WO2007063289A2

WO2007063289A2 - Transgenic plant overexpressing a bhlh transcription factor

Info

Publication number: WO2007063289A2
Application number: PCT/GB2006/004442
Authority: WO
Inventors: Brigitte Poppenberger; Tobias Sieberer
Original assignee: The University Of York
Priority date: 2005-11-30
Filing date: 2006-11-29
Publication date: 2007-06-07
Also published as: GB0524382D0; WO2007063289A3

Abstract

We describe a transgenic plant that has altered brassinosteroid levels and the phenotype of said plant.

Description

Transgenic Plant

The invention relates to a transgenic plant that has altered brassinosteroid levels and the phenotype of said plant.

There is an ongoing need to develop plants with increased productivity. The demand for plant based materials is ever increasing and methods to increase plant biomass are highly desirable. This may be achieved by the selection of desirable genetic traits by breeding plant varieties with increased biomass, for example increased seed yield, the application of chemicals with growth promoting properties, for example plant hormones, or by genetic modification.

For example, WO01/64928 describes the genetic modification of plants with nucleic acid molecules encoding ADP glucose pyrophosphorylase. The transgenic plants have a number of desirable traits when compared to a non-transgenic control plant. The traits include increased seed yield, both seed number and weight and an overall increase in plant weight. WO04/090143 describes the production of transgenic plants which have altered cytokinin levels, in particular, altered cytokinin levels in female organs and seeds. This is achieved by the temporal and spatial modulation of genes that affect the levels of cytokinins by utilising seed specific promoters, for example eepl and eep2. WO04/0101767 describes the modulation of the floral specific gene Pt M3. The phenotype of transgenic plants that over-express this gene have a number of valuable traits, for example accelerated early flowering, increased flower/fruit production and increased seed production. The ablation of PtM3 also has an interesting phenotype; the plants are partially or completely sterile. A further example is provided by US2004023801 which describes the use of neonicotinoid compounds to increase the yield and/or vigour of a plant.

WO98/59039 discloses the modulation of Bin 1, a plant steroid receptor. Bin 1 is a receptor that binds plant hormones of the class brassinosteroids. Brassinosteroids have economic importance as plant protectants. For example it is known that these hormones can act as natural insecticides. Brassinosteroids function to regulate the reproductive cycle in plants and can increase or decrease the reproductive process. For example, it may be desirable to eliminate flowering in certain agronomically important plants to promote the production of other tissues, (e.g. leaves, bulbs). Brassinosteroids also seem to promote root growth.

We describe a mutant Arabidopsis thaliana that over-expresses a gene that encodes a transcription factor suspected to be involved in the regulation of genes that control the biosynthesis of brassinosteroids. The gene is a basic Helix-loop Helix (bHLH) transcription factor. bHLH transcription factors constitute 162 members in A. thaliana, which makes them one of the largest family of transcription factors in this plant (Bailey et al., 2003). The proteins are defined by the presence of the bHLH signature domain, which consists of ~ 60 amino acids with two functionally distinct regions. Whereas the basic region, a stretch of ~ 15 amino acids located at the N- terminal end of the domain, is involved in DNA binding, the HLH region, at the C- terminal end functions as a dimerization domain. To date only ~10% of the Arabidopsis bHLH proteins have been characterised. They have been found to be involved in developmental processes, including floral organogenesis, trichome development, hormone responses and light signalling (reviewed by Toledo-Ortiz et al., 2003). The mutant plants have some interesting phenotypic traits, such as increased vegetative growth and increased seed production.

According to a further aspect of the invention there is provided a transgenic plant cell wherein the genome of said cell comprises a nucleic acid molecule selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence represented in

Figure 1; ii) a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor 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_n, 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 65⁰C 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: Ix 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 55⁰C for 20-30 minutes each.

In a preferred embodiment of the invention said transcription factor is encoded by a nucleic acid molecule consisting of the nucleic acid sequence illustrated in Figure 1, or a nucleic acid molecule that hybridises to the nucleic acid molecule illustrated in Figure 1, under stringent hybridisation conditions.

In a preferred embodiment of the invention said transcription factor activity is increased. Preferably said activity is increased by at least about 2-fold above a basal level of activity. More preferably said activity is increased by at least about 5 fold; 10 fold; 20 fold, 30 fold, 40 fold, 50 fold. Preferably said activity is increased by between at least 50 fold and 100 fold. Preferably said increase is greater than 100- fold.

