NOVEL PLANT GENES AND USES THEREOF

STATEMENT OF CORRESPONDING APPLICATIONS

This application is based on the Provisional specification filed in relation to New Zealand Patent Application Number 545621 , the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to novel plant genes and uses thereof. In particular, the present invention relates to novel plant genes involved in plant development and cell differentiation. Most preferably the invention relates to genes and polypeptides capable of: affecting the growth and shape of lateral shoot organs in plants or plant cells; or altering the arrest of meristemoid cell division during organ development.

BACKGROUND ART

Economic importance of plant lateral shoot organ size and shape:

Much of the economic value of crop plants comes either directly or indirectly from the growth and form of the lateral shoot organs: the leaves, the flowers (parts = sepal, petals, anthers and carpels), and the fruit/seeds/seed pods that develop from the flowers. Some of the economic value derived from variation in plant organ size or shape is obvious. Larger fruit or seed may represent a greater harvest index, more grain or fruit per plant or per crop area. The value of plant ornamentals is often because of unusual leaf, flower or seed pod shapes. Horticulturalists have been collecting "odd" plants because of their commercial value for centuries. Other economic impacts of variability in organ size and shape may not be so obvious. For example the leaf blade is the main site of photosynthesis and respiration in higher plants and leaf size and shape are key factors influencing these processes. Plants with large leaves grown in hot dry climates lose a lot of water by transpiration. Narrow curled leaves maybe a means of avoiding excessive transpiration

(i.e. less water use). Larger leaves on short stems can result in a higher grain yield, due to more carbon etc being made available for seed development instead of vegetative growth. These benefits are both indirect. They come from improvements in environment adaptation or the redirection of nutrient resources within the plant.

So the economic value of differences in the size and shape of plant shoot lateral organs are many and varied. The benefits of being able to manipulate leaf/flower/fruit size and shape are greatest if an invention is applicable to a wide range of crops. There is additional value if an invention allows independent manipulation of the size and/or shape of individual organ types in a plant without affecting the other organs.

Flexibility in growth of the plant organ laminal plane:

All plant shoot lateral organs have a number of planes of growth. One of these planes is the adaxial (upper or adjacent to the shoot meristem)-abaxial (lower or away from the shoot meristem) axis. Generally this plane determines the thickness of a leaf. Another is the proximal (base)-distal (tip) axis. This axis contributes to the length of the organ. Finally there is the medial (midrib)-lateral (margin) axis or blade. Growth of the blade contributes to both the length and width of the organ. The blade or lamina is most obvious in a leaf but the same laminal plane exists in flower sepals, petals, anthers and seed pods. There is substantial evidence that these other organs have evolved from leaves. Notably it is the leaves and seed pods that have the longest duration of growth and expand most after the initial shape is determined by development of the organ primordia. The leaves and seed pods also have the greatest flexibility in size of the lateral shoot organs. Under optimal growth conditions the leaves and fruit increase most in size whereas flowers are less flexible in size.

For ease of reference, development of the leaf will be discussed as an example. However it should be appreciated that many of the same processes occur during development of the other shoot lateral organs. During normal leaf development a wave of cell cycle arrest moves from the tip to the base of the developing organ and behind this arrest front most

cells differentiate and expand to form the mature leaf blade. However, immediately behind the front of general primordial cell division arrest, there is a short transition phase where a mixture of expanding differentiated cells and stem cell-like meristematic cells (termed meristemoids) co-exist. The meristemoids undergo asymmetric cell division and the progeny cells all differentiate into cell types appropriate to the tissue layer in which they occur. Meristemoid cell division is subsequently arrested and all cells then differentiate. Clearly meristemoid division contributes more cells to the developing organ, but neither the influence of meristemoid cell division on organ shape and size nor the genetic control of meristemoid cell division arrest is currently known.

The present invention is derived from the discovery of a novel Arabidopsis thaliana type of gene, named PEAPOD (PPD) that controls the arrest of meristemoid cell division during , organ development. Alterations in the number of copies of this gene or the level of PPD transcription, controls the timing of meristemoid cell division arrest. Fewer copies of PPD or less PPD transcription prolongs meristemoid cell division resulting in a larger organ blade. In the case of a complete loss of PPD function blade growth exceeds the expansion capacity of the margin and the organ develops a bell shape (referred to as positive Gaussian curvature) rather than the normal flat plane (zero Gaussian curvature). Higher levels of PPD transcription restrict organ size (and the flexibility of organ size) but the organs retain wild type shape. The PPD gene encodes a novel protein. Homologous genes are present in a wide range of eudicot plants and analysis of the primary amino acid structure of these proteins indicates the presence of a highly conserved novel plant specific domain present only in the PEAPOD-like proteins.

Other genes that influence shoot lateral organ size or curvature-the literature:

It has been proposed that the AINTEGUMENTA (ANT) gene, encoding an AP2-like transcription factor, controls organ size by regulating meristematic competence of cells within the lateral organ primordia. Mutations in the ANT gene of Arabidopsis disrupt ovule and floral organ growth and reduce the size of lateral organs by decreasing cell number.

Ectopic expression of the ANT gene (expression in most tissues rather than the restricted

tissue expression normally found in wild type plants), enlarges embryonic and all shoot organs without altering superficial morphology by increasing cell number. However, cells expressing ANTm mature organs exhibit neoplastic growth producing calli and adventitious shoots and roots. The ANT gene therefore acts to promote cell division. (Mizukami & Fischer, 2000. PNAS 97: 942-947).

