiquitin E3 ligases and optionally variants of an ICE1 transcription factor in crop seeds and plants, and to crop plants and seeds comprising genetic variations of said gene(s) resulting in reduced/increased expression. Methods enabling the identification of plants with modified expression of a ubiquitin E3 ligase and/or an ICE1 transcription factors.

Background of the Invention Agriculture is extremely vulnerable to environmental factors. Crop yields are significantly affected by increasing temperatures and adverse weather conditions, and with an ever increasing human population, methods are urgently required to permit efficient use of agricultural land guaranteeing high yields and so providing global food security for future generations. Hence, plant breeders continuously strive to develop crop varieties which are less susceptible to environmental factors such as drought or pests to increase yield and prevent losses.

A key feature of plant adaptive fitness is the ability to synchronise the onset of vegetative and reproductive development with seasonal changes in the environment. The commencement of vegetative development is controlled by a period of quiescence in the mature seed known as seed dormancy. During dormancy, seed germination does not occur and the period of dormancy of many plant seeds is terminated by environmental signals including light, temperature and nutrient availability, a system adapted to the promotion of germination only when conditions are optimal for seedling establishment and reproductive success. In particular the role of light and temperature in the promotion of germination in dormant seeds is highly conserved among seed plants from angiosperms to gymnosperms, demonstrating the importance of germination control as a vital adaptive trait in plants.

Dormancy and seed germination is a hormonally regulated process and the major players are two phyto-hormones, abscisic (ABA) and giberrelic acid (GA). ABA synthesis and GA catabolism is known to promote dormancy whilst GA synthesis and ABA catabolism stimulate dormancy breaking. Genetic screens have also identified a number of loci such as ABI3, FUS3, LEC1 , DOG1 , RD04, MFT and FLC important in dormancy regulation. A role for the floral repressor FLC in regulating dormancy at low temperatures has been proposed as dormancy levels were reduced in seeds expressing high levels of FLC that were matured l at 10 °C, and expression levels of FLC were positively correlate with germination levels when seeds were imbibed at low temperature. The increase in germination was linked to increased expression of the cytochrome P450 CYP707A2 and GA20ox1 expression, suggesting that levels of ABA and GA may be altered in these seeds. Low dormancy levels, a desired trait in commercially available crops, ensure fast and uniform germination and seedling establishment which can lead to high crop yields. However, premature loss of dormancy, known as pre-harvest sprouting can reduce the grain quality of crops significantly resulting in substantial economic losses. Temperature is an important factor in germination and plants respond to temperature in different ways as cold can promote germination in one species but have inhibitory effects in another one. Many annual plants require periods of cold to break dormancy and allow germination. However, the role of temperature in regulating seed dormancy and germination are not well understood.

Plants respond to cold stress by inducing a variety of genes which enables the plant to survive this adverse condition. In the model plant Arabidopsis three transcription factors known as C-repeat (CRT)-binding factor (CBF1 -3) activate many of the cold responsive genes, and expression of CBF increases transiently with exposure to low temperatures. Inducer of CBF expression 1 (ICE1 ) controls the cold induction of CBF3, and HOS1 (high expression of osmotically responsive gene 1 ), a protein with ubiquitin E3 ligase activity, was found to negatively regulate the CBF regulon. Loss of function hosl mutant plants showed enhanced cold induction of CBFs, and overexpression of CBFs confers freezing tolerance. HOS1 overexpression confers increased sensitivity to freezing stress.

US2002/0148008 discloses the development of genetically modified wheat seed, in which the expression levels of Viviparousl (VP1 ) are modulated to regulate seed dormancy. VP1 is a transcriptionally regulated gene essential for formation of seed dormancy; it is a transcription factor which acts in the ABA signalling system. Site-directed mutagenesis of the gene results in a protein which comprises an amino acid sequence having deletions, substitutions or additions. By introducing mutants of this gene into varieties of wheat, the expression of its protein can be altered, consequently the degree of ABA-sensitivity of the seeds in those varieties can be altered and thus the degree of dormancy of the seed.

WO02/077163 describes the over expression of the gene ABI5 in plants such as Arabidopsis, to prevent precocious seed germination. ABI5 encodes a putative transcription factor of the basic leucine zipper (bZIP) family. ABI5 has been shown to confer an enhanced response to exogenous ABA during germination. As a key component in ABA-triggered processes, ABI5 protein accumulation, phosphorylation, stability and activity are highly regulated by ABA during germination and early seedling growth. Plants which over express ABI5 are hypersensitive to ABA and therefore respond to very low levels of this phytohormone, which would have no effect on wild type plants.

We disclose that a reduction in expression levels or deletion of a functional HOS1 locus (or an increase in ICE1 expression or modification of ICE1 loci to confer constitutive gain-of- function) confers high seed vigour even when temperatures are decreased during seed development. This phenotype is maternally inherited and therefore seeds with high seed vigour can be produced for growing the crop; however, if the same plant is crossed with a plant containing paternal alleles that confer low vigour, this then provides crops that produce seeds with low vigour, preventing pre-harvest germination.