It will be apparent that means to increase the activity of a polypeptide encoded by a nucleic acid molecule axe known to the skilled artisan. For example, and not by limitation, increasing the gene dosage by providing a cell with multiple copies of said gene. Alternatively, or in addition, a gene(s) may be placed under the control of a powerful promoter sequence or an inducible promoter sequence to elevate expression of mRNA encoded by said gene. The modulation of mRNA stability is also a mechanism used to alter the steady state levels of an mRNA molecule, typically via alteration to the 5' or 3' untranslated regions of the mRNA.

m a preferred embodiment of the invention said cell is modified so that it has reduced transcription factor activity. Preferably said activity is reduced by at least 10%. Preferably said activity is reduced by between about 10%-90%. More preferably said activity is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80% or at least 90% when compared to anon-transgenic or genetically modified reference cell.

A number of techniques have been developed in recent years which purport to specifically ablate genes and/or gene products. A recent technique to specifically ablate gene function is through the introduction of double stranded RNA, also referred to as inhibitory RNA (RNAi), into a cell which results in the destruction of mRNA complementary to the sequence included in the RNAi molecule. The RNAi 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 RNAi molecule is typically derived from exonic or coding sequence of the gene which is to be ablated. Surprisingly, only a few molecules of RNAi are required to block gene expression which implies the mechanism is catalytic. The site of action appears to be nuclear as little if any RNAi is detectable in the cytoplasm of cells indicating that RNAi exerts its effect during mRNA synthesis or processing.

An alternative embodiment of RNAi involves the synthesis of so called stem loop RNAi molecules which are synthesised from expression cassettes carried in vectors. The DNA molecule encoding the stem-loop RNA is constructed in two parts, a first part which is derived from a gene the regulation of which is desired. The second part is provided with a DNA sequence which is complementary to the sequence of the first part. The cassette is typically under the control of a promoter which transcribes the DNA into RNA. The complementary nature of the first and second parts of the RNA molecule results in base pairing over at least part of the length of the RNA molecule to form a double stranded hairpin RNA structure or stem-loop. The first and second parts can be provided with a linker sequence. Stem loop RNAi has been successfully used in plants to ablate specific mRNA's and thereby affect the phenotype of the plant , see Smith et al (2000) Nature 407, 319-320.

In a preferred embodiment of the invention said cell is transfected with a nucleic acid molecule comprising an expression cassette which cassette comprises a nucleic acid sequence which encodes at least part of a gene which encodes a polypeptide with the specific transcription factor activity associated with a polypeptide encoded by the spatula gene wherein said cassette is adapted such that both sense and antisense nucleic acid molecules are transcribed from said cassette.

In a preferred embodiment of the invention said cassette is provided with at least two promoters adapted to transcribe sense and antisense strands of said nucleic acid molecule. In a further preferred embodiment of the invention said cassette comprises a nucleic acid molecule wherein said molecule comprises a first part linked to a second part wherein said first and second parts are complementary over at least part of their sequence and further wherein transcription of said nucleic acid molecule produces an RNA molecule which forms a double stranded region by complementary base pairing of said first and second parts.

Ih a preferred embodiment of the invention said first and second parts are linked by at least one nucleotide base. In a further preferred embodiment of the invention said first and second parts are linked by 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide bases. In a yet further preferred embodiment of the invention said linker is at least 10 nucleotide bases.

In a further preferred embodiment of the invention the length of the RNA molecule is between 10 nucleotide bases (nb) and lOOOnb. Preferably said RNA molecule is lOOnb; 200nb; 300nb; 400nb; 500nb; 600nb; 700nb; 800nb; 900nb; or lOOOnb in length. More preferably still said RNA molecule is at least lOOOnb in length. Preferably still said RNA molecule is 21nb in length.

According to an aspect of the invention there is provided a transgenic plant cell wherein the genome of said cell comprises a nucleic acid molecule encoding a transcription factor wherein said nucleic acid molecule comprises a nucleic acid sequence as represented in Figure 1; or a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor activity.

Preferably said vector is adapted for the over-expression of said nucleic acid molecule encoding said transcription factor.

Suitable vectors can be constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: Laboratory Manual: 2^nd edition, Sambrook et al. 1989, Cold Spring Habor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. Eds., John Wiley & Sons, 1992.

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).

Preferably the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, (e.g. bacterial), or plant cell. The vector may be a bi- functional expression vector which functions in multiple hosts. In the case of GTase genomic DNA this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.

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 MoI. 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 Serial 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.

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 Ih2-2 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- Ia 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) MoI. 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) MoI. Gen. Genet. 254(3): 337-343; Russell et al. (1997) Transgenic Res. 6(2): 157-168; Rinehart et al. (1996) Plant Physiol. 112(3): 1331-1341; Van Camp et al. (1996) Plant Physiol. 112(2): 525-535; Canevascni et al. (1996) Plant Physiol. 112(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 MoI. Biol. 23(6): 1129-1138; 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 an inducible promoter or a developmentally 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 upon plants are described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Lab fax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral- derived vectors (see e.g. EP-A-194809).