The JAGGED (JAG) gene of Arabidopsis appears to have a similar effect. Loss-of-function mutations of the JAG gene of Arabidopsis result in abnormal lateral organs including small serrated leaves, narrow floral organs, and petals with fewer larger cells. The JAG gene encodes a protein with a single C2H2 zinc-finger domain (transcription factor). Misexpression of JAG results in leaf fusion and the development of ectopic leaf-like outgrowth from both leaf and floral tissue. (Ohno et al., 2004. Development 131 : 1111- 1122).

ARGOS is another Arabidopsis gene that is involved in organ size control. Over expression of ARGOS in Arabidopsis increases cell proliferation resulting in larger organs, reduced expression reduces growth and results in smaller organs. It is proposed that ARGOS regulates growth through ANT during development. (Hu et al., 2003. Plant Cell 15: 1951- 1961 ).

It is noteworthy that loss-of-function for all these genes reduces growth, whereas miss expression increases organ size, with some distortion in morphology. It is probable that all the above genes act to promote primordial cell division. There is no indication that they influence meristemoid cell division.

In Antirrhinum (snapdragon), two members of the TCP family of transcription factors, encoded by Cl NCI NNATA (CIN) and CYCLOIDEA (CYC), appear to affect organ shape by promoting cell differentiation. CIN controls growth of the leaf blade and is required to produce flat leaves. Mutations in the CIN gene result in excessive growth at the periphery of the leaf blade, resulting in wavy leaves (negative Gaussian curvature). CIN appears to prevent excessive growth by sensitising peripheral cells to the cell cycle arrest front that

moves from the tip to the base. (Nath et al., 2003. Science 299: 14011407). CYC suppresses growth of dorsal floral structures producing asymmetric flowers and also appears to regulate growth by altering the cell cycle. (Luo et al., 1996. Nature 383:794- 799).

PPD is distinct in its predicted protein sequence, mode of action, loss-of-function phenotype, and elevated expression phenotype, from any gene previously described. The protein appears to act by restricting meristemoid cell division during growth of shoot organ laminal tissue. The result of this action is to co-ordinate growth of the organ blade and margin so that a "normal" curvature is maintained. Because the level of PPD transcription (or gene dosage) modulates the timing of meristemoid cell cycle arrest (this could be either by promoting differentiation or inhibiting cell division) the PPD gene appears to be a key regulator of plant shoot organ size flexibility.

All references, including any patents or patent applications cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents form part of the common general knowledge in the art, in New Zealand or in any other country.

It is acknowledged that the term 'comprise' may, under varying jurisdictions, be attributed with either an exclusive or an inclusive meaning. For the purpose of this specification, and unless otherwise noted, the term 'comprise' shall have an inclusive meaning - i.e. that it will be taken to mean an inclusion of not only the listed components it directly references, but also other non-specified components or elements. This rationale will also be used when the term 'comprised' or 'comprising' is used in relation to one or more steps in a method or process.

It is an object of the present invention to address the foregoing problems or at least to provide the public with a useful choice.

Further aspects and advantages of the present invention will become apparent from the ensuing description which is given by way of example only.

DISCLOSURE OF INVENTION

According to one aspect of the present invention there is provided an isolated nucleic acid molecule having a nucleotide sequence comprising:

a) a sequence selected from SEQ ID NOs. 1 , 2, 4 . 5, or 7;

b) a complement of a sequence in a);

c) a functional fragment or variant of a sequence in a) or b); or

d) a homolog or an ortholog of a sequence in a), b), or c).

According to another aspect of the present invention there is provided an isolated polypeptide having an amino acid sequence comprising:

a) a sequence selected from SEQ ID NOs. 3 , 6, or 8; or

b) a functional fragment or variant of a sequence in a); or

c) a homolog or an ortholog of a sequence in a) or b).

According to another aspect of the present invention there is provided an isolated nucleic acid molecule encoding a domain having conserved amino acids:

SXLXKPLXXLTXXDISQXTREDCRXXLKXKGMRXPSWNKSQAIQQVXXXKXLXE

wherein X = any amino acid.

According to another aspect of the present invention there is provided an isolated polypeptide encoding a domain having a conserved amino acid sequence:

SXLXKPLXXLTXXDISQXTREDCRXXLKXKGMRXPSWNKSQAIQQVXXXKXLXE

wherein X = any amino acid.

According to a further aspect of the present invention there is provided the use of a probe to a nucleic acid molecule encoding a conserved amino acid domain substantially as described above.

According to a further aspect of the present invention there is provided a nucleic acid molecule encoding a polypeptide substantially as described herein, comprising an amino acid sequence substantially as set forth in the sequence listing; or a functional fragment or variant thereof; or a homolog or ortholog thereof.

According to another aspect of the present invention there is provided a use of a nucleic acid molecule substantially as described above to alter a plant or plant cell.

According to another aspect of the present invention there is provided a use of a nucleic acid molecule substantially as described above to alter the growth and/or shape of lateral shoot organs in plants or plant cells.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are eudicots.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are Trifolium repens.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are Arabidopsis.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are cotton.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are soya bean.

According to another aspect of the present invention there is provided the use of a nucleic acid molecule substantially as described above wherein the plants or plant cells are Nicotiana.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above to alter the growth in plants or plant cells.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above to alter the growth and/or shape of lateral shoot organs in plants or plant cells.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant or plant cells are eudicots.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant or plant cells are Trifolium repens.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant cells are Arabidopsis.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant cells are cotton.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant cells are soya beans.

