Enhanced Plant Growth

The invention relates to plants with enhanced growth and altered growth cycle characteristics.

"Plant biomass" refers to living and recently dead plant tissue and lignocellulose [e.g. lignin, cellulose and hemicellulose] materials that comprise the plant and includes plant organs (e.g. stems, leaves, fruits, flowers, roots, seeds). Various approaches have been taken to alter plant traits that result in improvements to plant biomass yield. These can be via plant breeding and also by genetically modifying a plant to express a gene or genes that confer desirable traits. The improvements have utility in various aspects for example agricultural production of food for humans and animals, horticulture, biomass conversion in the creation of biofuels and industries involved in the production of paper, plant enzymes and plant derived metabolites. The ability to be able to modulate plant growth characteristics in a controlled fashion is highly desirable. Modulation means alteration of plant form, for example the size and stature of the plant or the number and/or size of organs [e.g. increase in flower stem length or numbers of flowers, increase in seed or fruit yield]. Alternatively, modulation can mean alteration of the normal growth cycle of the plant to enable growth in for example adverse or stressful environmental conditions [e.g. cold stress, drought stressor altered seasonal growth.

Plant derived products are currently being widely adopted both as industrial feedstock and as replacement fuels. So called first generation biofuels are either based on bioethanol or biodiesel. Bioethanol production relies on the process of fermentation using microbial organisms to produce ethanol. The ethanol is then used mainly as fuel for transportation. The feedstock for this microbial fermentation is typically sugar obtained from sugar cane or sugar beet or derived from starch obtained from cereal crops such as maize or wheat. Bioethanol production from sugarcane, sugar beet and cereal grains such as maize, wheat and barley feedstock has been widely adopted. Crops used to produce feedstock for biodiesel production include soybean, castor bean, sunflower, rapeseed, Jatropha and palm. The first generation biofuels are clearly unsustainable both because their production adds stress to world food supplies and because life cycle analyses reveal that their production often comes with a substantial net carbon emission. Second generation biofuels produced from cheap and abundant plant biomass are seen as an attractive solution to this problem but a number of technical hurdles must be overcome before their potential is realized. The ability to

produce second generation biofuels from plant biomass will be greatly facilitated by producing larger plants that have the ability to grow over an extended growth cycle [e.g. through autumn and/or winter and/or spring].

Genes that encode proteins that enhance the growth characteristics of a plant are well known in the art.

For example WO92/09685, describes the plant homologue of the yeast cell-cycle control gene cdc2 referred to as p34Cd 2 and is an important regulator of cell proliferation, particularly in leaf tissue. WO2005/085452, describes the shoot specific expression of cyclin D3, a cell growth regulator and the enhancement of plant yield. WO2004/087929, describes the expression of the CCS52 gene, a gene that encodes a cell-cycle regulatory protein and the enhancement of plant size and increased organ size and number. WO2005/083094 describes a D-type cyclin dependent kinase which when over- expressed results in increased seed yield. Further examples include WO2005/030966 over expression of transcription factors to improve biomass and cold tolerance. Prior attempts to improve biomass yield or alter growth characteristics involve transformation and over expression of selected plant genes.

In our co-pending application WO2007/049036 we disclose a transcription factor involved in regulating seed germination. The basic helix-loop-helix transcription factor called SPATULA (SPT) is involved in the control of the germination of dormant seeds by light and temperature. SPT is a multifunctional transcription factor, acting as a light stable repressor of GA3ox expression controlling seed responses to cold stratification and to a lesser extent red light. A mutational variant of SPT is disclosed in WO2007/049036 called spt-2 which has reduced germination and is not responsive to cold stratification. The spt-2 mutation has a semi-dominant effect on seed germination. We have surprisingly found that a deletion of SPT far from being deleterious results in enhanced growth and improved growth cycle characteristics.

According to an aspect of the invention there is provided a transgenic plant cell wherein the genome of said plant cell is modified which modification is to a nucleic acid molecule that encodes a transcription factor encoded by a nucleic acid sequence selected from the group consisting of: i) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 1a;

ii) a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleic acid sequence in Figure 1a and encodes a polypeptide with transcription factor activity; characterized in that said modification is an addition, deletion or substitution of at least one nucleotide which provides a transcription factor which has reduced transcription factor activity when compared to a non-transgenic reference cell of the same plant species.

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 Harbour Laboratory Press, Cold Spring

Harbor, NY, 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular

Biology — Hybridization with Nucleic Acid Probes Part I ₁ 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 65 ⁰ 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.

