TECHNICAL FIELD

The present invention relates methods for producing plants with increased root biomass.

BACKGROUND ART

The roots of vascular plants have multiple functions. These include: 1) absorption of water and inorganic nutrients, 2) anchoring of the plant to the ground, and supporting it, 3) storage of food and nutrients, and 4) vegetative reproduction. In response to the concentration of nutrients, roots also synthesise cytokinin, which acts as a signal influencing how fast the shoots can grow.

Plants with increased root biomass would therefore potentially have a number of advantages including better anchorage, more efficient water uptake, more efficient nutrient uptake, and improved drought tolerance. A combination of these features may also result in improved yield, including increased grain or fruit biomass and/or increased leaf biomass.

At present there is limited understanding of the genetic mechanisms controlling root biomass in plants.

It would be beneficial to have available alternative methods for controlling root biomass in plants.

It is therefore an object of the invention to provide methods and materials for increasing the production of root biomass in plants, and/or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

Previously, White (2006) discovered two adjacent homologous genes in Arabidopsis (named PEAPOD, PPDl and PPD2) that regulate the cell proliferation of meristemoids during the late stages of leaf and seed pod development. Deletion of these genes in Arabidopsis resulted in enlarged leaves and wide seed pods while over expression of PPDl resulted in a reduction in the size of the leaves and siliques (White, 2006).

The applicants have now surprisingly shown that the over-expression of PEAPOD genes in plants results in an increase in the production of root biomass.

The applicant's invention therefore relates to a method for increasing root biomass in plants by ectopic expression of PEAPOD. In particular the invention relates to ectopic expression of PEAPOD proteins that are characterized by presence of consensus amino acid motifs common to all PEAPOD proteins disclosed from a wide range of plant species.

Methods

In the first aspect the invention provides a method for increasing root biomass in a plant, the method comprising the step of increasing the expression of at least one PEAPOD protein in the plant.

In one embodiment root biomass is increased relative to that in a control plant, of the same species or variety.

In one embodiment the increased expression of the at least one PEAPOD protein is a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein.

In a further embodiment, the plant is transgenic for at least one polynucleotide encoding and expressing the PEAPOD protein.

In a further aspect the invention provides a method for producing a plant increased root biomass, the method comprising the step of increasing the expression of at least one PEAPOD protein in the plant.

In one embodiment the plant is transformed with at least one polynucleotide encoding a PEAPOD protein.

In a further embodiment the method comprises the step of transforming the plant, or transforming a plant cell which is regenerated into the plant, with a polynucleotide encoding the PEAPOD protein.

In one embodiment the method includes the additional step of testing or assessing the plant for increased root biomass.

In a further embodiment the method includes the step producing further plants with increased root biomass by asexually or sexually multiplying the plants tested for increased biomass.

PEAPOD proteins

In one embodiment the PEAPOD protein is a polypeptide comprising the sequence of at least one of SEQ ID NO: 28, 29, 31, 32, 34 and 35.

In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 28. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 29. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 31. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 32. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO: 34. In a further embodiment the PEAPOD protein comprises the sequence of SEQ ID NO : 35.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to any one of SEQ ID NO : 1 to 26.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence selected from any one of SEQ ID NO : 1 to 26.

In a further embodiment the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to SEQ ID NO: 1.

In a further embodiment the PEAPOD protein is a polypeptide comprising the sequence of SEQ ID NO : 1.

Expressing PEAPOD

Methods for expressing proteins in plants are well known to those skilled in the art, and are described herein. All of such methods are included within the scope of the invention.

Increasing expression of PEAPOD by introducing a polynucleotide

In one embodiment expression is increased by introducing at least one polynucleotide into the plant cell or plant.

In a preferred embodiment the polynucleotide encodes a PEAPOD protein as herein defined.

In a further embodiment the polynucleotide comprises a sequence with at least 70% identity to the coding sequence of any one of SEQ ID NO : 83-107.

In a further embodiment the polynucleotide comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises the coding sequence of any one of SEQ ID NO: 83-107.

In a further embodiment the polynucleotide comprises the sequence of any one of SEQ ID NO : 83-107.

In a further embodiment the polynucleotide comprises a fragment of the sequences described above, that is capable of encoding a polypeptide with the same function as a PEAPOD protein. In one embodiment the fragment encodes a polypeptide capable of increasing root biomass.

Expressing PEAPOD via an expression construct

In a preferred embodiment the polynucleotide is introduced into the plant as part of an expression construct.

In a preferred embodiment the expression construct comprises a promoter operatively linked to the polynucleotide.