According to another aspect of the present invention there is provided the use of a polypeptide substantially as described above wherein the plant cells are Nicotiana.

According to another aspect of the present invention there is provided a vector or construct including a nucleotide sequence substantially as described herein.

According to a further aspect of the present invention there is provided a cell transformed with a vector or construct substantially as described above.

According to a yet still further aspect of the present invention there is provided a cell which has been altered from the wild type to include a nucleic acid molecule substantially described herein.

According to a still further aspect of the present invention there is provided a probe comprising at least 20 or more contiguous nucleotides selected from a sequence substantially as set forth in the sequence listing; or a functional fragment or variant thereof; or a homolog or ortholog thereof.

According to a still further aspect of the present invention there is provided a plant which has been altered from the wild type to include a nucleic acid molecule substantially as described above.

According to a still further aspect of the present invention there is provided a progeny of a plant cell or a plant substantially as described above.

According to a still further aspect of the present invention there is provided a clone of a plant cell or plant substantially as described above.

According to a still further aspect of the present invention there is provided a use of a nucleic acid molecule substantially as described herein to control the arrest of meristemoid cell division during organ development.

According to a still further aspect of the present invention there is provided a use of a polypeptide substantially as described herein to control the arrest of meristemoid cell division during organ development.

According to a further aspect of the present invention there is provided a primer for the conserved PEAPOD domain which has the following nucleotide sequence:

5' GAY ATH WSN CAR BTN CAN MGN GAR 3'.

Preferably, the primer may have the sequence:

5' GAT/C ATA/C/T T/AC/GN CAA/G C/T/GTN ACN C/AGN GAA/G 3'.

According to another aspect of the present invention there is provided a primer for the conserved PEAPOD domain which has the following nucleotide sequence:

5 ¹ NAC YTG YTG DAT NGC YTG NSW YTT RTT 3'.

Preferably, the found primer may have the sequence:

5 ¹ NAC T/CTG T/CTG T/G/AAT NGC TICTG NG/CT/A T/CTT G/ATT 3'.

In order to achieve altered levels of PEAPOD gene expression one or more of the following prior art techniques may be employed:

Over expression - refer He, S.S., Liu, J., Xie, Z., O'Neill, D., Dotson, S. (2004) Plant Molecular Biology 56: 171-184.

RNAi gene silencing - refer Stoutjesdijk, P.A., Singh, S.P., Liu, Q., Hurlston, CJ. ,

Waterhouse, P.A., Green, A.G. (2002) Plant Physiology 129: 1723-1731.

Gene deletion - refer Lloyd, A., Plaisier, C.L., Carroll, D., Drews, G.N. (2005) PNAS 102: 2232-2237.

Mutation - refer Perry, J.A., Wang, T.L., Welham, TJ. , Gardner, S., Pike, J. M., Yoshida, S., Parniske, M. (2003) Plant Physiology 131 : 866-871.

Other suitable techniques as known to a person skilled in the art may also be employed.

The term "nucleic acid molecule" as used herein may be an RNA, cRNA, genomic DNA or

cDNA molecule, and may be single- or doublestranded. The nucleic acid molecule may also optionally comprise one or more synthetic, non-natural or altered nucleotide bases, or combinations thereof.

The term "isolated" means substantially separated or purified away from contaminating sequences in the cell or organism in which the nucleic acid naturally occurs and includes nucleic acids purified by standard purification techniques as well as nucleic acids prepared by recombinant technology, including PCR technology, and those chemically synthesised.

The term 'variant' as used herein refers to a nucleic acid molecule or polypeptide wherein the nucleotide or amino acid sequence exhibits substantially 70, 80, 95, or 99% homology with the nucleotide or amino acid sequence as set forth in the sequence listing - as assessed by GAP or BESTFIT (nucleotides and peptides), or BLASTP (peptides), or BLASTX (nucleotides). It should be appreciated that the variant may result from a modification of the native nucleotide or amino acid sequences, or by modifications including insertion, substitution or deletion of one or more nucleotides or amino acids. Where such a variant is desired, the nucleotide sequence of the native DNA may be altered appropriately for example by synthesis of the DNA de novo, or by modification of the native DNA, for example by site-specific or cassette mutagenesis. Preferably, where portions of the cDNA or genomic DNA require sequence modifications, site-specific primer directed mutagenesis is employed using techniques standard in the art. Alternatively, a variant may be naturally occurring. The term variant also encompasses homologous sequences which hybridise under stringent conditions to the sequences of the invention.

The term 'variant' also encompasses "conservative substitutions" wherein the alteration of the nucleotide or amino acid sequences, as set out in the sequence listing of this specification, results in the substitution of a functionally similar amino acid residue (Creighton T. E. 'Proteins Structure and Molecular Properties.' WH Freeman and Co. 1984).

The term 'fragment nucleic acid molecule' as used herein refers to a nucleic acid molecule

which represents a portion of the nucleic acid molecule of the present invention and is therefore less than full length and comprises at least a minimum sequence capable of hybridising stringently with a nucleic acid molecule of the present invention (or a sequence complementary thereto).

A "fragment" of a polypeptide of the present invention is a portion of the polypeptide that is less than full length. Preferably the polypeptide fragment has at least approximately 60% identity to a polypeptide of the present invention, more preferably at least approximately an 80% identity, and most preferably at least approximately a 90% identity. Preferably the fragment has size of at least 10 amino acids, more preferably at least 15 amino acids, and most preferably at least 20 amino acids.