If desired, selectable genetic markers maybe included in the construct, such as those that confer selectable phenotypes such as resistance to antibodies or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate).

Plants transformed with a DNA construct of the invention may be produced by standard techniques known in the art for the genetic manipulation of plants. DNA can be introduced into plant cells using any suitable technology, such as a disarmed

Ti-plasmid vector carried by Agrobacterium exploiting its natural gene transferability

(EP-A-270355, EP-A-Ol 16718, NAR 12(22): 8711-87215 (1984), Townsend et al.,

US Patent No. 5,563,055); particle or microprojectile bombardment (US Patent No. 5,100,792, EP-A-444882, EP-A-434616; Sanford et al, US Patent No. 4,945,050;

Tomes et al. (1995) "Direct DNA Transfer into Intact Plant Cells via Microprojectile

Bombardment", in Plant Cell,, Tissue and Organ Culture: Fundamental Methods, ed.

Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988)

Biotechnology 6: 923-926); microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. 91987) Plant Tissue and Cell Culture, Academic

Press, Crossway et al. (1986) Biotechniques 4:320-334); electroporation (EP 290395,

WO 8706614, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606; DΗalluin et al. 91992). Plant Cell 4:1495-1505) other forms of direct DNA uptake (DE 4005152, WO 9012096, US Patent No. 4,684,611, Paszkowski et al. (1984) EMBO J. 3:2717-2722); liposome-mediated DNA uptake (e.g. Freeman et al (1984) Plant Cell Physiol, 29:1353); or the vortexing method (e.g. Kindle (1990) Proc. Nat. Acad. Sci. USA 87:1228). Physical methods for the transformation of plant cells are reviewed in Oard (1991) Biotech. Adv. 9:1-11. See generally, Weissinger et al. (1988) Ann. Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Sciences and Technology 5:27-37; Christou et al. (1988) Plant Physiol. 87:671-674; McCabe et al. (1988) Bio/Technology 6:923-926; Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P:175-182; Singh et al. (1988) Theor. Appl. Genet. 96:319-324; Datta et al. (1990) Biotechnology 8:736-740; Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85: 4305-4309; Klein et al. (1988) Biotechnology 6:559-563; Tomes, US Patent No. 5,240,855; Buising et al. US Patent Nos. 5,322, 783 and 5,324,646; Klein et al. (1988) Plant Physiol 91: 440-444; Fromm et al (1990) Biotechnology 8:833-839; Hooykaas-Von Slogteren et al. 91984). Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349; De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues ed. Chapman et al. (Longman, New- York), pp. 197-209; Kaeppler et al. (1990) Plant Cell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566; Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407- 413;Osjoda et al. (1996) Nature Biotechnology 14:745-750, all of which are herein incorporated by reference.

Agrobacterium transformation is widely used by those skilled in the art to transform dicotyledonous species. Recently, there has been substantial progress towards the routine production of stable, fertile transgenic plants in almost all economically relevant monocot plants (Toriyama et al. (1988) Bio/Technology 6: 1072-1074;

Zhang et al. (1988) Plant Cell rep. 7379-384; Zhang et al. (1988) Theor. Appl. Genet.

76:835-840; Shimamoto et al. (1989) Nature 338:274-276; Datta et al. (1990) Bio/Technology 8: 736-740; Christou et al. (1991) Bio/Technology 9:957-962; Peng . et al (1991) International Rice Research Institute, Manila, Philippines, pp.563-574; Cao et al. (1992) Plant Cell Rep. 11: 585-591; Li et al. (1993) Plant Cell Rep. 12: 250-255; Rathore et al. (1993) Plant MoI. Biol. 21:871-884; Fromm et al (1990) Bio/Technology 8:833-839; Gordon Kamm et al. (1990) Plant Cell 2:603-618; DΗalluin et al. (1992) Plant Cell 4:1495-1505; Walters et al. (1992) Plant MoI. Biol. 18:189-200; Koziel et al. (1993). Biotechnology 11194-200; Vasil, IK. (1994) Plant MoI. Biol. 25:925-937; Weeks et al (1993) Plant Physiol. 102:1077-1084; Somers et al. (1992) Bio/Technology 10:1589-1594; WO 92/14828. In particular, Agrobacterium mediated transformation is now emerging also as a highly efficient transformation method in monocots. (Hiei, et al. (1994) The Plant Journal 6:271- 282). See also, Shimarnoto, K. (1994) Current Opinion in Biotechnology 5:158-162; Vasil, et al. (1992) Bio/Technology 10:667-674; Vain, et al. (1995) Biotechnology Advances 13(4):653-671; Vasil, et al. (1996) Nature Biotechnology 14: 702).