In a preferred embodiment of the invention said modification is to the nucleic acid sequence that encodes the amino acid sequence of said transcription factor.

In a further preferred embodiment of the invention said modification is to a nucleic acid sequence comprising a regulatory region that is associated with said nucleic acid molecule.

A regulatory region is understood to include promoter regions and including enhancers, untranslated regions of mRNA that function to control the translation and/or stability of an mRNA encoding said transcription factor polypeptide. Regulatory regions may also include intronic regions that control mRNA processing.

According to a further aspect of the invention there is provided a transgenic plant cell wherein the genome of said plant cell is modified which modification is to a nucleic acid molecule that encodes a transcription factor comprising an amino acid sequence wherein said amino acid sequence is represented in Figure 1b, or a variant amino acid sequence that varies from the amino acid sequence in Figure 1b by addition, deletion or substitution of at least one amino acid residue wherein said variant polypeptide substantially lacks transcription factor activity.

A variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, truncations that may be present in any combination. Among 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. The following non-limiting list of amino acids are considered conservative replacements (similar): a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or enhance the same biological function and activity as the reference polypeptide from which it varies.

In addition, the invention features polypeptide sequences having at least 60-75% identity with the polypeptide sequences as herein disclosed, or fragments and functionally equivalent polypeptides thereof. In one embodiment, the polypeptides have at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, still more preferably at least 97% identity, and most preferably at least 99%

identity with the amino acid sequences illustrated herein.

In a preferred embodiment of the invention said plant transgenic cell is modified to reduce the expression of said nucleic acid molecule or polypeptide activity wherein transcription factor activity is reduced by at least 10% when compared to a non- transgenic reference cell of the same species.

Preferably said activity is reduced by between about 10%-99%. More preferably said activity is reduced by at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95% or at least 99%.

In a preferred embodiment of the invention said transgenic plant cell is null for a nucleic acid molecule comprising a sequence selected from the group consisting of: i) the nucleic acid molecule comprising a sequence as represented by Figure 1a; and ii) nucleic acid molecules that hybridize under stringent hybridization conditions to the sequence of (i) above and which have transcription factor activity.

Null refers to a cell that includes a non-functional copy of the nucleic acid sequence described above wherein the activity of the polypeptide encoded by said nucleic acid is ablated. Methods to provide such a cell are well known in the art and include the use of anti-sense genes to regulate the expression of specific targets; insertional mutagenesis using T-DNA; the introduction of point mutations and small deletions into open reading frames and regulatory sequences and double stranded inhibitory RNA (RNAi).

A number of techniques have been developed in recent years that 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 that 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 that 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 that are synthesised from expression cassettes carried in vectors. The DNA molecule encoding the stem-loop RNA is constructed in two parts, a first part that is derived from a gene the regulation of which is desired. The second part is provided with a DNA sequence that is complementary to the sequence of the first part. The cassette is typically under the control of a promoter that 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 haiφin 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 the inhibition of transcription factor activity is inducible. Preferably the inducible inhibition of transcription factor activity in inducible by regulating expression of an anti-sense or siRNA directed to a mRNA encoding said transcription factor.

In a further preferred embodiment of the invention said transgenic plant is further transformed with a nucleic acid molecule encoding a polypeptide with lignocellulose modifying activity wherein said nucleic acid molecule is operably linked to a promoter that controls expression of said nucleic acid molecule.

Lignocellulose modifying enzymes generally belong to the category of enzymes involved in the degradation or turn-over of cellulose, lignin and hemicellulose. Non-limiting examples of these enzymes include endo-rhamnogalacturonan hydrolases, endo- rhamnogalacturonan lyases, endo-polygalacturonases, endo-pectate lyases, endo-pectin lyases, endo-galactanases, endo-arabinanases, endo-xyloglucanases, endo- glucanases, xylanases, arabi- noxylanases, xylogalacturonases, arabinofuranosidases, galactosidases, fucosidases, exo-galacturonases, xylosidases., β-mannanase, β-1,4- glucanase, expansin, xyloglucan, endotransglycosidases, polygalacturonases, endoglucanase, exoglucanase, cellbiohydrolase, β glucosidase, xylanase, and β xylosidase. Expression of these genes in biomass crops will result in cell wall loosening

and cell wall breakdown which will be valuable for biomass utilisation by either making the cell walls more available to further breakdown to component sugars by additional enzymes or releasing sugars that can be used as feedstocks for fermentation directly.