The disclosure relates to the identification of alleles of HOS1 and ICE1 for the identification of seed with high seed vigour, but minimising pre-harvest sprouting. Methods for the production of such seeds and plants are also disclosed as the variation in the environment during seed production can have profound effects on the quality of seed produced for sale. In many cases the mother plant has an important role in sensing the environment and using the information to modify the behaviour of seeds. By understanding the molecular mechanisms through which the mother plant controls seed quality we can devise new strategies for the control of seed quality through the use of novel alleles in fast track breeding programmes. Here we used a genetic screen to identify Arabidopsis mutants with high seed vigour that is insensitive to the seed production environment. This showed that 3 alleles of the locus known previously as HIGH EXPRESSION OF OSMOTICALLY SENSTIVE GENES 1 (HOS1 ) showed high seed vigour even when temperature is reduced during seed development, a condition that causes low germination in wild type seeds. Importantly, genetic studies show that HOS1 acts to control seed behaviour from the maternal genome (Figure 2). This conclusion is further supported by experiments with applied hormones that control the seed germination process. Hos1 mutant seeds show normal responses to these hormones, suggesting that HOS1 does not act directly in seed tissues to control dormancy. Our work suggests that hosl may be a useful maternal genotype for the production of high quality seed, and for high quality F1 hybrid seed. In this way F1 seed could be produced with high vigour in multiple seed production environments, but the seed vigour of the crop is left unaltered. This may have uses when low crop seed vigour is desirable, for instance to prevent pre-harvest sprouting.

Statements of the Invention

According to an aspect of the invention there is provided a plant wherein said plant is not of the genus Arabidopsis and further wherein said plant expresses a nucleotide sequence variant of a gene comprising a nucleotide sequence that encodes a polypeptide that has ubiquitin E3 ligase activity and which plant produces seed that has increased vigour and/or low seed dormancy.

Seed vigour is defined as the ability of the seed to germinate at speed and establish with high frequency to form healthy strong-growing stands of plants under favourable and also less favourable environmental conditions. Seed as herein described with improved/increased seed vigour have at the time of germination a reduced response to sub-opitmal conditions such as temperature fluctuations, but still germinating efficiently and producing healthy seedlings. Low seed dormancy is an important determinant of vigour and low dormant seeds are less dependent of environmental factors which are, depending on the plant species, typically required to promote rapid germination.

In a preferred embodiment of the invention said sequence variant encodes an ubiquitin E3 ligase with reduced enzyme activity.

In an alternative preferred embodiment of the invention said plant is modified to provide an inactive ubiquitin E3 ligase.

In a preferred embodiment of the invention said ubiquitin E3 ligase is encoded by a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46;

a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);

iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 , 2, 10 or 19 wherein said nucleic acid molecule encodes a ubiquitin E3 ligase;

iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 49, 50, 51 , 52, 53, 55, 56, 57, 58, 59, 60, 61 , 62, 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 or 90;

a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 47, 48, 54 or 63 wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and that has ubiquitin E3 ligase activity. Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001 ); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology— Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The T_m is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:

Very High Stringency (allows sequences that share at least 90% identity to hybridize) Hybridization: 5x SSC at 65°C for 16 hours

Wash twice: 2x SSC at room temperature (RT) for 15 minutes each

Wash twice: 0.5x SSC at 65°C for 20 minutes each

High Stringency (allows sequences that share at least 80% identity to hybridize)

Hybridization : 5x-6x SSC at 65°C-70°C for 16-20 hours Wash twice: 2x SSC at RT for 5-20 minutes each

Wash twice: 1 x SSC at 55°C-70°C for 30 minutes each

Low Stringency (allows sequences that share at least 50% identity to hybridize)

Hybridization: 6x SSC at RT to 55°C for 16-20 hours

Wash at least twice: 2x-3x SSC at RT to 55°C for 20-30 minutes each. A modified polypeptide as herein disclosed may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination with reference to SEQ ID NO: 47, 48, 54 or 63. Preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid by another amino acid of like characteristics.

In one embodiment, the variant polypeptides have at least 30% identity, even more preferably at least 35% identity, still more preferably at least, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% identity, and at least 99% identity with most or the full length amino acid sequence illustrated herein.

In a preferred embodiment of the invention said ubiquitin E3 ligase is encoded by a nucleotide sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46.

In a preferred embodiment of the invention said plant has enhanced expression of a variant gene comprising a nucleotide sequence that encodes an ICE1 transcription factor that regulates the expression of one or more transcription factors.

In a preferred embodiment of the invention said ICE1 transcription factor comprises a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);

iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 91 , 92, 101 , 104 or 105 wherein said nucleic acid molecule encodes a transcription factor that regulates the expression of one or more transcription factors;

iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 109, 1 10, 1 1 1 , 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122, 123, 124 or 125;

v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has transcription factor activity that regulates the expression of a transcription factor. In a preferred embodiment of the invention said transcription factor is encoded by a nucleotide sequence as set forth in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108.

In a preferred embodiment of the invention said plant is a transgenic plant engineered to reduce or abrogate the expression of said ubiquitin E3 ligase activity and optionally is engineered to enhance the expression of a transcription factor. In a preferred embodiment of the invention said transgenic plant is transformed with a transcription cassette wherein said cassette encodes all or part of a nucleotide sequence that encodes an ubiquitin E3 ligase and is adapted for expression by provision of at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette.