Promoter for increasing expression of PEAPOD

In one embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide constitutively in all tissues of the plant.

In a further embodiment the promoter is a tissue-preferred promoter.

In a further embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide in the below ground tissues of the plant.

In one embodiment the promoter is a below ground tissues-preferred promoter.

In a further embodiment the promoter is a below ground tissue-specific promoter.

In one embodiment the promoter is a light-repressed promoter.

In a further embodiment the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide in the roots of the plant.

In one embodiment the promoter is a root- preferred promoter.

In a further embodiment the promoter is a root-specific promoter.

Source of polynucleotides and polypeptides

The polynucleotides and variants of polynucleotides of the invention, or used in the methods of the invention, may be derived from any species. The polynucleotides and variants may also be synthetically or recombinantly produced, and also may be the products of "gene shuffling" approaches.

The polypeptides and variants of polypeptides of the invention, or used in the methods of the invention, may be derived from any species. The polypeptides

and variants may also be recombinantly produced and also may also be expressed from the products of "gene shuffling' approaches.

In one embodiment the polynucleotide, polypeptide or variant, is derived from a plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from gymnosperm plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from an angiosperm plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a dicotyledonous species.

In a preferred embodiment the polynucleotide, polypeptide or variant, is derived from a eudicot species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a eudicot plant species.

In a further embodiment the polynucleotide, polypeptide or variant, is derived from a monocotyledonous species. Preferred monocot plants include: palm, banana, duckweed and orchid species.

Plant cells and plants to be transformed

The plant cells and plants of the invention, or used in the methods of the invention, are from any plant species.

In one embodiment the plant cells or plants are from gymnosperm plant species.

In a further embodiment the plant cells or plants are from angiosperm plant species.

In a further embodiment the plant cells or plants are from a dicotyledonous species.

Preferred monocotyledonous genera include: Agropyron, Allium, Alopecurus, Andropogon, Arrhenatherum, Asparagus, Avena, Bambusa, Bothrichloa, Bouteloua, Bromus, Calamovilfa, Cenchrus, Chloris, Cymbopogon, Cynodon, Dactylis, Dichanthium, Digitaria, Eleusine, Eragrostis, Fagopyrum, Festuca, Helianthus, Hordeum, Lolium, Miscanthis, Miscanthus x giganteus, Oryza, Panicum, Paspalum, Pennisetum, Phalaris, Phleum, Poa, Saccharum, Secale, Setaria, Sorgahastum, Sorghum, Triticum, Vanilla, X Triticosecale Triticale and Zea.

Preferred monocotyledonous species include: Agropyron cristatum, Agropyron desertorum, Agropyron elongatum, Agropyron intermedium, Agropyron smithii, Agropyron spicatum, Agropyron trachycaulum, Agropyron trichophorum, Allium ascalonicum, Allium cepa, Allium chinense, Allium porrum, Allium schoenoprasum, Allium fistulosum, Allium sativum, Alopecurus pratensis, Andropogon gerardi, Andropogon Gerardii, Andropogon scoparious, Arrhenatherum elatius, Asparagus officinalis, Avena nuda, Avena sativa, Bambusa vulgaris, Bothrichloa barbinodis, Bothrichloa ischaemum, Bothrichloa saccharoides, Bouteloua curipendula, Bouteloua eriopoda, Bouteloua gracilis, Bromus erectus, Bromus inermis, Bromus riparius, Calamovilfa longifilia, Cenchrus ciliaris, Chloris gayana, Cymbopogon nardus, Cynodon dactylon, Dactylis glomerata, Dichanthium annulatum, Dichanthium aristatum, Dichanthium sericeum, Digitaria decumbens, Digitaria smutsii, Dioscorea rotundata, Dicsorea alata, Dicscorea opposita, Dicscorea bulbifera, Dioscorea esculenta, Dioscorea trifida, Eleusine coracan, Elymus angustus, Elymus junceus, Eragrostis curvula, Eragrostis tef, Fagopyrum esculentum, Fagopyrum tataricum, Festuca arundinacea, Festuca ovina, Festuca pratensis, Festuca rubra, Helianthus annuus sunflower, Hordeum distichum, Hordeum vulgare, Lolium multiflorum, Lolium perenn, Miscanthis sinensis, Miscanthus x giganteus, Oryza sativa, Panicum italicium, Panicum maximum, Panicum miliaceum, Panicum purpurascens, Panicum virgatum, Panicum virgatum, Paspalum dilatatum, Paspalum notatum, Pennisetum clandestinum, Pennisetum glaucum, Pennisetum purpureum, Pennisetum spicatum, Phalaris arundinacea, Phleum bertolinii, Phleum pratense, Poa fendleriana, Poa pratensis, Poa. nemoralis, Saccharum officinarum, Saccharum robustum, Saccharum sinense, Saccharum spontaneum, Secale cereale, Setaria sphacelata, Sorgahastum nutans, Sorghastrum nutans, Sorghum dochna, Sorghum halepense, Sorghum sudanense, Sorghum vulgare, Sorghum vulgare, Triticum aestivum, Triticum dicoccum, Triticum durum, Triticum monococcum, Vanilla fragrans, X Triticosecale and Zea mays.