In a preferred embodiment of the invention said nucleic acid molecule is part of a vector. "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. Of particular of interest in the present context are nucleic acid constructs which operate as plant vectors. For example those described by Guerineau and Mullineaux (1993) (Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Cray RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148. Suitable vectors may include plant viral-derived vectors (see e.g. EP194809). 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.

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-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 McNeINs 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.

In a further preferred embodiment of the invention said promoter sequence is a senescence inducible promoter sequence.

Senescence specific promoters are known in the art. For example, WO0070061; US2004025205; WO2006102559; US6, 359, 197; WO2006025664 the contents of which are incorporated by reference in their entirety, describe various plant promoters that become activated when senescence is induced. The present disclosure also describes two promoters that control the expression of genes involved in triacylglycerol metabolism. The genes that encode ACX 1 and KAT 2 are both induced during the induction of senescence and are therefore considered a least in part, senescence inducible.

Vectors may also include a selectable genetic marker 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 transgenic plant comprising a plant cell according to the invention.

In a preferred embodiment of the invention said plant cell is selected from the group consisting of: In a preferred embodiment of the invention said plant is selected from the group consisting of: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), flax

(LJnum usitatissimum), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale),

sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annus), 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 biomass crops (switchgrass, alfalfa, willow, poplar, eucalyptus, miscanthus, wheat, maize or barley), other 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 oilseed rape, sugar beet, maize, sunflower, soybean, sorghum, and flax (linseed). Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassica including cabbage, broccoli, and cauliflower. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper.

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

According to a further aspect of the invention there is provided a method to enhance the growth of a plant comprising: i) providing a transgenic plant cell is wherein said plant is modified which modification is to a nucleic acid molecule that encodes a transcription factor encoded by a nucleic acid sequence selected from the group consisting of: a) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 1a; b) a nucleic acid molecule that hybridizes under stringent hybridization conditions to the nucleic acid sequence in Figure 1a and encodes a polypeptide with transcription factor activity wherein said modification is an addition, deletion or substitution of at least one nucleotide which provides a substantially inactive

transcription factor when compared to a non-transgenic reference cell of the same plant species; ii) cultivating said cell to produce a plant; and optionally iii) harvesting said plant or part thereof.

In a preferred method of the invention said transgenic plant cell is part of a seed.

In a further preferred method of the invention said cultivation is conducted at sub-optimal temperature conditions.

In a preferred embodiment of the invention said cultivation is conducted between 10-20° C; preferably between 10-15 ⁰ C.

In a preferred method of the invention said plant is a biomass crop selected from the group consisting of: switchgrass, alfalfa, willow, poplar, eucalyptus, miscanthus, wheat, maize or barley.

According to a further aspect of the invention there is provided the use of a gene encoded by a nucleic acid molecule as represented by the nucleic acid sequence in Figure 1a, or a nucleic acid molecule that hybridizes to the sequence in Figure 1a and encodes a polypeptide with transcription factor activity as a means to identify a locus wherein said locus is associated with reduced expression or activity of said transcription factor.

Mutagenesis as a means in 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].

According to a further aspect of the invention there is provided a method to produce a plant variety that has reduced expression of a transcription factor comprising the steps of: i) mutagenesis of wild-type seed from a plant that does express said transcription factor;

ii) cultivation of the seed in i) to produce first and subsequent generations of plants; iii) obtaining seed from the first generation plant subsequent generations of plants ; iv) determining if the seed from said first and subsequent generations of plants has reduced expression of said transcription factor; v) obtaining a sample and analysing the nucleic acid sequence of a nucleic acid molecule selected from the group consisting of: a) a nucleic acid molecule comprising a nucleic acid sequence as represented in Figure 1a; b) a nucleic acid molecule that hybridises to the nucleic acid molecule in a) under stringent hybridisation conditions and that encodes a polypeptide with transcription factor activity; and optionally vi) comparing the sequence of the nucleic acid molecule in said sample to a nucleic acid sequence of a nucleic acid molecule of a plant that has reduced expression of said the transcription factor.

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; i) forming a preparation comprising the amplified nucleic acid and nucleic acid extracted from wild-type seed to form heteroduplex nucleic acid; ii) 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 iii) determining the site of the mismatch in said nucleic acid heteroduplex.

In a preferred method of the invention said plant variety does not express said transcription factor.