In a preferred embodiment of the invention said cassette is adapted such that both sense and antisense ribonucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part or all of their length to form a inhibitory RNA or short hairpin RNA. A technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as small inhibitory/interfering RNA (siRNA) or short hairpin RNA [shRNA], into a cell which results in the destruction of mRNA complementary to the sequence included in the siRNA/shRNA molecule. The siRNA molecule comprises two complementary strands of RNA (a sense strand and an antisense strand) annealed to each other to form a double stranded RNA molecule. The siRNA molecule is typically derived from exons of the gene which is to be ablated. The mechanism of RNA interference is being elucidated. Many organisms respond to the presence of double stranded RNA by activating a cascade that leads to the formation of siRNA. The presence of double stranded RNA activates a protein complex comprising RNase III which processes the double stranded RNA into smaller fragments (siRNAs, approximately 21 -29 nucleotides in length) which become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave mRNA complementary to the antisense strand of the siRNA thereby resulting in destruction of the mRNA.

In an alternative preferred embodiment of the invention said cassette is adapted to express an antisense RNA wherein said antisense RNA is adapted to anneal to a mRNA sequence that encodes a ubiquitin E3 ligase.

In a preferred embodiment of the invention said inhibitory RNA or antisense RNA is designed with reference to a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46 ;

a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 , 2, 10 or 19 wherein said nucleic acid molecule encodes a ubiquitin E3 ligase.

In a preferred embodiment of the invention said cassette is part of an expression vector adapted for expression in a plant cell.

In a preferred embodiment of the invention said transcription factor is encoded by a nucleotide sequence which is part of an expression vector wherein said nucleotide sequence is operably linked to a nucleotide sequence comprising a transcription promoter. In a preferred embodiment of the invention said promoter confers constitutive expression on said transcription factor.

In an alternative preferred embodiment of the invention said promoter confers regulated expression on said transcription factor.

In a preferred embodiment of the invention said regulated expression is tissue or developmental^ regulated expression.

In a further alternative embodiment of the invention said regulated expression is inducible expression.

Preferably the nucleic acid molecule in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a plant cell. The vector may be a bi-functional expression vector which functions in multiple hosts. By "promoter" is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in plant cells comprised in plants depending on design. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.

Constitutive promoters include, for example CaMV 35S promoter (Odell et al. (1985) Nature 313, 9810-812); rice actin (McElroy et al. (1990) Plant Cell 2: 163-171 ); ubiquitin (Christian et al. (1989) Plant Mol. Biol. 18 (675-689); pEMU (Last et al. (1991 ) Theor Appl. Genet. 81 : 581 -588); MAS (Velten et al. (1984) EMBO J. 3. 2723-2730); ALS promoter (U.S. Application Seriel No. 08/409,297), and the like. Other constitutive promoters include those in U.S. Patent Nos. 5,608,149; 5,608,144; 5,604,121 ; 5,569,597; 5,466,785; 5,399,680, 5,268,463; and 5,608,142, each of which is incorporated by reference.

Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter, where application of the chemical induced gene expression, or a chemical-repressible promoter, where application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize ln2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre-emergent herbicides, and the tobacco PR-1 a promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, for example, the glucocorticoid-inducible promoter in Schena et al. (1991 ) Proc. Natl. Acad. Sci. USA 88: 10421 -10425 and McNellis et al. (1998) Plant J. 14(2): 247-257) and tetracycline-inducible and tetracycline-repressible promoters (see, for example, Gatz et al. (1991 ) Mol. Gen. Genet. 227: 229-237, and US Patent Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

Where enhanced expression in particular tissues is desired, tissue-specific promoters can be utilised. Tissue-specific promoters include those described by Yamamoto et al. (1997) Plant J. 12(2): 255-265; Kawamata et al. (1997) Plant Cell Physiol. 38(7): 792-803; Hansen et al. (1997) Mol. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157- 168; Rinehart et al. (1996) Plant Physiol. 1 12(3): 1331 -1341 ; Van Camp et al. (1996) Plant Physiol. 1 12(2): 525-535; Canevascni et al. (1996) Plant Physiol. 1 12(2): 513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35(5): 773-778; Lam (1994) Results Probl. Cell Differ. 20: 181 -196; Orozco et al. (1993) Plant Mol. Biol. 23(6): 1 129-1 138; Mutsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90 (20): 9586-9590; and Guevara-Garcia et al (1993) Plant J. 4(3): 495-50.

"Operably linked" means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is "under transcriptional initiation regulation" of the promoter. In a preferred aspect, the promoter is a tissue specific promoter, an inducible promoter or a developmental^ regulated promoter.

Particular of interest in the present context are nucleic acid constructs which operate as plant vectors. Specific procedures and vectors previously used with wide success in plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121 -148. Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). If desired, selectable genetic markers may be included in the construct, such as those that confer selectable phenotypes such as resistance to herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

According to a further aspect of the invention there is provided a plant according to the invention for use in the improvement of seed vigour and/or seed dormancy.