A preferred family of monocotyledonous plants is poaceae family.

Preferred poaceae subfamilies include the: Anomochlooideae, Pharoideae, Puelioideae, Bambusoideae, Pooideae, Ehrhartoideae, Aristidoideae, Arundinoideae, Chloridoideae, Panicoideae, Danthonioideae, and Micrairoideae.

A preferred poaceae family is the subfamily pooideae. Preferred pooideae plants include wheat, barley, oats, brome grass and reed grass.

Another preferred poaceae family is the subfamily Ehrhartoideae. Preferred ehrhartoideae plants include rice.

Another preferred poaceae family is the subfamily panicoideae. Preferred panicoideae plants include panic grass, maize, sorghum, sugar cane, energy cane, millet, fonio and bluestem grasses.

Another preferred poaceae family is the subfamily Arundinoideae. Preferred Arundinoideae plants include Arundo donax.

Another preferred poaceae family is the subfamily Bambusoideae. Preferred Bambusoideae plants include bamboo.

Preferred poaceae species include those form the Lolium genera. Preferred Lolium species include Lolium longiflorum, Lolium multiflorum, Lolium perenne, Lolium westerwoldicum, Lolium temulentum, and Lolium hybridum.

Other preferred poaceae species include those form the Festuca genera. Preferred Festuca species include Festuca arundinacea, Festuca ovina, Festuca pratensis and Festuca rubra.

Preferably the plant cells or plants are from a dicotyledonous species.

Preferred dicotyledonous genera include: Amygdalus, Anacardium, Arachis, Brassica, Cajanus, Cannabis, Carthamus, Carya, Ceiba, Cicer, Cocos, Coriandrum, Coronilla, Cossypium, Crotalaria, Dolichos, Elaeis, lycine, Gossypium, Helianthus, Lathyrus, Lens, Lespedeza, Linum, Lotus, Lupinus, Macadamia, Medicago, Melilotus, Mucuna, Olea, Onobrychis, Ornithopus, Papaver, Phaseolus, Phoenix, Pistacia, Pisum, Prunus, Pueraria, Ribes, Ricinus, Sesamum, Theobroma, Trifolium, Trigonella, Vicia and Vigna.

Preferred dicotyledonous species include: Amygdalus communis, Anacardium occidentale, Arachis hypogaea, Arachis hypogea, Brassica napus Rape, Brassica. nigra. Brassica campestris, Cajanus cajan, Cajanus indicus, Camelina sativa, Cannabis sativa, Carthamus tinctorius, Carya illinoinensis, Ceiba pentandra, Cicer arietinum, Cocos nucifera, Coriandrum sativum, Coronilla varia, Cossypium hirsutum, Crotalaria juncea, Dolichos lablab, Elaeis guineensis, Gossypium arboreum, Gossypium nanking, Gossypium barbadense, Gossypium herbaceum, Gossypium hirsutum, Glycine max, Glycine ussuriensis, Glycine gracilis, Helianthus annus, Jatropha cuneata, Jatropha curcas, Lupinus angustifolius, Lupinus luteus, Lupinus mutabilis, Lespedeza sericea, Lespedeza striata, Lotus uliginosus, Lathyrus sativus, Lens culinaris, Lespedeza stipulacea, Linum usitatissimum, Lotus corniculatus, Lupinus albus, Lupinus angustifolius, Lupinus luteus, Medicago arborea, Medicago falcate, Medicago hispida, Medicago officinalis, Medicago. sativa Alfalfa, Medicago tribuloides, Macadamia integrifolia, Medicago arabica, Melilotus albus, Millettia pinnata, Mucuna pruriens, Olea europaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus, Prunus cerasifera, Prunus cerasus, Phaseolus coccineus, Prunus domestica, Phaseolus lunatus, Prunus. maheleb, Phaseolus mungo, Prunus. persica, Prunus. pseudocerasus, Phaseolus vulgaris, Papaver somniferum, Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisum sativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana, Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamum indicum, Solanum tuberosum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Trifolium incarnatum, Trifolium ingrescens, Trifolium pratense, Trifolium repens, Trifolium resupinatum, Trifolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonella foenumgraecum, Vernicia fordii, Vicia angustifolia, Vicia atropurpurea, Vicia calcarata, Vicia dasycarpa, Vicia ervilia, Vaccinium oxycoccos, Vicia pannonica, Vigna sesquipedalis, Vigna sinensis, Vicia villosa, Vicia faba, Vicia sative and Vigna angularis.