As used herein, a "substantially identical" amino acid sequence is an amino acid sequence which differs only by conservative amino acid substitutions. For example, substitution of one amino acid for another of the same class (e.g., valine for glycine, arginine for lysine and so forth) or by one or more non-conservative substitutions, deletions, or insertions located at positions of the amino acid sequence which do not destroy the function of the protein. Preferably, such a sequence is at least 85%, more preferably 90% and more preferably 95% identical to amino acid level to the sequence of the protein or peptide to which it is being compared.

A "substantially identical" nucleotide sequence codes for a substantially identical amino acid sequence as defined above. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.

The term "ortholog", "orthologous gene", or "orthologous polypeptide" refers to a functionally equivalent yet distinct corresponding nucleotide or amino acid sequence that may be derived from another plant. In general an ortholog may have a substantially identical nucleotide or amino acid sequence to the sequences of the present invention as set forth in the sequence listing.

The term "construct" as used herein refers to an artificially assembled or isolated nucleic acid molecule which includes the gene or nucleic acid molecule of interest. In general a construct may include the gene or genes of interest and appropriate regulatory sequences. It should be appreciated that the inclusion of regulatory sequences in a construct is optional for example, such sequences may not be required in situations where the regulatory sequences of a host cell are to be used. The term construct includes vectors but should not be seen as being limited thereto.

The term "vector" as used herein encompasses both cloning and expression vectors. Vectors are often recombinant molecules containing nucleic acid molecules from several sources.

The cloning vector selected may depend on the host and host cell as used. In general the vector may:

• self replicate;

• include a marker gene for selection of transformed cells;

• have a nucleic acid sequence capable of being cleaved by an endonuclease

Some examples of suitable vectors may include:

• General cloning - pGEMT

• Binary plant transformation vectors - pRD410, pHZbar, pKR10, pGREEN, pBin19.

In most cases the nucleic acid molecules of the present invention may be expressed via a control sequences such as a promoter operably linked thereto. Other control sequences may also be employed and may include origins of replication, enhancer and transcriptional terminator sequences.

The term "clone" as used herein refers to a population of cells derived from a single cell.

The term "stable clone" refers to a host cell which incorporates and expresses the exogenous nucleic acid or polypeptide molecule introduced via a vector or construct.

The term "transformed cell" as used herein refers to a cell into which (or into an ancestor of which) there has been introduced, by means of recombinant DNA techniques, a nucleic acid molecule of interest. The nucleic acid of interest will typically encode a peptide or protein. The transformed cell may express the sequence of interest or may be used only to propagate the sequence. The term "transformed" may be used herein to embrace any method of introducing exogenous nucleic acids including, but not limited to, transformation, transfection, electroporation, microinjection, viral-mediated transfection, and the like.

The term "probe" as used herein refers to a single-stranded nucleic acid molecules with a known nucleotide sequence which are labelled in some way (for example, radioactively, fluorescently or immunologically), or are otherwise detectable, and which are used to find and mark a target DNA or RNA sequence by hybridizing to it.

The term "protein (or polypeptide or peptide)" refers to a protein encoded by the nucleic acid molecules of the invention, including fragments, mutations and homologs, orthologs, or analogs having the same biological activity. The protein or polypeptide or peptide of the invention can be isolated from a natural source, produced by the expression of a recombinant nucleic acid molecule, or can be chemically synthesized.

The term 'plant' refers to the plant in it's entirety or a part thereof including selected portions of the plant during the plant life cycle such as the plant seeds, shoots, leaves, bark, pods, roots, flowers, stems and the like, or parts thereof.

The term 'plant cell' refers to any plant cell(s) from any stage of the plant life cycle, including plant seed cells, shoot cells, leave cells, bark cells, root cells, flower cells, pod cells, stem cells and the like.

For ease of reference the gene sequence of the present invention will now simply be referred to as PEAPOD, however, this should not be seen as limiting.

Preferred embodiments of the present invention may be applied in the following non-limiting ways:

• Adding 1 or more copies of the PEAPOD gene (from Arabidopsis or the target crop) to a crop or ornamental plant by transformation to reduce leaf and/or seed pod size. Here the intact gene with its own promoter may be added.

• Deleting 1 or more copies of the PEAPOD gene from a crop or ornamental plant by mutagenesis or site directed recombination to increase leaf and/or seed pod size. This includes removal of any part of the peapod gene.

• Reducing the level of PEAPOD gene expression by partial or full inactivation of its mRNA by introducing an antisense or RNA interference construct of the gene to increase or alter the shape of plant shoot organs. This construct to be expressed from either the peapod promoter or any other promoter active in plants. The inactivation construct might also be expressed under the control of a tissue specific or inducible or tissue-specific inducible promoter (see below).

• Increasing the level of PEAPOD gene expression by over expression of a transgene introduced into a crop or ornamental plant resulting in reduced organ size or alter organ shape. This involves introducing the genomic or cDNA coding portion of the gene fused to a promoter active in plants as above.

• Identifying and/or altering the wild type activity of orthologous genes in other plants.

Thus, the present invention may be useful in achieving genetically directed increases or decreases in the level of PEAPOD gene expression (this extends to orthologous genes where ever they are found in plants) to either increase or decrease the size of shoot organs. Altering the expression includes changes to the number of PEAPOD genes in the genome, gene silencing, mutating the PEAPOD gene, modification of the level of peapod gene transcription, steady state levels of mRNA or activity of the PEAPOD protein by alterations to the "PEAPOD Domain" conserved amino acid sequence.

Similarly, the invention may be used to genetically direct increases or decreases in the level of PEAPOD gene expression to alter the shape of plant shoot organs.