According to a further aspect of the invention there is provided a method for the preparation of F1 hybrid seed and the formation of an F1 hybrid plant comprising the steps: i) providing a first male sterile female plant cultivar wherein said plant encodes a plant ubiquitin E3 ligase variant and crossing said plant with a second male plant cultivar; and

ii) obtaining F1 hybrid seed from said cross wherein said seed has improved seed vigour and/or low seed dormancy.

In a preferred method of the invention said ubiquitin E3 ligase variant is inactive or has reduced enzyme activity.

In a preferred method of the invention said male sterile female plant is genetically male sterile.

In an alternative method of the invention said male sterile female plant is treated with an agent that induces male sterility.

Improving crop yield by crossing two inbred plant lines is a common plant breeding technique, which can result in heterosis. Male plant sterility, the inability to produce viable pollen and so inhibit self-crossing, is often necessary to produce F1 hybrids efficiently on an economical scale. There are several methods to introduce plant male sterility such as chemical hybridising agents such as 2-chloroethylphosphonic acid, sodium 1 -(p- chlorophenyl)-1 ,2-dihydro-4,6-dimethyl-2-oxonicotinate,3-(p-chlorophenyl)-6-methoxy-s- triazine-2,4 (1 H,3H) dione-triethanolamine, 2,7-diamino-10-ethyl-6-phenylantridium bromide, or utilising plant lines comprising genes causing cytoplasmic male sterility, genetic male sterility or cytoplasmic-genetic male sterility. According to a further aspect of the invention there is provided the use of a gene comprising a nucleic acid molecule that encodes a plant ubiquitin E3 ligase as a means to identify a plant with altered expression of said ubiquitin E3 ligase wherein said plant has improved seed vigour and/or seed dormancy.

According to a further aspect of the invention there is provided a method to produce a plant that has altered expression of an ubiquitin E3 ligase comprising the steps of: i) mutagenesis of wild-type seed from a plant that expresses a ubiquitin E3 ligase;

ii) cultivation of the seed in i) to produce first and subsequent generations of plants;

iii) obtaining seed from the first generation plant and subsequent generations of plants;

iv) determining if the seed from said first and subsequent generations of plants has altered nucleotide sequence and/or altered expression of a ubiquitin E3 ligase polypeptide variant;

v) obtaining a sample and analysing the nucleic acid sequence of a nucleic acid molecule that encodes said ubiquitin E3 ligase variant; and optionally vi) comparing the nucleotide sequence of the nucleic acid molecule that encodes an ubiquitin E3 ligase in said sample to a nucleotide sequence of a nucleic acid molecule of the original wild-type plant.

In a preferred method of the invention said nucleic acid molecule is analysed by a method comprising the steps of: i) extracting nucleic acid from said mutated plants;

ii) amplification of a part of said nucleic acid molecule by a polymerase chain reaction;

iii) forming a preparation comprising the amplified nucleic acid and nucleic acid extracted from wild-type seed to form heteroduplex nucleic acid; iv) incubating said preparation with a single stranded nuclease that cuts at a region of heteroduplex nucleic acid to identify the mismatch in said heteroduplex; and

v) determining the site of the mismatch in said nucleic acid heteroduplex. Mutagenesis as a means to induce phenotypic changes in organisms is well known in the art and includes but is not limited to the use of mutagenic agents such as chemical mutagens [e.g. base analogues, deaminating agents, DNA intercalating agents, alkylating agents, transposons, bromine, sodium azide] and physical mutagens [e.g. ionizing radiation, psoralen exposure combined with UV irradiation].

In a preferred method of the invention said plant encodes an ubiquitin E3 ligase variant that has reduced ubiquitin E3 ligase expression and/or activity.

In a preferred method of the invention said ubiquitin E3 ligase is encoded by a nucleotide sequence selected from the group: i) a nucleotide sequence as represented by the sequence in SEQ ID

NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46 ;

ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 , 2, 10 or 19wherein said nucleic acid molecule encodes a ubiquitin E3 ligase.

In an alternative method of the invention said plant is analysed to determine expression of an ICE1 transcription factor variant that regulates expression of one or more transcription factors.

In a preferred method of the invention said transcription factor is selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108; ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);

iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 91 , 92, 101 , 104 or 105 wherein said nucleic acid molecule encodes a transcription factor that regulates the expression of one or more transcription factors. According to a further aspect of the invention there is provided a screening method that detects the expression of an ubiquitin E3 ligase and/or an ICE1 transcription factor in a plant comprising the steps: i) providing an isolated sample from a plant to be assessed for expression and extracting nucleic acid from said sample;

ii) preparing cDNA from said extracted nucleic acid; iii) forming a preparation comprising said cDNA and an oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising a nucleotide sequence that encodes a ubiquitin E3 ligase and/or an ICE1 transcription factor; a thermostable DNA polymerase, deoxynucleotide triphosphates and co -factors; iv) providing polymerase chain reaction conditions sufficient to amplify all or part of said nucleic acid molecule; v) analysing the amplified products of said polymerase chain reaction for the presence of a nucleic acid molecule comprising a nucleotide sequence that encodes an ubiquitin E3 ligase and/or an ICE1 transcription factor; and optionally vi) comparing the amplified product with a normal matched control.