Microprojectile bombardment, electroporation and direct DNA uptake are preferred where Agrobacterium is inefficient or ineffective. Alternatively, a combination of different techniques maybe employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium-co&ted microparticles (EP-A- 486234) or microprojectile bombardment to induce wounding followed by co- cultivation with Agrobacterium (EP-A-486233).

In a preferred embodiment of the invention said seed is produced from a plant selected from the group consisting of: corn {Zea mays), canola {Brassica napus, Brassica rapa ssp.), flax (Linum usitatissimum), alfalfa (Medicago sativά), rice {Oryza sativa), rye (Secale cerale), sorghum {Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), wheat {Tritium aestivum), soybean {Glycine max), tobacco {Nicotiana tabacum), potato {Solarium tuberosum), peanuts {Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato {Iopmoea batatus), cassava {Manihot esculenta), coffee (Cofea spp.), coconut {Cocos nucifera), pineapple (Anana comosus), citris tree (Citrus spp.) cocoa {TJieobroma 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 occidental^), macadamia {Macadamia inter grifolia), almond {Prunus amygdalus), sugar beets {Beta vulgaris), oats, barley, vegetables.

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, sorghum, and flax (linseed).

Horticultural plants to which the present invention may be applied may include herbs, lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower. The present invention, may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper. Also included are ornamental plants e.g Agastache, Ageratum, Althea rosea, Alyssum, Amaranthus, Antirrhinum, Asclepias, Asters, ornamental forms of Asparagus, Balsam, Basil (ornamental), Begonia semperflorens, Begonia elatior, Begonia tuberous, Bidens, , Calceolaria rugosa, Calendula, Callistephus, Canna, Capsicum, Carnation, Carthamus, Celosia, Centaurea, Chrysanthemum, Cineraria maritima, Cleome, Coleus, Coreopsis, Cosmos, Cosmos sulphureurn, Cuphea, Cynoglossum, Dahlia,Dianthus barbatus, DianthuscariophylluS_jDianthusplumarius, Dianthus sinensis, Delphinium, Diasca, Didiscus, Echium, Euphorbia, Exacum, Ficoides, Flower Kale, Fuchsia, Gazania, Geranium, Gerbera, Godetia, Grasses (ornamental), Helianthus, Heliotrope, Helichrysum, ϋnpatiens, ϋnpatiens New Guinea, Ipomea, Lagerstroemia, Larkspur, Lavender, Lavatera, Leucanthemum, Lilium, Linaria, Lisianthus, Lobelia, Lobelia speciosa, Marigold, African, Marigold, French, Matthiola, Mesambrianthemum, Mimulus, Molucella, Nasturtium, Nemesia, Nicotiana, Nierembergia, Oxypetalum, Papaver, ornamental, Pelargonium, Pentas, Pepper, ornamental, Petunias, Petunia double, Petunia gigantiflora, Petunia grandiflora, Petunia milliflora, Petunia multiflora, Phlox, Pinks, Platycodon, Portulacca, Primula, Poinsettia, Rhododendron, Rosa, Ricinus, Rudbeckia, Sanvitalia, Salvia, Salvia coccinea, Salvia farinacea, Salvia patens, Salvia splendens, Schizanthus, Snapdragon, Solatium, Statice, Stocks, Sweet Peas, Swiss Chard, T.E.P. (Tagetes Erecta x Patula), Tagetes erecta, Tagetes patula, Tagetes signata, Thlaspi, Tithonia, Tobacco, ornamental, Verbascum, Verbena, Vinca, Zinniapetunia.

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 been, lentils, chickpea, etc.

According to a yet further aspect of the invention there is provided a transgenic plant comprising a cell according to the invention.

According to a further aspect of the invention there is provided a transgenic plant seed comprising a cell according to the invention.

According to a further aspect of the invention there is provided the use of a plant according to the invention to increase seed production.

An increase in seed production may be construed as an increase in seed number and/or an increase in seed weight and/or size.

According to a further aspect of the invention there is provided the use of a plant according to the invention for modulating said plants brassinosteroid levels.

Ih a preferred embodiment of the invention said brassinosteroid levels are increased when compared to a non-transgenic reference plant of the same species. In an alternative preferred embodiment of the invention said brassinosteroid levels are decreased when compared to a non-transgenic reference plant of the same species.

According to a further aspect of the invention there is provided the use of a plant according to the invention to modulate said plants reproductive cycle.

In a preferred embodiment of the invention said plant has altered flowering times when compared to a non-transgenic reference plant of the same species.