Plants and plant parts

In a further aspect the invention provides a plant that has increased root biomass as a result of having increased expression a PEAPOD protein, or fragment thereof.

In one embodiment expression of the PEAPOD protein, or fragment thereof, is increased as a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein, or fragment thereof.

In a further embodiment plant is transgenic for a polynucleotide expressing the PEAPOD protein, or fragment thereof.

In a further embodiment the the polynucleotide or fragment thereof is operatively linked polynucleotide to a tissue-preferred promoter.

In a further embodiment the promoter is a root-preferred promoter.

In a further embodiment the promoter is a root-specific promoter.

In a further embodiment the PEAPOD protein is as herein defined.

In a further embodiment the polynucleotide, encoding the PEAPOD protein, is as herein defined.

In a further aspect the invention provides a cell, part, propagule or progeny of the plant that is transgenic for at least one of:

a) the polynucleotide, and

b) the polynucleotide and operatively linked promoter.

DETAILED DESCRIPTION

In this specification where reference has been made to patent specifications, other external documents, or other sources of information, this is generally for the purpose of providing a context for discussing the features of the invention. Unless specifically stated otherwise, reference to such external documents is not to be construed as an admission that such documents, or such sources of information, in any jurisdiction, are prior art, or form part of the common general knowledge in the art.

The term "comprising" as used in this specification means "consisting at least in part of". When interpreting each statement in this specification that includes the term "comprising", features other than that or those prefaced by the term may also be present. Related terms such as "comprise" and "comprises" are to be interpreted in the same manner.

Increased root biomass

A plant with "increased root biomass" produces more root biomass than does a control plant of the same type and age. Thus "increased" means increased relative to a control plant of the same type and age.

Preferably the plant with "increased root biomass" produces at least 10%, preferably at least 20%, more preferably at least 30%, more preferably at least 40%, more preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 100%, more preferably at least 150%, more preferably at least 200%, more preferably at least 300%, more preferably at least 400% more root biomass than does a control plant of the same type and age.

In one embodiment the plant with "increased root biomass" has at least one of: larger roots, more roots, or a more extensive root system, than does a control plant.

Root biomass

The term root biomass refers to total mass of root tissue produced by the plant. This can be assessed by dry weight or wet weight.

Root

The term root as used herein means the same as standard usage of the term. The term root encompasses the primary root, secondary roots, adventitious roots, root branches and root hairs. Roots are generally below ground, but the term also encompasses aerial roots. In one embodiment the term root encompasses non-leaf, non-node bearing parts of the plant.

Increased drought tolerance

In one embodiment the plant with "increased root biomass" also has increased drought tolerance. Again "increased" means increased relative to a control plant of the same type and age.

The term "increased drought tolerance" is intended to describe a plant, or plants, which perform more favourably in any aspect of their growth and development under sub-optimal hydration conditions than do suitable control plants in the same conditions.

Control plant

In one embodiment the control plant is a wild-type plant. In a further embodiment the control plant is a non-transformed plant. In a further embodiment the control plant is a plant that has not been transformed with a PEAPOD polynucleotide. In a further embodiment the control plant is a plant that has not been transformed with a construct. In a further embodiment the control plant is a plant that has been transformed with a control construct. In one embodiment the construct is an empty vector construct.

Ectopic expression

The term "ectopic expression" is intented to be interpreted broadly. The term refers to expression of a polynucleotide or polypeptide in any one of:

• a cell, organ, tissue or plant where it is not normally expressed,

• a cell, organ, tissue or plant at a time, or developmental stage, when it is not normally expressed, and

• a cell, organ, tissue or plant at a level higher than it is normally expressed in that cell, organ, tissue or plant.

Tissue preferred promoters

In certain embodiments, the PEAPOD protein encoding polynucleotides are expressed under the control of tissue preferred promoters. The term "preferred" with respect to tissue preferred promoters means that the promoter primarily drives expression in that tissue. Thus, for example, a root- preferred promoter drives a higher level of expression of an operably linked polynucleotide in root tissue than it does in other tissues or organs or the plant.