In a preferred method of the invention said ubiquitin E3 ligase is encoded by a nucleotide sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46.

In an alternative preferred method of the invention said ICE1 transcription factor is encoded by a nucleotide sequence as set forth in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108

In a preferred method of the invention said method is a real time PCR method for the detection and quantification of a nucleic acid encoding all or part of a nucleotide sequence that encodes said ubiquitin E3 ligase and/or an ICE1 transcription factor. According to an alternative aspect of the invention there is provided a screening method that detects the expression of an ubiquitin E3 ligase and/or an ICE1 transcription factor in a plant comprising the steps i) providing an isolated sample from a plant to be assessed for expression;

ii) forming a preparation comprising said sample and an antibody, or antibodies, that specifically bind an ubiquitin E3 ligase and/or ICE1 transcription factor polypeptide in said sample to form an antibody/polypeptide complex;

iii) detecting the complex; and

iv) comparing the expression of said polypeptide^] with a normal matched control.

In a preferred method of the invention said ubiquitin E3 ligase polypeptide is represented by the amino acid sequence set forth in SEQ ID NO: 49, 50, 51 , 52, 53, 55, 56, 57, 58, 59, 60, 61 , 62, 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 or 90.

In a preferred method of the invention said ICE1 transcription factor polypeptide is represented by the amino acid sequence set forth in SEQ ID NO: 109, 1 10, 1 1 1 , 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122, 123, 124 or 125.

In a preferred method of the invention said isolated plant samples comprise an array of samples isolated from a plurality of plants to be tested for expression of said ubiquitin E3 ligase and/or ICE1 transcription factor nucleic acid or polypeptide.

In a preferred method of the invention said method further includes DNA sequencing of genomic DNA isolated from a plant variant obtained by said method[s] to determine a variant sequence encoding or controlling expression of a ubiquitin E3 ligase variant.

In a further preferred method of the invention said method further includes DNA sequencing of genomic DNA isolated from a plant variant obtained by the method[s] to determine the variant sequence encoding or controlling expression of a ICE1 transcription factor variant. It will be apparent that the methods of the invention will generate novel sequence variants of ubiquitin E3 ligase and ICE1 transcription factor genomic sequences with altered expression and/or activity. The sequence variants may be modifications to the coding sequences to create variant proteins with altered activity. The sequence variants may be modifications to expression control sequences [i.e. promoter sequences] which alter the expression of the respective genes.

According to a further aspect of the invention there is provided a plant obtained by the method according to the invention. According to an aspect of the invention there is provided a plant wherein said plant comprises a viral vector that includes all or part of a gene comprising a nucleotide sequence that encodes an ubiquitin E3 ligase.

In a preferred embodiment of the invention said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 3, 4,

5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46 ;

ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i);

iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 , 2, 10 or 19wherein said nucleic acid molecule encodes a ubiquitin E3 ligase.

According to a further aspect of the invention there is provided a viral vector comprising all or part of a nucleic acid molecule according to the invention. According to an aspect of the invention there is provided the use of a viral vector according to the invention in viral induced gene silencing of a plant ubiquitin E3 ligase.

Virus induced gene silencing [VIGS] is known in the art and exploits a RNA mediated antiviral defence mechanism. Plants that are infected with an unmodified virus induces a mechanism that specifically targets the viral genome. However, viral vectors which are engineered to include nucleic acid molecules derived from host plant genes also induce specific inhibition of viral vector expression and additionally target host mRNA. This allows gene specific gene silencing without genetic modification of the plant genome and is essentially a non-transgenic modification.

The invention is typically applicable to crop plants and includes cereals, oils seed plants, fruits and ornamentals.

In particular, the invention is applicable to, for example, corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avacado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.

Preferably, plants of the present invention are crop plants (for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops). Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum.

Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, rye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.

Throughout the description and claims of this specification, the words "comprise" and "contain" and variations of the words, for example "comprising" and "comprises", means "including but not limited to", and is not intended to (and does not) exclude other moieties, additives, components, integers or steps. "Consisting essentially" means having the essential integers but including integers which do not materially affect the function of the essential integers.

Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

An embodiment of the invention will now be described by example only and with reference to the following figures: Figure 1 shows that in Arabidopsis thaliana, hosl mutants have a strong germination vigour even when the temperature during seed production is lowered and the germination of wild type seeds is strongly inhibited. Therefore deleting HOS1 in plants causes germination vigour that is less sensitive to the temperature conditions during seed production;

Figure 2 shows that HOS1 acts through the genome of the mother plant to control seed vigour;

Figure 3 shows that hormone sensitivity in mature imbibed seeds is not very different in hosl mutants compared to wild type. This is consistent with HOS1 acting during seed production to control vigour;

Figure 4 shows that the seed coats of hosl mutants are more permeable to the metabolic dye tetrazolium. Permeability is known to be controlled by the biochemistry of tannins in the seed coat which is a tissue derived from the mother plant;