Preferably said plant has delayed flowering times when compared to a non-transgenic reference plant of the same species.

In an alternative preferred embodiment said flowering times are accelerated.

According to a further aspect of the invention there is provided the use of a plant according to the invention to modulate senescence in said plant.

According to a still further aspect of the invention there is provided the use of a plant according to the invention to increase the biomass of said plant.

Preferably senescence is delayed when compared to a non-transgenic reference plant of the same species.

According to a further aspect of the invention there is provided a method to increase seed yield in a plant of comprising the steps of: i) providing a cell/seed according to the invention; and ii) regenerating said cell/seed into a plant; and optionally iii) monitoring seed yield in said regenerated plant. According to a further aspect of the invention there is provided a method to modulate brassinosteroid levels in a plant of comprising the steps of: i) providing a cell/seed according to the invention; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the levels of brassinosteroids in said regenerated plant.

According to a further aspect of the invention there is provided a method to modulate the flowering time in a plant comprising the steps of: i) providing a cell/seed according to the invention; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the flowering of isaid regenerated plant.

According to a further aspect of the invention there is provided a method to modulate senescence in a plant comprising the steps of: i) providing a cell/seed according to the invention; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the senescence of said regenerated plant.

According to a further aspect of the invention there is provided a method to increase the biomass of a plant comprising the steps of: i) providing a cell/seed according to the invention; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the growth of said regenerated plant.

In a preferred method of the invention said plant has increased numbers of secondary rosettes. Ih a further preferred method of the invention said plant has increased leaf number.

According to a further aspect of the invention there is provided a method to screen for a plant for altered brassinosteroid levels comprising detecting and determining the sequence structure of a gene that encodes the specific transcription factor activity associated with a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 1; or a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor activity; and comparing said structure with the sequence structure of a reference gene which encodes the same said activity.

Ih a preferred method of the invention the level of expression of said nucleic acid molecule is compared between said plant and said reference plant.

An embodiment of the invention will now be described by example only and with reference to the following figures:

Figure 1 is the nucleic acid sequence of CES coding sequence;

Figure 2 shows the ces phenotype and the mutants molecular characterisation, a, ces and wild-type Coϊ-0 plants grown for 30 days under long day conditions (16 hours light/ 8 hours dark), b, ces grown for 40 days under LD conditions, c, To analyse the molecular nature of the mutant phenotype DNA flanking both the right and left boarder of the T-DNA were cloned by plasmid rescue. Sequencing of the boarder regions revealed that in ces the T-DNA is inserted in the 5'UTR of a putative bHLH transcription factor (Atlg25330), 152 bp upstream of the ATG. The 4x35S enhancer faces the start codon. d, 2 week old seedlings of both wt and ces were analysed for expression of Atlg25330 using semi-quantitative RT-PCR. Whereas in wt no transcript could be detected, the gene is over-expressed in ces; and Figure 3 illustrates that ces rescues the Bit-deficient phenotype of UGT73C5 over- expression (oe) to wild-type. Plants were grown for 25 days under long day conditions. From left to right: wild-type CoI-O, ces, UGT73C5oe, ces x UGT73C5oe.

MATERIALS AND METHODS

Plant materials and growth conditions

Arabidopsis thaliana ecotype Columbia (Col) was used as the wild-type. General plant handling and transformation protocols followed standard procedures (Weigel and Glazebrook, 2001).

For germination experiments mother plants were cultivated in a controlled environment of 16h/8h light-dark cycle (150 μmol m^"2 sec^'1 white light) at a constant temperature of 21 °C (±1 ). Seeds of all lines used in one experiment were harvested at the same time, sterilized, plated on standard MS growth medium supplemented with 1.0% phytagar (Life Technologies) and subjected to a 4 day dark treatment at 4⁰C to synchronize germination. Plates were then either directly transferred to 21⁰C keeping seeds in the dark or subjected to a 2 hour light impuls before incubation in the dark depending on the experiment. Germination phenotypes were generally analysed after 4 days.

Analysis of the adult ces phenotype was conducted under defined conditions using different light conditions with varying day-length as well as light intensity (60 to 180 μmol m^"2 sec^"1 white light), but at a consistent temperature of 21⁰C (±1) and a consistent type of white light (warm white light fluorescent tubes).

Mutant screen and genetic analysis

ces was originally isolated in the eirl-1 background (Luschnig et al., 1999) when screening a collection of T-DNA insertional mutants, generated with the T-DNA construct pSK115 (Weigel et al., 2000) corresponding to approximatly 14.000 iπdependant transformants (Sieberer et al., 2003). When backcrossed into wild-type CoI-O the ces phenotype was found to segregate in a 3:1 ratio. AU further analysis was performed in the CoI-O background.

ces was crossed with 1319/2-5 (Poppenberger et al., 2003) and double mutants resistant to both selection markers were selected for phenotypic analysis.