Root preferred promoters

A root-preferred promoter drives a higher level of expression of an operably linked polynucleotide in root tissue than it does in other tissues or organs or the plant.

Root-preferred promoters may include non-photosynthetic tissue preferred promoters and light-repressed regulated promoters.

Non-photosynthetic tissue preferred promoters

Non-photosynthetic tissue preferred promoters include those preferentially expressed in non-photosynthetic tissues/organs of the plant.

Non-photosynthetic tissue preferred promoters may also include light repressed promoters.

Light repressed promoters

An example of a light repressed promoter is found in US 5,639,952 and in US 5,656,496.

Root specific promoters

An example of a root specific promoter is found in US 5,837,848; and US

2004/0067506 and US 2001/0047525.

The term "preferentially expressed" with respect to a promoter being preferentially expressed in a certain tissue, means that the promoter is expressed at a higher level in that tissue than in other tissues of the plant.

The term "tissue specific" with respect to a promoter, means that the promoter is expressed substantially only in that tissue, and not other tissues of the plant.

In one embodiment the root- preferred promoter is a root-specific promoter.

The term "gene" as used herein means an endogenous genomic sequence which includes a coding sequence which encodes a polypeptide or protein. The coding sequence may be interrupted by one or more introns. A gene typically also includes a promoter sequence, 5' untranslated sequence, 3' untranslated sequence, and a terminator sequence. Genomic sequences that regulate expression of the protein may also be considered part of the gene.

Polynucleotides and fragments

The term "polynucleotide(s)," as used herein, means a single or double-stranded deoxyhbonucleotide or ribonucleotide polymer of any length but preferably at least 15 nucleotides, and include as non-limiting examples, coding and non-coding sequences of a gene, sense and antisense sequences complements, exons, introns, genomic DNA, cDNA, pre-mRNA, mRNA, rRNA, siRNA, miRNA, tRNA, ribozymes, recombinant polypeptides, isolated and purified naturally occurring DNA or RNA sequences, synthetic RNA and DNA sequences, nucleic acid probes, primers and fragments.

A "fragment" of a polynucleotide refers to a contiguous subsequence of larger a polynucleotide sequence. Preferably the fragment is at least 15 nucleotides preferably at least 16 nucleotides, more preferably at least 17 nucleotides, more preferably at least 18 nucleotides, more preferably at least 19 nucleotides, more preferably at least 20 nucleotides, more preferably at least 21 nucleotides, more preferably at least 22 nucleotides, more preferably at least 23 nucleotides, more preferably at least 24 nucleotides, more preferably at least 25 nucleotides, more preferably at least 26 nucleotides, more preferably at least 27 nucleotides, more preferably at least 28 nucleotides, more preferably at least 29 nucleotides, more preferably at least 30 nucleotides, more preferably at least 31 nucleotides, more preferably at least 32 nucleotides, more preferably at least 33 nucleotides, more preferably at least 34 nucleotides, more preferably at least 35 nucleotides, more preferably at least 36 nucleotides, more preferably at least 37 nucleotides, more preferably at least 38 nucleotides, more preferably at least 39 nucleotides, more preferably at least 40 nucleotides, more preferably at least 41 nucleotides, more preferably at least 42 nucleotides, more preferably at least 43 nucleotides, more preferably at least 44 nucleotides, more preferably at least 45 nucleotides, more preferably at least 46 nucleotides, more preferably at least 47 nucleotides, more preferably at least 48 nucleotides, more preferably at least 49 nucleotides, more preferably at least 50 nucleotides, more preferably at least 51 nucleotides, more preferably at least 52 nucleotides, more preferably at least 53 nucleotides, more preferably at least 54 nucleotides, more preferably at least 55 nucleotides, more preferably at least 56 nucleotides, more preferably at least 57 nucleotides, more preferably at least 58 nucleotides, more preferably at least 59 nucleotides, more preferably at least 60 nucleotides, more preferably at least 61 nucleotides, more preferably at least 62 nucleotides, more preferably at least 63 nucleotides, more preferably at least 64 nucleotides, more preferably at least 65 nucleotides, more preferably at least 66 nucleotides, more preferably at least 67 nucleotides, more preferably at least 68 nucleotides, more preferably at least 69 nucleotides, more preferably at least 70 nucleotides, more preferably at least 71 nucleotides, more preferably at least 72 nucleotides, more preferably at least 73 nucleotides, more preferably at least 74 nucleotides, more preferably at least 75 nucleotides, more preferably at least 76 nucleotides, more preferably at least 77 nucleotides, more preferably at least 78 nucleotides, more preferably at least 79 nucleotides, more preferably at least 80 nucleotides, more preferably at least 81 nucleotides, more preferably at least 82 nucleotides, more preferably at least 83 nucleotides, more preferably at least 84 nucleotides, more preferably at least 85 nucleotides, more preferably at least 86 nucleotides, more preferably at least 87 nucleotides, more preferably at least 88 nucleotides, more preferably at least 89 nucleotides, more preferably at least 90 nucleotides, more preferably at least 91 nucleotides, more preferably at least 92 nucleotides, more preferably at least 93 nucleotides, more preferably at least 94 nucleotides, more preferably at least 95 nucleotides, more preferably at least 96 nucleotides, more preferably at least 97 nucleotides, more preferably at least 98 nucleotides, more preferably at least 99 nucleotides, more preferably at least 100 nucleotides, more preferably at least 150 nucleotides, more preferably at least 200 nucleotides, more preferably at least 250 nucleotides, more preferably at least 300 nucleotides, more preferably at least 350 nucleotides, more preferably at least 400 nucleotides, more preferably at least 450 nucleotides and most preferably at least 500 nucleotides of contiguous nucleotides of a polynucleotide disclosed. A fragment of a polynucleotide sequence can be used in antisense, RNA interference (RNAi), gene silencing, triple helix or ribozyme technology, or as a primer, a probe, included in a microarray, or used in polynucleotide-based selection methods of the invention.