Furthermore, PEAPOD genes can be reasonably predicted to be capable of manipulating; leaves, seed pods, fruits and flower petals.

BRIEF DESCRIPTION OF DRAWINGS

Further aspects of the present invention will become apparent from the following description which is given by way of example only and with reference to the accompanying drawings in which:

Figure 1 Leaves of peapod mutant plants are enlarged and have a bell-shaped curvature rather than the flat blade of wild type plants. The seed pods of peapod plants have a wide and flattened shape rather than the long tubular shape of wild type plants;

Figure 2 Replica SEM of the leaf surface of wild type and peapod mutant leaves 14 days after germination;

Figure 3 Cyclin index during development of leaves and siliques;

Figure 4 Chromosomal map position of the peapod locus;

Figure 5 Independent T-DNA insertions lines obtained for both PPD1 & PPD2

Figure 6a Q RT-PCR relative PEAPOD mRNA levels in wild type and complemented transgenic peapod inflorescences;

Figure 6b Mutant peapod plants are complemented by transformation with either PPD1 or PPD2 cloned genes (genomic); and

Figure 7 Multiple alignment of PPD-like proteins.

Figure 8 Phenotypes of Arabidopsis peapod mutant plants are complemented by transformation with TrPPD cloned cDNA.

Figure 8a Mature leaves of peapod mutant (left) and three different complemented transgenic plants (right).

Figure 8b Siliques of peapod (second left), wild type (left) and three different

TrPPD cDNA transgenic plants (right).

BRIEF DESCRIPTION OF SEQUENCE LISTING

Sequence Name Corresponding sequence

SEQ ID No. 1 At4g14713 genomic DNA SEQ ID No. 2 At4g14713.1 cDNA

SEQ ID No. 3 At4g 14713.1 protein

SEQ ID No. 4 At4g14720 genomic DNA

SEQ ID No. 5 At4g14720.1 cDNA

SEQ ID No. 6 At4g 14720.1 protein SEQ ID No. 7 TrPPD cDNA

SEQ ID No. 8 TrPPD protein

BEST MODES FOR CARRYING OUT THE INVENTION

Experiment 1 : Arabidopsis PEAPOD gene

Experimental:

Screening for morphological mutants:

The plant having the mutant peapod gene was identified during a screen for morphological mutants of ca 3,500 M2 seedlings grown from a commercially supplied Arabidopsis thaliana Landsberg erecta ecotype population obtained from M1 plants grown from seed treated by fast neutron mutagenesis. Fast neutrons typically cause mutations by deletion of segments of the genome. A single plant with the peapod mutant phenotype (large bell shaped leaves,

short wide pea pod-like siliques, and reduced trichome branching) was identified. All M3 and M4 seed (self) collected and germinated from this plant retained the mutant phenotype.

Growth conditions:

Arabidopsis plants were germinated after stratification at 4C for 5 days in 0.1 % agarose and grown in seedling soil mix either in an Environ cabinet, 2OC, 16 hour day length, or in a heated greenhouse with supplemented artificial light as required to give a 16 day length.

Genetic analysis:

The peapod plant was backcrossed five times to wild type Landsberg erecta plants (peapod X wild type F1 selfed each time with selection of peapod homozygotes from the F2 before repeating the backcross to wild type i.e. a total of 10 generations). For mapping purposes peapod Ler (recipient) was outcrossed with wild type Columbia ecotype (pollen donor). F1 plants where selected by the dominant CoI phenotype and confirmed using a molecular CAPS marker that distinguishes Ler X CoI heterozygotes. F2 (self) progeny where screened to identify peapod homozygote segregants (those with all the original characteristics). Inheritance of peapod was determined in BC5 self progeny by scoring leaf curvature, trichome branching, and measuring silique width.

Phenotypic analysis:

Leaf dimension measurements of first and fourth leaves collected at the floral bolt stage where made by flattening the leaves between two microscope slides, scanning to produce a computer image together with an internal mm rule, and then calculating length, width, area and perimeter using a public domain image analysis programme (ImageJ). Mature silique dimensions where determined by measuring at least 30 siliques from each genotype using a digital micrometer. To detect the affect of peapod on cell division a cyc1At::GUS reporter gene was introgressed into wild type Ler and peapod Ler genetic backgrounds. Detection of GUS activity was carried out as described by Donnelly et al., 1999, Developmental

Biology 215: 407-419. For scanning electron microscopy silique samples were fixed in half

strength buffer and processed directly. For SEM replicas leaf segments were coated with polyvinylsiloxane dental impression material to make a mould. The leaf material was removed and replaced with Spurr's resin. The resin was polymerised at 6OC overnight and then coated for SEM examination.

Mapping of the PEAPOD genes:

DNA extracted from individual F2 peapod homozygous segregants was bulked into a single sample and analysed using a set of CAPS molecular markers suitable for detecting heterozygote loci (and map homozygote recombination positions) in a CoI X Ler population. The set of CAPS markers used are spaced throughout the Arabidopsis genome such that they can be used to distinguish the general chromosome arm position of a homozygous mutant locus. (Note: At the mutant locus all sequences flanking peapod are Ler. Linked markers have a higher than 50% chance of being Ler homozygotes. In bulk segregant analysis linkage is indicated by little or no apparent heterozygosity of a marker). Linkage was then confirmed by testing individual DNA samples with the CAPS marker identified by bulk segregant analysis. To fine map the position of the peapod locus DNA from individuals of a larger population of homozygous mutant F2 segregants was analysed with a series of CAPS, PCR or InDeI markers spanning regular intervals along the mapped chromosome arm. Once linkage distance and direction of the peapod locus from markers was established it became apparent that a known trichome branching gene was possibly collocated. PCR analysis using primers specific for genes located adjacent to the trichome branching gene At4g 14750 established the site and extent of chromosomal deletion in the peapod mutant.