Cloning of CES

The ces phenotype is genetically linked to the BASTA resistance locus of a single T- DNA insertion. To further verify the molecular nature of the mutant phenotype genomic DNA flanking both the right and left boarder of the T-DNA were cloned by plasmid rescue (Weigel et al., 2000) using Xhol (right border) and BamHI (left border) for digestion and the AMP resistance marker for selection. The 15.5 kb Xhol rescued plasmid (DS39/XhoI-l) was used to sequence the right border junction of the insertion with a primer located in the right border region of the T-DNA (SOER2, 5'- GCAGGCATGCAAGCTTATCGATATCTAGA-3'). A BLAST search identified genomic sequence on BAC clone F4F7, 152 bp upstream of the ATG of a putative bHLH transcription factor (Atlg25330). The DS39/XhoI-l plasmid contains the whole ORF of the gene encoding the putative bHLH protein and 8.57 kb of 3' genomic sequence. The BamHI rescued plasmid (DS39/BamHI-7) is 9.8 kb and was used to sequence the left boarder junction (SOEL2, 5'- TGATGTGATATCTAGATCCGAAACTATCA-3').

Reverse Transcription (RT)-PCR Analysis

Total RNA was isolated from plant tissue ground in liquid nitrogen with Trizol

Reagent as recommended by the manufacturers (Gibco BRL Life Technologies). After RNA quality control cDNA was synthesized from 1 μg of total RNA with 500 ng of a 18-mer oligo(dT) and the reverse transcriptase Superscript (Life Technologies). PCR was performed with 2 μl of the 1:20 diluted cDNA using primers (CESRT-fw 5'-CTCAGAAGCCAAAAGATGT-S'; CESRT-rv 5'- TCAAAAGGGTAATGTTGAA-3') that amplify a 390 bp large fragment located in the C-terminal part of the gene. UBQ5 was used as an internal template control.

EXAMPLES

cesta, a mutant with a BR over-accumulation phenotype

We isolated cesta (ces) from a collection of activation-tagged Arabidopsis thaliana T- DNA insertional mutants (generated by Sieberer et al., 2003). The constitutive phenotype of ces is already visible in seedlings, which have elongated hypocotyls, but . becomes most pronounced after plants have developed first rosette leaves (about 20 ^' days after germination, DAG, under long day, LD, conditions). The name for the mutant was chosen due to the adult morphology of its rosette-leaves, which have elongated petioles, display a proximo-distal lengthening and are outwardly curving as well as epinastic (Figure 2a), giving them a cesta-like appearance (the cesta, Spanish for basket, is used in the Basque ball game pelota as a throwing and catching tool). Adult ces plants are furthermore characterised by prolonged vegetative development of axillary shoot-meristems. Secondary rosettes are formed in the axils of rosette- leaves in a basal-apical direction, which consequently results in a markely increased number of rosette-leaves as well as influorescenses, as secondary rosettes undergo conversion from the vegetative to the reproductive phase (Figure 2b). ces influorescenses are longer than those of wild-type and seem to be defective in their positive phototropic response. Ih contrast to wild-type the ces mutant continues to grow beyond 35 DAG with flowering and senescence being delayed and" its seed yield being increased. Most of these phenotypic features have previously been described to be characteristic for mutant plants that either over-accumulate brassinosteroids (BRs) (Choe et al., 2001) or exhibiting a constitutive BR-signalling response (Wang et al., 2001 ; Yin et al., 2002; Mora-Garcia et al., 2004). To determine whether BRs are increased in ces we performed a cross with a plant line over-expressing the UDP-glucosyltransferase UGT73C5 which, as our current data suggests, glucosylates BRs, leading to biological inactivation of those compounds in planta (Popperiberger et al., unpublished). We could show that an over-accumulation of UGT73C5 rescues the ces phenotype in the double mutant (ces x 1319/2-5) in all stages of the life cycle. In seedlings, hypocotyl length is restored to wild-type (data not shown). Adult double mutant plants show reduced petiole and infiuorescense length, leave curving as well as their proximo-distal lengthening is released and the wild-type rosette physiology as well as the normal phototropic response of the shoot is restored (Figure 3). This result clearly supports the hypothesis that an increased amount of active BRs present in ces is the cause for its' sever phenotype.