In one embodiment the fragment encodes a polypeptide that performs, or i capable of performing, the same function as the polypeptide encoded by th larger polynucleotide that the fragment is part of.

The term "primer" refers to a short polynucleotide, usually having a free 3ΌΗ group that is, or can be, hybridized to a template and used for priming polymerization of a polynucleotide complementary to the target.

The term "probe" refers to a short polynucleotide that is, or can be, used to detect a polynucleotide sequence that is complementary to the probe, in a hybridization-based assay. The probe may consist of a "fragment" of a polynucleotide as defined herein.

Polypeptides and fragments

The term "polypeptide", as used herein, encompasses amino acid chains of any length but preferably at least 5 amino acids, including full-length proteins, in which amino acid residues are linked by covalent peptide bonds. Polypeptides of the present invention, or used in the methods of the invention, may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.

A "fragment" of a polynucleotide refers to a contiguous subsequence of a larger polynucleotide sequence. Preferably the fragment

A "fragment" of a polypeptide refers to a contiguous subsequence of larger a polypeptide. Preferably the fragment is at least 5, more preferably at least 10, more preferably at least 20, more preferably at least 30, more preferably at least 40, more preferably at least 50, more preferably at least 100, more preferably at least 120, more preferably at least 150, more preferably at least 200, more preferably at least 250, more preferably at least 300, more preferably at least 350 , more preferably at least 400.

In one embodiment the fragment performs, or is capable of performing, the same function as the polypeptide that the fragment is part of.

Preferably the fragment performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide.

The term "isolated" as applied to the polynucleotide or polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular environment. In one embodiment the sequence is separated from its flanking sequences as found in nature. An isolated molecule may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.

The term "recombinant" refers to a polynucleotide sequence that is synthetically produced or is removed from sequences that surround it in its natural context. The recombinant sequence may be recombined with sequences that are not present in its natural context.

A "recombinant" polypeptide sequence is produced by translation from a "recombinant" polynucleotide sequence.

The term "derived from" with respect to polynucleotides or polypeptides of the invention being derived from a particular genera or species, means that the polynucleotide or polypeptide has the same sequence as a polynucleotide or polypeptide found naturally in that genera or species. The polynucleotide or polypeptide, derived from a particular genera or species, may therefore be produced synthetically or recombinantly.

Variants

As used herein, the term "variant" refers to polynucleotide or polypeptide sequences different from the specifically identified sequences, wherein one or more nucleotides or amino acid residues is deleted, substituted, or added . Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides and polynucleotides disclosed herein possess biological activities that are the same or similar to those of the disclosed polypeptides or polypeptides. The term "variant" with reference to polypeptides and polynucleotides encompasses all forms of polypeptides and polynucleotides as defined herein.

Polynucleotide variants

Variant polynucleotide sequences preferably exhibit at least 50%, more

preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% identity to a sequence of the present invention. Identity is found over a comparison window of at least 20 nucleotide positions, preferably at least 50 nucleotide positions, more preferably at least 100 nucleotide positions, and most preferably over the entire length of a polynucleotide of the invention.

Polynucleotide sequence identity can be determined in the following manner. The subject polynucleotide sequence is compared to a candidate polynucleotide sequence using BLASTN (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq (Tatiana A. Tatusova, Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174: 247-250), which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). In one embodiment the default parameters of bl2seq are utilized. In a further except the default parameters of bl2seq are utilized, except that filtering of low complexity parts should be turned off.