Insertion lines:

Plant lines containing T-DNA insertions in At4g14713 or At4g14720 in the Columbia (CoI) ecotype background were identified by searching the TAIR Arabidopsis web site. Insertion lines for At4g14713 (SALKJ 49924, SALK_057237) and At4g14720 (SALKJ 4698) were obtained from the Arabidopsis Biological Resource Center, Ohio State University,

Columbus, OH, USA and verified by PCR analysis with T-DNA left border and gene specific primers, together with sequence analysis of the amplified fragment. Identification of homozygous T-DNA insertion plants was confirmed by co-segregation of the T-DNA and mutant phenotype.

Complementation:

The At4g14713 and At4g14720 genes were cloned from wild type CoI ecotype genomic DNA by Hi Fidelity PCR using gene specific primers for sequences immediately at the end of the 3' UTR and about 1.5 Kb upstream of the beginning of the 5' UTR. The At4g14713 and At4g14720 gene PCR fragments were each cloned first into pGEMT and confirmed by PCR and sequence analysis. The gene specific primers each incorporated a terminal Not 1 restriction enzyme site. Clones were excised from pGEMT by Not 1 digestion and subcloned into the unique Not 1 site of pHZbar, a binary Agrobacterium transformation vector. Plasmids pHZbAt4g14713 and pHZbAt4g14720 were transferred to Agrobacterium tumefaciens strain AGL1. Mutant peapod plants were transformed by using an /tørojbactera/m-mediated floral dip infiltration method. (Clough & Bent. 1998. Plant J 16: 735-743). Soil grown transgenic T1 plants were selected by repeated (3X) spraying with a solution of Buster (phosphothicin herbicide, 375 ug/L). Transgenic plants were confirmed by PCR analysis; with a combination of transgene specific and T-DNA primers, the absence of PCR products for other genes in the deleted region and the mutant unbranched trichome phenotype.

Quantitative RT-PCR:

Total RNA was extracted from the first 12 green siliques closest to the apical meristem of wild type, wild type appearance transgenic and abnormally small (leaf/silique) transgenic plants by using RNAEasy kits (Qiagen). First strand cDNA was synthesized by using poly(dT) primer and Moloney murine leukaemia virus reverse transcriptase (Promega) according to the manufacturers protocol. Quantitative RT-PCR assays for the relative level of At4g14713 gene transcript were performed on a Bio-Rad MyIQ colour Real Time PCR

instrument (Bio-Rad) using Ornithine Transfer Carboxylase (OTC) as an internal control. One of the gene-specific primers used to detect each gene were designed to span an intron boundary thereby eliminating detection of any contaminating genomic DNA.

Results and discussion:

The discovery leading to this invention is based on the identification of an Arabidopsis mutant, peapod, with enlarged and altered curvature of the laminal plane (mediolateral axis or blade) of lateral shoot organs. The peapod mutant has enlarged bell shaped instead of flat leaves. The seed pods of peapod plants have a wide and flattened shape rather than the long tubular shape of wild type plants (Figure 1 ). Trichomes (hairs) on the mutant have only two branches rather than the wild type 3-4 branches.

The initial approach taken was to compare the lateral shoot organ development of peapod mutant and wild type plants. Results, from experiments measuring organ dimensions and patterns of cell division and differentiation during development, indicate how loss-of- function of the PEAPOD gene(s) leads to modifications to organ shape and size. For example, peapod leaves have a greater length, width and lamina area, but the same perimeter as wild type leaves - refer Table 1.

Table 1 Measurements of mature wild type and peapod leaf laminae. Mean mm (SD) from 14 leaves; Leaf Genotype Length Width Area Perimeter

1 Wild type 7.77(0.9) 6.69 (0,7) 40,58 (6,7) 22.48 (2.1) Peapod 10.67(1.1) 7.96 (0.7) 53.22 (8.5) 22.55 (2.1)

4 Wild type 15,84 (0.9) 10.51 (0.6) 126.46 (11.3) 41.23 (2.9) Peapod 25.00 (2,5) 13.31 (1.2) 196.24 (25.3) 46.52 (4.2)

Therefore, the bell shape curvature of peapod leaves occurs because the additional lamina area of the mutant is constrained by the margin of the blade. Both scanning electron

microscopy and expression of an introduced reporter gene that allows histological detection of dividing cells were used to monitor cell differentiation during leaf and seed pod development. There are two phases of cell division during plant organ development; division of founder cells in the undifferentiated primordia, and division of stem cell like meristemoid cells during the differentiation phase. In peapod the pattern and timing of primordia cell division arrest is not altered, but there is a significant delay in the arrest of meristemoid cell division. This leads to many more cells being produced in the laminal plane (Figures 2 & 3). When these additional cells differentiate and expand they result in the larger blade dimensions observed in the mutant. The additional meristemoid divisions occur in all cell layers of the lamina, in their regular pattern, and hence the general lamina structure is not altered. There is simply more lamina, particularly in the leaves and seed pod walls.

Genetic analysis established that the peapod mutation is inherited as a single, semi- dominant locus. This means that the loss of one copy in the presence of a functional copy of the gene results in a distinct mutant phenotype - refer Table 2.