CES encodes a basic helix-loop-helix transcription factors that is overexpressed in the mutant

The cesta phenotype was found to be genetically linked to the BASTA resistance locus of a single T-DNA insertion and to be dominant to wild-type. The genomic DNA flanking both right and left border of the T-DNA was cloned by plasmid rescue and border regions were sequenced. This revealed that the T-DNA has inserted on Chromosome I in the 5'UTR of a putative bHLH transcription factor (Atlg25330), 152 bp upstream of the ATG with the 35S enhancer element facing the start codon (Figure 2c). 6 bp of the genomic region were the T-DNA has inserted (5'-CTTAAC- 3') were found to be deleted. Semi-quantitative RT-PCR analysis revealed that expression of this putative transcription factor is significantly up-regulated in the mutant compared to wild-type (Figure 2d). Expression of two other genes on both sides of the T-DNA (Atlg25320 and Atlg25340) was also analysed, but was found not to be altered in ces (data not shown).

CES (bHLH075) has been assigned to subfamily XII of the bHLH-family (Heim et al., 2003). Interestingly, its closest homologues BEEl (BR Enhanced Expression 1) and BEE3, have been identified as BR early response genes and are thought to be positive regulators of BR-signalling (Friedrichsen et al., 2002). Whereas the expression of BEEl, BEE3 as well as BEE2, another member of the same bHLH subfamily, was shown to be up-regulated by BL two- to three-fold within 30 minutes of treatment, CES was not BL-induced and was therefore not included in a further genetic analysis, beel bee2 bee3 triple mutants were generated and characterised, but showed only subtle phenotypes, such as a reduced responsiveness to BRs as well as weak seedling and floral phenotypes characteristic of other known BR mutants. The subtlety of the triple knockout mutant phenotype compared to that of bril mutants was said to suggest that the BR signalling pathway either does not absolutely require the function of the BEEs, or that other additional redundant factors may exist (Friedrichsen et al., 2002).

The regulation of genes involved in BR-biosynthesis is altered in ces

We analysed the expression of the BR biosynthetic genes CPD and DWF4 in the ces mutant, both of which are known to be subjected to feedback regulation, and found that the genes are up-regulated in the mutant compared to wild-type (data not shown). This result was highly interesting since ces seams to over-accumulates BRs, yet feedback-control appeared not to be operating. Those results led to our current hypothesis that CES may be involved in the regulation of BR-synthesis possibliy by controlling the transcription of biosynthetic enzymes.

References

Bailey, P.C., Martin, C, Toledo-Ortiz, G., Quail, P.H., Huq, E., Heim, M.A., Jakoby, M., Werber, M. and Weisshaar, B. (2003) Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell 15: 2497-2502. Choe, S., Fujioka, S., Noguchi, T., Takatsuto, S., Yoshida, S. and Feldmann, K.A. (2001) Overexpression of DWARF4 in the brassinosteroid biosynthetic pathway results in increased vegetative growth and seed yield in Arabidopsis. Plant J. 26: 573- 82.

Friedrichsen, D.M., Nemhauser, J., Muramitsu, T., Maloof, J.N., Alonso, J., Ecker, J.R., Furuya, M., and Chory J. (2002) Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth. Genetics 162: 1445-1456.

Heim, M.A., Jakoby, M., Werber, M., Martin, C, Weisshaar, B., and Bailey, P.C. (2003). The basic helix-loop-helix transcription factor family in plants: a genome- wide study of protein structure and functional diversity. MoI. Biol. Evol. 20: 735- 747.

Luschnig, C, Gaxiola, R.A., Grisafϊ, P., Fink, G.R. (1988) EIRl, a root-specific protein involved in auxin transport, is required for gravitropism in Arabidopsis thaliana. Genes Dev. 12: 2175-87.

Mora-Garcia, S., Vert, G., Yin, Y., Cano-Delgado, A., Cheong, H. and Chory, J. (2004) Nuclear protein phosphatases with. Kelch-repeat domains modulate the response to brassinosteroids in Arabidopsis. Genes Dev. 18: 448-60.

Poppenberger, B., Berthiller, F., Lucyshyn, D., Sieberer, T., Schuhmacher, R., Krcka, R., Kuchler, K., Glδssl, J., Luschnig, C. and Adam, G. 2003. Detoxification of the Fusarium mycotoxin deoxynivalenol by UDP-glycosyltransferases from Arabidopsis thaliana. J. Biol. Chem. 278: 47905-47914.

Sieberer, T., Hauser, M.-T., Seifert, G. and Luschnig, C. 2003. PROPORZl, a putative Arabidopsis transcriptional adaptor protein, mediates auxin and cytokinin signals in the control of cell proliferation. Curr. Biol. Toledo-Ortiz, G., Huq, E., and Quail, P.H. 2003. The Arabidopsis basic/helix-loop- helix transcription factor family. Plant Cell 15: 1749-1770.