Polynucleotide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs (e.g. Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453). A full implementation of the Needleman-Wunsch global alignment algorithm is found in the needle program in the EMBOSS package (Rice,P. Longden,I. and Bleasby,A. EMBOSS : The European Molecular Biology Open Software Suite, Trends in Genetics June 2000, vol 16, No 6. pp.276-277) which can be obtained from http://www.hgmp.mrc.ac.uk/Software/EMBOSS/. The European Bioinformatics Institute server also provides the facility to perform EMBOSS-needle global alignments between two sequences on line at http:/www. ebi.ac.uk/emboss/align/.

Alternatively the GAP program may be used which computes an optimal global alignment of two sequences without penalizing terminal gaps. GAP is described in the following paper: Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.

A preferred method for calculating polynucleotide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

Polynucleotide variants of the present invention also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences and which could not reasonably be expected to have occurred by random chance. Such sequence similarity with respect to polypeptides may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp://ftp.ncbi.nih.aov/blast/').

Alternatively, variant polynucleotides of the present invention hybridize to the specified polynucleotide sequences, or complements thereof under stringent conditions.

The term "hybridize under stringent conditions", and grammatical equivalents thereof, refers to the ability of a polynucleotide molecule to hybridize to a target polynucleotide molecule (such as a target polynucleotide molecule immobilized on a DNA or RNA blot, such as a Southern blot or Northern blot) under defined conditions of temperature and salt concentration. The ability to hybridize under stringent hybridization conditions can be determined by initially hybridizing under less stringent conditions then increasing the stringency to the desired

stringency.

With respect to polynucleotide molecules greater than about 100 bases in length, typical stringent hybridization conditions are no more than 25 to 30o C (for example, 10ο C) below the melting temperature (Tm) of the native duplex (see generally, Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Ausubel et al., 1987, Current Protocols in Molecular Biology, Greene Publishing,). Tm for polynucleotide molecules greater than about 100 bases can be calculated by the formula Tm = 81. 5 + 0. 41% (G + C-log (Na+). (Sambrook et al., Eds, 1987, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press; Bolton and McCarthy, 1962, PNAS 84: 1390). Typical stringent conditions for polynucleotide of greater than 100 bases in length would be hybridization conditions such as prewashing in a solution of 6X SSC, 0.2% SDS; hybridizing at 65oC, 6X SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in IX SSC, 0.1% SDS at 65o C and two washes of 30 minutes each in 0.2X SSC, 0.1% SDS at 65oC.

With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10ο C below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 bp is reduced by approximately (500/oligonucleotide length)o C.

With respect to the DNA mimics known as peptide nucleic acids (PNAs) (Nielsen et al., Science. 1991 Dec 6; 254(5037) : 1497-500) Tm values are higher than those for DNA-DNA or DNA-RNA hybrids, and can be calculated using the formula described in Giesen et al., Nucleic Acids Res. 1998 Nov 1; 26(21) : 5004-6. Exemplary stringent hybridization conditions for a DNA-PNA hybrid having a length less than 100 bases are 5 to 10ο C below the Tm.

Variant polynucleotides of the present invention also encompasses polynucleotides that differ from the sequences of the invention but that, as a consequence of the degeneracy of the genetic code, encode a polypeptide having similar activity to a polypeptide encoded by a polynucleotide of the present invention. A sequence alteration that does not change the amino acid sequence of the polypeptide is a "silent variation". Except for ATG (methionine) and TGG (tryptophan), other codons for the same amino acid may be changed by art recognized techniques, e.g., to optimize codon expression in a particular host organism.

Polynucleotide sequence alterations resulting in conservative substitutions of one or several amino acids in the encoded polypeptide sequence without significantly altering its biological activity are also included in the invention. A skilled artisan will be aware of methods for making phenotypically silent amino acid substitutions (see, e.g., Bowie et al., 1990, Science 247, 1306).

Variant polynucleotides due to silent variations and conservative substitutions in the encoded polypeptide sequence may be determined using the publicly available bl2seq program from the BLAST suite of programs (version 2.2.5 [Nov 2002]) from NCBI (ftp ://ftp.ncbi.nih.gov/blast/) via the tblastx algorithm as previously described.