Table 2 Measurements of wild type and peapod siliques. Mean mm (SD) from 30 siliques;

Genotype Length Width PEAPOD gene copy number

WT Ler 13.92 (0.4) 1.27 (0.0) 4 peapod Ler 8.12 (0.6) 3.13 (0.2) 0 peapod/WT F1 14.08 (0.3) 1.51 (0.0) 2

A combination of genetic mapping, molecular markers, chromosome walking and chromosome landing, was subsequently used to map the position of the peapod mutation,

identified as a 65 Kilobase deletion from the Arabidopsis genome (Figure 4). PCR analysis established that genes At4g14695 and At4g14760 are present but At4g14700, At4g14710, At4g14713, At4g14716, At4g14720, At4g14730, At4g14740, At4g14743, At4g14746, and At4g 14750 have all been deleted from the mutant. Such a large deletion raises the prospect that the mutation might be due to the loss of a combination of genes. However, within the deleted region there is a small direct tandem repeat where a pair of genes has been duplicated (i.e. 2+2). In each of the pair of duplicated genes, one gene is otherwise unique in the Arabidopsis genome (i.e. At4g14713, or At4g14720), whereas the other gene present in each respective pair of the repeat region (i.e. At4g14710, or At4g14716), are similar to members of a larger gene family and therefore might be expected to be functionally redundant in the genome. On this basis the "unique pair of genes" were chosen as candidates for PEAPOD. Introduction of either of the "PEAPOD" genes compensates (i.e. corrects) the peapod mutation. Co-inheritance of the PEAPOD gene with correction of the defect provided proof that the mutant phenotype is solely due to loss of the PEAPOD genes. Independent insertion mutations in individual peapod genes were identified and characterised. These produce the expected semi-dominant phenotype (equivalent to only one functional copy of the PEAPOD gene). Having a deletion of two duplicated genes together with mutations in either of the individual genes has allowed me to produce, by inter-crossing, a series of plants with different dosages of functional peapod gene (0, 1 , 2, 4 copies). Measurements of seed pod width indicated that lamina growth is directly influenced by PEAPOD gene dosage - refer Table 3 and Figure 5.

Table 3 Homozygous T-DNA insertions in individual peapod genes produce a semi- dominant peapod phenotype

Influence of PEAPOD gene copy number on silique dimensions. Mean mm (SD) from 30 siliques.

Genotype Length Width PEAPOD gene copy number

WT CoI 16.01 (0,5) 0.84 (0.1) 4 peapod CoI 10.69 (0.7) 2.20 (0,2) 0

Insert peapodi CoI horn 16.24 (0.3) 1.18 (0.1) 2

Insert peapod2 CoI horn 16.33 (0.3) 1.17 (0.0) 2

Insert ρeapod1/2 CoI net 16.30 (0.3) 1.17 (0,0) 2 peapod/insert peapodi hemi 15.28 (0.4) 1.28 (0.1) 1

These experiments also indicate that the two PEAPOD genes are functional equivalent.

During complementation experiments, where the intact wild type PEAPOD gene was transformed into peapod to correct the mutation, a few of the transgenic plants had particularly small leaves and seed pods. These plants over express the PEAPOD gene and have almost no meristemoid cell division. Therefore, high levels of peapod expression stop meristemoid cell division, whereas a loss of PEAPOD expression results in a significant delay in the arrest of meristemoid cell division (Figures 6a and 6b). This suggests that altered levels of PEAPOD gene expression are responsible for the temporal pattern of meristemoid cell division arrest, a key mechanism controlling the flexibility of organ size.

The expression pattern of the PEAPOD gene was established using mRNA in situ localisation in tissue sections. Expression of the peapod gene coincides with the onset of cell differentiation and the arrest of meristemoid cell division in developing leaves and seed pods. Expression is absent from tissues undergoing cell division (not shown).

Computer analysis of the predicted PEAPOD protein sequence indicates that it is both novel and plant specific. Homology comparisons identified a domain of the peapod predicted protein that is highly conserved in a range of other plants (Figure 7).

Further sequence information on At4g14713 and At4g14720 (cDNA, genomic, and predicted amino acid sequence) is given in the technical section. There is also a multiple

alignment with a wider group of proteins that have the "PEAPOD Domain" sequence runs at the 5' end of cDNA clones (i.e. EST's). However, these sequences are useful for determining the likely distribution of the PEAPOD-Wke genes in the plant kingdom.

Experiment 2: PEAPOD gene in other eudicot plants - Is PEAPOD gene function is conserved in other eudicot plants?

Experimental:

Cloning a PPD-like gene from white clover.

To identify possible legume PPD-like genes a public database of Medicago truncatula cDNA sequences was searched for homologs to the Arabidopsis PPD1 and PPD2 predicted protein sequences (http://tigrblast.tigr.org) using the blastX search programme. Potential corresponding sequences to the Arabidopsis PPD1 and PPD2 predicted protein sequences where located and PCR primers were designed to the N and COOH ends of a putative full length Medicago truncatula PPD-like gene sequence.