Wang, Z. Y., Seto, H., Fujioka, S., Yoshida, S. and Chory, J. (2001) BRIl is a critical component of a plasma-membrane receptor for plant steroids. Nature 410: 380-383.

Weigel, D., Ahn, J.H., Blazquez, M.A., Borevitz, J.O., Christensen, S.K., Fankhauser, C, Ferrandiz, C, Kardailsky, L, Malancharuvil, EJ., Neff, M.M. et al. 2000. Activation tagging in Arabidopsis. Plant Physiol. 122: 1003-1013.

Weigel, D. and Glazebrook, J. 2001. Arabidopsis: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Yin, Y., Wang, Z. Y., Mora-Garcia, S., Li, J., Yoshida, S., Asami, T. and Chory, J. (2002) BESl accumulates in the nucleus in response to brassinosteroids to regulate gene expression and promote stem elongation. Cell 109: 181-191.

Claims

1. A transgenic plant cell wherein the genome of said cell comprises a nucleic acid molecule encoding a transcription factor wherein said nucleic acid molecule comprises a nucleic acid sequence as represented in Figure 1; or a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor activity.

2. A transgenic plant cell according to Claim 1 wherein said transcription factor is encoded by a nucleic acid molecule consisting of the nucleic acid sequence represented in Figure 1, or a nucleic acid molecule that hybridises to the nucleic acid molecule represented in Figure 1, under stringent hybridisation conditions.

3. A transgenic cell according to Claim 1 or 2 wherein said transcription factor activity is increased.

4. A transgenic cell according to Claim 1 or 2 wherein said transcription factor activity is reduced.

5. A vector comprising an expression cassette wherein said cassette comprises a nucleic acid sequence that encodes a transcription factor selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence represented in Figure 1; ii) a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor activity.

6. A vector according to Claim 5 wherein said vector is adapted for the over- expression of said nucleic acid molecule encoding said transcription factor.

7. A transgenic plant comprising a cell according to any of Claims 1 -4.

8. A transgenic plant seed comprising a cell according to any of Claims 1-4.

9. The use of a plant according to Claim 7 to increase seed production.

10. The use of a plant according to Claim 7 for modulating brassinosteroid levels in said plant.

11. Use according to Claim 10 wherein said brassinosteroid levels are increased when compared to a non-transgenic reference plant of the same species.

12. Use according to Claim 10 wherein said brassinosteroid levels are decreased when compared to a non-transgenic reference plant of the same species_*

13. The use of a plant according to Claim 7 to modulate said plants reproductive cycle.

14. Use according to Claim 13 wherein said plant has altered flowering times when compared to a non-transgenic reference plant of the same species.

15. Use according to Claim 14 wherein said plant has delayed flowering times.

16. Use according to Claim 14 wherein said flowering times are accelerated.

17. The use of a plant according to Claim 8 to modulate senescence in said plant.

18. Use according to Claim 17 wherein senescence is delayed.

19. A method to increase seed yield in a plant comprising the steps of: i) providing a cell/seed according to any of Claims 1-4 or 8; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring seed yield in said regenerated plant.

20. A method to modulate brassinosteroid levels in a plant comprising the steps of: i) providing a cell/seed according to any of Claims 1-4 or 8; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the levels of brassinosteroids in said regenerated plant.

21. A method to modulate the flowering time in a plant comprising the steps of: i) providing a cell/seed according to any of Claims 1-4 or 8; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the flowering of said regenerated plant.

22. A method to modulate senescence in a plant comprising the steps of: i) providing a cell/seed according to any of Claims 1-4 or 8; ii) regenerating said cell into a plant; and optionally iii) monitoring the senescence of said regenerated plant.

23. A method to increase the biomass of a plant comprising the steps of: i) providing a cell/seed according to any of Claims 1 -4 or 8; ii) regenerating said cell/seed into a plant; and optionally iii) monitoring the growth of said regenerated plant.

24. A method according to Claim 23 wherein said plant has increased numbers of secondary rosettes.

25. A method according to Claim 23 or 24 wherein said plant has increased leaf number.

26. A method to screen for a plant for altered brassinosteroid levels comprising:

i) detecting and determining the sequence structure of a gene in a plant to be tested that encodes the specific transcription factor activity associated with a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 1; or a nucleic acid molecule that hybridises under stringent hybridisation conditions to the sequence in Figure 1 and has transcription factor activity; and ii) comparing said structure with the sequence structure of a gene in a reference plant that encodes the same said activity.

27. A method according to claim 26 wherein the level of expression of said nucleic acid is compared between said plant and said reference plant.