Figure 5 shows that the gene expression of enzymes in the tannin biosynthetic pathway is lower in hosl mutants than wild type, and that decreasing the temperature during seed production increases seed tannin biosynthetic gene expression. This is consistent with temperature and HOS1 acting to control seed coat biochemistry. Because seed coat tannin content is known to be associated with vigour, this represents a mechanism trherough which HOS1 may control seeed vigour from the maternal genome;

Figure 6 describes the germination vigour of mutants of the Arabidopsis thaliana ICE1 locus. The HOS1 protein is known to interact physically with ICE1 in plant cells, and hosl mutants are known to have increased levels of the ICE1 protein. ICE1 loss-of-function mutants have low germination vigour and icel gain-of-function mutants have high vigour. This data is consistent with the control of seed vigour by HOS1 being mediated by ICE1 ; and Figure 7 shows that icel loss-of-function mutants have visibly darkened seed coats, consistent with increased tannin levels.

Materials and Methods

Seed dormancy assays Seeds were produced from plants growing at ^~\ 6°C in a Percival growth cabinet under long days in white light. Germination of mature seeds took place at 22 °C on water agar plates in white light for 7 days. Data shown for germination experiments are mean and standard errors of five biological replicate seed batches (from 5 individual plants).

Hormone sensitivity assays Seeds were sown on water agar plates with the indicated concentration of abscisic acid (ABA) or the gibberellin biosynthesis inhibitor paclobutrazol (PAC; Greyhound Chromatography, Liverpool, UK). Seeds were then cold stratified for 3 days before germinating at 22 °C for 7 days and scoring germination freqeuency.

Tetrazolium assays The tetrazolium staining protocol was based on Debeaujon et al. (2000 dx.doi. org/10.1 104/pp.122.2.403). Freshly harvested seeds were incubated in water or an 1 % (w/v) aqueous solution of 2,3,5-Triphenyltetrazolium chloride (Sigma-Aldrich cat# T8877- 5G) in 96-well plates in the dark at 30 °C. At 24, 48 and 72 hours after the start of incubation, aliquots of seed were transferred to acetate paper, the liquid was removed from them and the seeds were scanned using a tabletop scanner at 12000 pixel resolution.

RNA-seq data

RNA was harvested from green cotyledon stage Arabidopsis seeds and first strand cDNA synthesised using standard methods. Sequencing took place on a illumina hiseq platform, data was processed by TopHat and Cuff Links with reference to the Arabidopsis TAIR8 genome annotation. Reads numbers were normalised by transcript length to provide the indicated expression values.

Examples

Figure 1 shows that in Arabidopsis thaliana, hosl mutants have a strong germination vigour even when the temperature during seed production is lowered and the germination of wild type seeds is strongly inhibited. Therefore deleting HOS1 in plants causes germination vigour that is less sensitive to the temperature conditions during seed production. Figure 2 shows that HOS1 acts through the genome of the mother plant to control seed vigour. Figure 3 shows that hormone sensitivity in mature imbibed seeds is not very different in hosl mutants compared to wild type. This is consistent with HOS1 acting during seed production to control vigour. Figure 4 shows that the seed coats of hosl mutants are more permeable to the metabolic dye tetrazolium. Permeability is known to be controlled by the biochemistry of tannins in the seed coat which is a tissue derived from the mother plant. Figure 5 shows that the gene expression of enzymes in the tannin biosynthetic pathway is lower in hosl mutants than wild type, and that decreasing the temperature during seed production increases seed tannin biosynthetic gene expression. This is consistent with temperature and hosl acting to control seed coat biochemistry. Because seed coat tannin content is known to be associated with vigour, this represents a mechanism trherough which HOS1 may control seeed vigour fromt he maternal genome. Figure 6 describes the germination vigour of mutants of the Arabidopsis thaliana ICE1 locus. The HOS1 protein is known to interact physically with ICE1 in plant cells, and hosl mutants are known to have increased levels of the ICE1 protein. ICE1 loss-of-function mutants have low germination vigour and icel gain-of-function mutants have high vigour. This data is consistent with the control of seed vigour by HOS1 being mediated by ICE1 . Figure 7 shows that icel loss-of-function mutants have visibly darkened seed coats, consistent with increased tannin levels.

Together our data confirm that HOS1 and ICE1 both act to couple seed vigour to the environmental conditions during seed production, and that by mutating either gene we can uncouple the process and produce seeds of predictably high or low vigour regardless of the environment during seed production. This invention, the manipulation of seed vigour by ICE1 and HOS1 , can be used in commercial seed production to increase germination vigour and to cause predictable vigour when seeds are produced at different sites or at the same site but in a varying climate.

Claims

1 . A plant wherein said plant is not of the genus Arabidopsis and further wherein said plant expresses a nucleotide sequence variant of a gene comprising a nucleotide sequence that encodes a polypeptide that has ubiquitin E3 ligase activity and which plant produces seed that has increased vigour and/or low seed dormancy.

2. The plant according to claim 1 wherein said sequence variant encodes a ubiquitin E3 ligase with reduced enzyme activity.