According to a further aspect of the invention there is provided a plant obtained by the method according to the invention wherein said plant is modified wherein said modification is transformation with a nucleic acid molecule encoding a polypeptide with

lignocellulose modifying activity wherein said nucleic acid molecule is operably linked to a promoter that controls expression of said nucleic acid molecule.

In a preferred embodiment of the invention said plant is a biomass plant selected from the group consisting of: switchgrass, alfalfa, willow, poplar, eucalyptus, miscanthus, wheat, maize or barley.

According to a further aspect of the invention there is provided a method to prepare plant biomass for saccharification comprising: i) forming a preparation comprising a plant biomass material comprising a plant identified by the method according to the invention and optionally including at least one lignocellulose modifying enzyme ; and ii) incubating said preparation under conditions that modify said plant biomass material to hydrolyse and/modify said material.

In a preferred method of the invention said hydrolysed and/or modified material is further processed by saccharification to sugar.

In a further preferred method of the invention said sugar is used as a feedstock in the production of ethanol by microbial fermentation.

Microorganisms used in the process according to the invention are grown or cultured in the manner with which the skilled worker is familiar, depending on the host organism. As a rule, microorganisms are grown in a liquid medium comprising a carbon source (e.g. sugar as formed during the saccharification process), a nitrogen source, usually in the form of organic nitrogen sources such as yeast extract or salts such as ammonium sulfate, trace elements such as salts of iron, manganese and magnesium and, if appropriate, vitamins, at temperatures of between 0°C and 100 ⁰ C, preferably between 10 ⁰ C and 60 ⁰ C, while gassing in oxygen.

The pH of the liquid medium can either be kept constant, that is to say regulated during the culturing period, or not. The cultures can be grown batch wise, semi-batchwise or continuously. Nutrients can be provided at the beginning of the fermentation or fed in semi-continuously or continuously. The products produced can be isolated from the organisms as described above by processes known to the skilled worker, for example by extraction or distillation. In this process, the pH value is advantageously kept between pH 4 and 12, preferably between pH 6 and 9, especially preferably between pH 7 and 8.

The culture medium to be used must suitably meet the requirements of the strains in question. Descriptions of culture media for various microorganisms can be found in the textbook "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington D.C., USA ₁ 1981).

As described above, these media which can be employed in accordance with the invention usually comprise one or more, nitrogen sources, inorganic salts, vitamins and/or trace elements.

Nitrogen sources are usually organic or inorganic nitrogen compounds or materials comprising these compounds. Examples of nitrogen sources comprise ammonia in liquid or gaseous form or ammonium salts such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources such as cornsteep liquor, soya meal, soya protein, yeast extract, meat extract and others. The nitrogen sources can be used individually or as a mixture.

Inorganic salt compounds which may be present in the media comprise the chloride, phosphorus and sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

Inorganic sulfur-containing compounds such as, for example, sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, or else organic sulfur compounds such as mercaptans and thiols may be used as sources of sulfur for the production of sulfur- containing fine chemicals, in particular of methionine.

Phosphoric acid, potassium dihydrogenphosphate or dipotassium hydrogenphosphate or the corresponding sodium-containing salts may be used as sources of phosphorus.

Chelating agents may be added to the medium in order to keep the metal ions in solution. Particularly suitable chelating agents comprise dihydroxyphenols such as catechol or protocatechuate and organic acids such as citric acid.

The fermentation media used according to the invention for culturing microorganisms usually also comprise other growth factors such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenate and pyridoxine. Growth factors and salts are frequently derived from complex media components such as yeast extract, molasses, cornsteep liquor and the like. It is moreover possible to add suitable precursors to the culture medium. The exact

composition of the media compounds heavily depends on the particular experiment and is decided upon individually for each specific case. Information on the optimization of media can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Editors P.M. Rhodes, P.F. Stanbury, IRL Press (1997) pp. 53-73, ISBN 0 19 963577 3). Growth media can also be obtained from commercial suppliers, for example Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) and the like.

All media components are sterilized, either by heat (20 min at 1.5 bar and 121 "C) or by filter sterilization. The components may be sterilized either together or, if required, separately. All media components may be present at the start of the cultivation or added continuously or batch wise, as desired.