The Medicago truncatula primer sequences used to amplify the white clover PPD-like gene are:

Med PPD ATG-ATGAACGGCGGAAGCACCGTTTCCTT Med PPD 3 end~GCATTCTTGAACATCTTTATCATTCA

These primers were used to PCR amplify a DNA fragment from reverse transcribed RNA isolated from shoot tips of white clover (Trifolium repens L. cultivar Huia) plants. Using a similar cloning strategy to the above Arabidopsis cloning strategy described above, an amplified DNA fragment was cloned in pGEMT vector and the DNA sequence of the clone encoded a putative white clover PPD-like gene (TrPPD). The TrPPD cDNA fragment was subcloned to produce a gene cassette incorporating the regulatory regions of the Arabidopsis PPDI gene (λfPPDpromoter - 5 ^' UTR- TrPPD cDHA-AtPPD1 3' UTR). The TrPPD cDNA expression cassette was subsequently subcloned into the Not1 restriction site

of the Agrobacterium binary vector pHZbar and transformed into peapod mutant Arabidopsis plants using the floral dip method (Clough & Bent. 1998. Plant J 16: 735-743)

Silencing TrPPD in white clover

To silence the TrPPD gene in white clover a RNA interference construct was prepared using a 165 base pair DNA fragment spanning the start codon and predicted PPD Domain. The DNA fragment was amplified from the cloned TrPPD cDNA sequence by PCR using sequence specific primers incorporating EcoR1, Xho1, BamHI, or Xbal restriction enzyme sites.

The sequence amplified was:

5'atgaacggcggaagcaccgtttccttccgatccatcctcgacaaaccccttaaccagc tcaccgaagatgacattt ctcaactcactcgtgaagactgtcgcagattcctcaaagataaagggatgcgcaggcctt cctggaacaaatctcag gcgatccagcaa -3 ^'

This encoded the following predicted amino acid sequence;

5 ^' - MNGGSTVSFRSILDKPLNQLTEDDISQLTREDCRRFLKDKGMRRPSWNKSQAIQQ -S ^' .

Equivalent 5 ^' BamH1/Xba1-3' and 5εcoR1/Xho1 3' fragments were cloned into RNAi vector pRNA 69 to produce a expression cassette composed of CaMV 35S promoter - reverse TrPPD fragment -intron - forward TrPPD fragment - OCS 3' terminator. This TrPPD RNAi cassette was subcloned into the Not1 site of Agrobacterium plant transformation vector pHZbar. The contruct was used to transform white clover, cultivar Huia, cotyledons using established tissue culture and clover transformation methods (White & Voisey 1994. Plant Cell Reports 13: 303-308; Voisey, White, Dudas, Appleby, Ealing, Scott 1994. Plant Cell Reports 13:309-314).

White clover leaf dimensions

Leaf and petiole length dimension measurements of the fourth fully expanded leaf from the shoot tip, and intemode length between the fourth and fifth observable nodes were

obtained using a digital micrometer (measured in mm). At least five samples were measured for each reported dimension.

Results and Discussion:

To test the hypothesis that PEAPOD-WB genes identified in other eudicot plants can have the same function as the Arabidopsis PEAPOD genes a PEAPOD homolog (TrPPD) was isolated from the forage legume, white clover (Tήfolium repens L). A clone of the white clover TrPPD cDNA sequence (SEQ ID No 7), fused to the regulatory sequences of the Arabidopsis PPD1 gene, and introduced into Arabidopsis null, PEAPOD loss-of-function plants, rescued the mutant phenotype and produced plants with wild type appearance (Figure 8). Over 90% of transgenic peapod plants transformed with the TrPPD cDNA gene construct had silique dimensions similar to wild type plants (Table 4).

Table 4 Silique sizes of Arabidopsis peapod plants complemented with a white clover cDNA gene construct.

The white clover PEAPOD-WWe protein coding sequence is therefore functionally equivalent to the Arabidopsis PPD protein coding sequence. Furthermore, experiments silencing the endogenous TrPPD gene in transgenic white clover plants established that the TrPPD gene also acts in white clover to limit leaf lamina size. When a TrPPD RNA interference gene construct was introduced into white clover some of the transgenic plants had abnormally enlarged leaves with positive Gaussian curvature and elongated petioles. An example, comparing some leaf and stem dimensions of wild type and RNAi transgenic plants, is given in Table 5.

Table 5 Leaf, petiole and stem dimensions of wild type and TrPPD RNAi plants. Measurements (mm) are the mean of 5 samples.

Therefore, the PPD gene function in two different eudicot plant species has been conserved.

Key findings for the PEAPOD genes include:

• The peapod mutant phenotype is due to the loss-of-function of a key gene controlling the arrest of meristemoid cell division during organ development

• The peapod mutation is due to a deletion of a ca 60 Kilobase segment of the Arabidopsis genome, located between ca 8,420Kb and 84800Kb.

• The peapod mutation is due to the deletion of a duplicated pair of genes, i.e. there are two adjacent peapod genes (At4g14713 & At4g 14720) that have to be inactivated to give the mutant phenotype.

• Either of these two PEAPOD genes can complement the mutation.

• The peapod mutation is semi-dominant.

• The two PEAPOD genes appear to be functionally equivalent.

• Function of the PEAPOD gene is dosage dependent (i.e. depends on the number of genes that are present and expressed).

• Over expression phenotype - no meristemoid cell division, normal development, but smaller lateral organs.

• Expression pattern of the PEAPOD gene (transcription) coincides with the arrest of cell cycling and the onset of cell differentiation.

• PEAPOD is a protein unlike any other previously described.

• PEAPOD has a protein domain conserved in a range of plants.

• The PEAPOD gene is only found in plants.

• PEAPOD is a key gene regulating flexibility in organ size.

• PEAPOD gene function is conserved in other eudicot plants.

Aspects of the present invention have been described by way of example only and it should be appreciated that modifications and additions may be made thereto without departing from the scope of the appended claims.