3. The plant according to claim 1 wherein said plant is modified to provide an inactive ubiquitin E3 ligase.

4. The plant according to any one of claims 1 to 3 wherein said ubiquitin E3 ligase is encoded by a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24,

25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46 ;

a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to a nucleotide sequence set forth in SEQ ID NO: 1 , 2, 10 or 19 wherein said nucleic acid molecule encodes a ubiquitin E3 ligase;

iv) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 49, 50, 51 , 52, 53, 55, 56, 57, 58, 59, 60, 61 , 62, 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 or 90; a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 47, 48, 54 or 63 wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has ubiquitin E3 ligase activity;

vi) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that has at least 30% amino acid sequence identity to the amino acid sequence represented in SEQ ID NO: 47, 48, 54 or 63 and which has ubiquitin E3 ligase activity.

5. The plant according to claim 4 wherein said ubiquitin E3 ligase is encoded by a nucleotide sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46. .

6 The plant according to any one of claims 1 to 5 wherein said plant has enhanced expression of a gene comprising a nucleotide sequence variant that encodes an ICE1 transcription factor.

7. The plant according to claim 6 wherein said transcription factor comprises a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108;

ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 91 , 92, 101 , 104 or 105 wherein said nucleic acid molecule encodes a transcription factor that regulates the expression of one or more transcription factors;

v) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence as represented in SEQ ID NO: 109, 1 18, 121 or 122 wherein said amino acid sequence is modified by addition deletion or substitution of at least one amino acid residue as represented in iv) above and which has ICE1 transcription factor activity;

vi) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence that has at least 30% amino acid sequence identity to the amino acid sequence represented in SEQ ID NO: 109, 1 18, 121 or 122 and which that regulates the expression of a transcription factor.

8. The plant according to claim 7 wherein said transcription factor is encoded by a nucleotide sequence as set forth in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 ,

102, 103, 104, 105, 106, 107 or 108.

9. The plant according to any one of claims 1 to 8 wherein said plant is a transgenic plant engineered to reduce or abrogate the expression of said ubiquitin E3 ligase activity and optionally is engineered to enhance the expression of an ICE1 transcription factor.

10. The plant according to claim 9 wherein said transgenic plant is transformed with a transcription cassette wherein said cassette encodes all or part of a nucleotide sequence that encodes an ubiquitin E3 ligase and is adapted for expression by provision of at least one promoter operably linked to said nucleotide sequence such that both sense and antisense molecules are transcribed from said cassette.

1 1 . The plant according to claim 10 wherein said cassette is adapted such that both sense and antisense ribonucleic acid molecules are transcribed from said cassette wherein said sense and antisense nucleic acid molecules are adapted to anneal over at least part or all of their length to form a inhibitory RNA or short hairpin RNA.

12. The plant according to claim 10 or 1 1 wherein said cassette is adapted to express an antisense RNA wherein said antisense RNA is adapted to anneal to a mRNA sequence that encodes a ubiquitin E3 ligase.

13. The plant according to any one of claims 10 to 12 wherein said inhibitory RNA or antisense RNA is designed with reference to a nucleotide sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID

NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46;

14. The plant according to any one of claims 10 to 13 wherein said cassette is part of an expression vector adapted for expression in a plant cell.

15. The plant according to claim 14 wherein said transcription factor is encoded by a nucleotide sequence which is part of an expression vector wherein said nucleotide sequence is operably linked to a nucleotide sequence comprising a transcription promoter.

16. A seed obtained from a plant according to any one of claims 1 to 1 5.

17. A plant or seed according to any one of claims 1 to 16 for use in the improvement of seed vigour and/or low seed dormancy.

18. A method for the preparation of F1 hybrid seed and formation of an F1 hybrid plant comprising the steps: i) providing a first male sterile female plant cultivar wherein said plant encodes a a plant ubiquitin E3 ligase variant and crossing said plant with a second male plant cultivar; and

19. The method according to claim 18 wherein said ubiquitin E3 ligase variant is inactive or has reduced enzyme activity.

20. The method according to claim 18 or 19 wherein said male sterile female plant is genetically male sterile.

21 . The method according to claim 18 or 19 wherein said male sterile female plant is treated with an agent that induces male sterility.

22. The use of a gene comprising a nucleic acid molecule that encodes aplant ubiquitin E3 ligase as a means to identify a plant with altered expression of a ubiquitin E3 ligase variant wherein said plant has seeds with improved seed vigour and/or low seed dormancy.

23. A method to produce a plant that has altered expression of an ubiquitin E3 ligase comprising the steps of: i) mutagenesis of wild-type seed from a plant that expresses a ubiquitin E3 ligase;

iv) determining if the seed from said first and subsequent generations of plants has altered nucleotide sequence and/or altered expression of a ubiquitin E3 ligase variant polypeptide;

v) obtaining a sample and analysing the nucleic acid sequence of a nucleic acid molecule that encodes said ubiquitin E3 ligase variant; and optionally

vi) comparing the nucleotide sequence of the nucleic acid molecule encoding a ubiquitin E3 ligase variant in said sample to a nucleotide sequence of a nucleic acid molecule of the original wild-type plant.