The culture temperature is normally between 15°C and 45°C, preferably at from 25 ^β C to 40 ⁰ C, and may be kept constant or may be altered during the experiment. The pH of the medium should be in the range from 5 to 8.5, preferably around 7.0. The pH for cultivation can be controlled during cultivation by adding basic compounds such as sodium hydroxide, potassium hydroxide, ammonia and aqueous ammonia or acidic compounds such as phosphoric acid or sulfuric acid. Foaming can be controlled by employing antifoams such as, for example, fatty acid polyglycol esters.

The fermentation broth can then be processed further. The biomass may, according to requirement, be removed completely or partially from the fermentation broth by separation methods such as, for example, centrifugation, filtration, decanting or a combination of these methods or be left completely in said broth.

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.

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 1a the nucleic acid sequence of SPT; Figure 1b is the amino acid sequence of SPT;

Figure 2 illustrates total leaf area of Arabidopsis wild type, spatula mutant and over expressing plants during vegetative growth at either 15°C or 25°C. Values represent the mean and standard error of up to ten individual plants;

Figure 3 illustrates the biomass of Arabidopsis plants grown for 37 days at either 15°C or 25°C. Values shown are the mean and standard error of up to ten individual plants; and

Figure 4 shows a western blot demonstrating SPATULA protein levels when expressed under the constitutive cauliflower mosaic virus 35S promoter in 3 week old Arabidopsis plants growing at either 15°C (15) or 25°C (25) at various times in the diurnal cycle. The SPATULA protein is detected using an antibody specific for the hemagglutenin (HA) epitope tag which was added to the C-terminus of the SPT protein (Penfield et al., 2005).

Materials and methods

Wild type and spt-2 seeds were obtained from NASC. We obtained one further allele from NASC designated WiscDsLox466B7 which we called spt-11. This allele contains a T-DNA insertion in the first exon and has the seedling, petal and fruit phenotypes described for other spt alleles (Penfield et al., 2005), and is in the Columbia (CoI) wild type background. Plants over expressing SPT under the control of the Cauliflower Mosaic Virus 35S promoter and fused to a hemagglutenin epitope tag have been described previously (Penfield et al., 2005; designated SPT-OX). SPT-OX and spt-2 are in the Landsberg erecta (Ler) wild type background.

Wild type Arabidopsis thaliana (CoI and Ler), spt-2, spt-11 and SPT-OX seeds were sown on tissue culture plates containing 1x Murashige and Skoog medium and incubated at either 15°C or 25 ⁰ C ₁ with 75umol light in 12 hour light, 12 hour dark photoperiod. After 7 days these plants were transferred to compost and grown for a further month under the previously indicated conditions. Total leaf area of whole plants was measured from standard photographs taken with a Livecam Optia AF webcam, and calculated using I mage J. Fresh (above ground) weight was measured 37 days after sowing and the mean and standard error calculated from up to ten individuals of each genotype.

Eξxample

Previously we have shown that the bHLH transcription factor known as SPATULA (SPT) is important for temperature-regulated seed germination control (Penfield et al., 2005). In order to investigate the affect of temperature on the growth of spt mutant (spt-2, spt-11) and over expressing lines (SPT-OX), we grew plants at either 15 ^β C or 25°C under controlled conditions. At 15°C wild type growth substantially suppressed compared to 25°C (Figure 1A, B). Notably, both spt mutants tested showed a significantly increased growth phenotype compared to wild type at 15°C (Figure 1A). This increased growth phenotype was obvious throughout vegetative development. Fresh weight measurements revealed that spt mutant plants accumulated nearly twice as much vegetative biomass as wild type by 37 days after sowing (Figure 2A). In contrast, over expression of SPT resulted in plants that grow very small compared to wild type at 15°C.

Next we examined spt mutant growth at 25 ^β C. The two spt mutants grew faster than wild type at 25°C, but the overall increase in growth rate was low compared to that at 15°C. Plants over expressing SPT also grew at a rate similar to that of wild type at 25 ⁰ C. This indicates that SPT expression levels are relatively unimportant for the control of growth rate at higher temperatures, and that SPT function is to regulate growth rate in plants grown at temperatures below those that are optimal for growth. Measurements of plant biomass after 37 days of growth confirmed that at 25°C spt mutants were only slightly faster than wild type at 25°C, and that SPT-OX plants were not significantly slower (Figure 2B). Therefore we concluded that plants with reduced SPT activity show increased growth under cool conditions, while leaving growth at warmer temperatures unaffected.

Reference

Penfield et al., 2005 Cold and light control seed germination through the bHLH transcription factor SPATULA. Curr Biol. 15(22): 1998-2006.