24. The method according to claim 23 wherein said nucleic acid molecule is analysed by a method comprising the steps of: i) extracting nucleic acid from said mutated plants;

iii) forming a preparation comprising the amplified nucleic acid and nucleic acid extracted from wild-type seed to form heteroduplex nucleic acid;

iv) incubating said preparation with a single stranded nuclease that cuts at a region of heteroduplex nucleic acid to identify the mismatch in said heteroduplex; and

v) determining the site of the mismatch in said nucleic acid heteroduplex.

25. The method according to claim 23or 24 wherein said plant encodes a ubiquitin E3 ligase variant which variant has reduced expression and/or activity.

26. The method according to any one of claims 23 to 25 wherein said ubiquitin E3 ligase is encoded by a nucleotide sequence selected from the group: i) a nucleotide sequence as represented by the sequence in

SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46;

27. The method according to any one of claims 23 to 26 wherein said plant is analysed to determine expression of an ICE1 transcription factor variant that regulates expression of one or more transcription factors.

28. The method according to claim 27 wherein said transcription factor is selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108 ;

ii) a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 91 , 92, 101 , 104 or 105 wherein said nucleic acid molecule encodes a transcription factor that regulates the expression of one or more transcription factors.

29. A screening method that detects the expression of an ubiquitin E3 ligase and/or an ICE1 transcription factor in a plant comprising the steps: i) providing an isolated sample from a plant to be assessed for expression and extracting nucleic acid from said sample; ii) preparing cDNA from said extracted nucleic acid; iii) forming a preparation comprising said cDNA and an oligonucleotide primer pair adapted to anneal to a nucleic acid molecule comprising a nucleotide sequence that encodes a ubiquitin E3 ligase and/or an ICE1 transcription factor; a thermostable DNA polymerase, deoxynucleotide triphosphates and co-factors; iv) providing polymerase chain reaction conditions sufficient to amplify all or part of said nucleic acid molecule; v) analysing the amplified products of said polymerase chain reaction for the presence of a nucleic acid molecule comprising a nucleotide sequence that encodes an ubiquitin E3 ligase and/or an ICE1 transcription factor; and optionally vi) comparing the amplified product with a normal matched control.

30. The method according to claim 29 wherein said ubiquitin E3 ligase is encoded by a nucleotide sequence as set forth in SEQ ID NO: 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46.

31 The method according to claim 29 wherein said ICE1 transcription factor is encoded by a nucleotide sequence as set forth in SEQ ID NO: 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 104, 105, 106, 107 or 108

32 The method according to any one of claims 29 to 31 wherein said method is a real time PCR method for the detection and quantification of a nucleic acid encoding all or part of a nucleotide sequence that encodes said ubiquitin E3 ligase and/or an ICE1 transcription factor.

33 A screening method that detects the expression of an ubiquitin E3 ligase and/or an ICE1 transcription factor in a plant comprising the steps i) providing an isolated sample from a plant to be assessed for expression;

iii) detecting the complex; and

34 The method according to claim 33 wherein said ubiquitin E3 ligase polypeptide is represented by the amino acid sequence set forth in SEQ ID NO: 49, 50, 51 , 52, 53, 55, 56, 57, 58, 59, 60, 61 , 62, 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 or 90.

35. The method according claim 33 or 34 wherein said ICE1 transcription factor polypeptide is represented by the amino acid sequence set forth in SEQ ID NO: 109, 1 10, 1 1 1 , 1 12, 1 13, 1 14, 1 15, 1 16, 1 17, 1 18, 1 19, 120, 121 , 122, 123, 124 or 125.

36. The method according to any one of claims 29 to 35 wherein said isolated plant samples comprise an array of samples isolated from a plurality of plants to be tested for expression of said ubiquitin E3 ligase and/or ICE1 transcription factor nucleic acid or polypeptide.

37 The method according to any one of claims 23 to 36 wherein said method further includes DNA sequencing of genomic DNA isolated from a plant variant obtained by said method[s] to determine a variant sequence encoding or controlling expression of a ubiquitin E3 ligase variant.

38. The method according to any one of claims 23 o 37 wherein said method further includes DNA sequencing of genomic DNA isolated from a plant variant obtained by the method[s] to determine the variant sequence encoding or controlling expression of a ICE1 transcription factor variant.

39. A plant obtained by the method according to any one of claims 23 to 38.

40. A plant wherein said plant comprises a viral vector that includes all or part of a gene comprising a nucleotide sequence that encodes an ubiquitin E3 ligase.

41 . The plant according to claim 40 wherein said gene is encoded by a nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of: i) a nucleotide sequence as represented by the sequence in SEQ ID NO: : 3, 4, 5, 6, 7, 8, 9, 1 1 ,12, 13, 14, 15, 16, 17, 18, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45 or 46;

a nucleotide sequence wherein said sequence is degenerate as a result of the genetic code to the nucleotide sequence defined in (i); iii) a nucleic acid molecule the complementary strand of which hybridizes under stringent hybridization conditions to the sequence in SEQ ID NO: 1 , 2, 10 or 19 wherein said nucleic acid molecule encodes an ubiquitin E3 ligase.

42. A viral vector comprising all or part of a nucleic acid molecule that encodes a plant ubiquitin E3 ligase.

43. The use of a viral vector according to claim 42 in viral induced gene silencing of a plant ubiquitin E3 ligase.