Polypeptide variants

The term "variant" with reference to polypeptides encompasses naturally occurring, recombinantly and synthetically produced polypeptides. Variant polypeptide sequences preferably exhibit at least 50%, more preferably at least 51%, more preferably at least 52%, more preferably at least 53%, more preferably at least 54%, more preferably at least 55%, more preferably at least 56%, more preferably at least 57%, more preferably at least 58%, more preferably at least 59%, more preferably at least 60%, more preferably at least 61%, more preferably at least 62%, more preferably at least 63%, more preferably at least 64%, more preferably at least 65%, more preferably at least 66%, more preferably at least 67%, more preferably at least 68%, more preferably at least 69%, more preferably at least 70%, more preferably at least 71%, more preferably at least 72%, more preferably at least 73%, more preferably at least 74%, more preferably at least 75%, more preferably at least 76%, more preferably at least 77%, more preferably at least 78%, more preferably at least 79%, more preferably at least 80%, more preferably at least 81%, more preferably at least 82%, more preferably at least 83%, more preferably at least 84%, more preferably at least 85%, more preferably at least 86%, more preferably at least 87%, more preferably at least 88%, more preferably at least 89%, more preferably at least 90%, more preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most

preferably at least 99% identity to a sequences of the present invention. Identity is found over a comparison window of at least 20 amino acid positions, preferably at least 50 amino acid positions, more preferably at least 100 amino acid positions, and most preferably over the entire length of a polypeptide of the invention.

Polypeptide sequence identity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.5 [Nov 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). In one embodiment the default parameters of bl2seq are utilized. In a further except the default parameters of bl2seq are utilized, except that filtering of low complexity parts should be turned off.

Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polynucleotide sequences using global sequence alignment programs. EMBOSS-needle (available at http:/www. ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity.

A preferred method for calculating polypeptide % sequence identity is based on aligning sequences to be compared using Clustal X (Jeanmougin et al., 1998, Trends Biochem. Sci. 23, 403-5.)

A variant polypeptide includes a polypeptide wherein the amino acid sequence differs from a polypeptide herein by one or more conservative amino acid substitutions, deletions, additions or insertions which do not affect the biological activity of the peptide. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g ., substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagines, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

Non-conservative substitutions will entail exchanging a member of one of these classes for a member of another class.

Analysis of evolved biological sequences has shown that not all sequence changes are equally likely, reflecting at least in part the differences in conservative versus non-conservative substitutions at a biological level. For example, certain amino acid substitutions may occur frequently, whereas others are very rare. Evolutionary changes or substitutions in amino acid residues can be modelled by a scoring matrix also referred to as a substitution matrix. Such matrices are used in bioinformatics analysis to identify relationships between sequences, one example being the BLOSUM62 matrix shown below (Table 1).

CLAIMS:

1. A method for increasing root biomass in a plant, the method comprising the step of increasing the expression of at least one PEAPOD protein, or fragment thereof, in the plant.

2. The method of claim 1 in which the increased expression of the at least one PEAPOD protein, or fragment thereof, is a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein, or fragment thereof.

3. The method of claim 1 or 2 in which the plant is transgenic for at least one polynucleotide encoding and expressing the PEAPOD protein, or fragment thereof.

4. A method for producing a plant increased root biomass, the method comprising the step of increasing the expression of at least one PEAPOD protein, or fragment thereof, in the plant.

5. The method of claim 4 in which the plant is transformed with at least one polynucleotide encoding a PEAPOD protein, or fragment thereof.

6. The method of claim 4 or 5 comprising the step of transforming the plant, or transforming a plant cell which is regenerated into the plant, with a polynucleotide encoding the PEAPOD protein.

7. The method of claim 6 which includes the additional step of testing or assessing the plant for increased root biomass.

8. The method of claim any one of claims 1 to 7 in which the PEAPOD protein, or fragment thereof, is a polypeptide comprising the sequence of at least one of SEQ ID NO : 28, 29, 31, 32, 34 and 35.

9. The method of claim any one of claims 1 to 8 in which the PEAPOD protein is a polypeptide comprising a sequence with at least 70% identity to any one of SEQ ID NO : 1 to 26.

10. The method of any one of claims 1 to 9 in which expression is increased by introducing a polynucleotide encoding a PEAPOD protein, or fragment thereof, into the plant cell or plant.

11. The method of claim 10 in which the polynucleotide comprises a sequence with at least 70% identity to the coding sequence of any one of SEQ ID NO : 83-107 or a fragment thereof.

12. The method of claim 10 in which the polynucleotide comprises a sequence with at least 70% identity to the sequence of any one of SEQ ID NO: 83-107 or a fragment thereof.

13. The method of any one of claims 10 to 12 in which the polynucleotide, or fragment thereof, is introduced into the plant as part of an expression construct.

14. The method of claim 13 in which the expression construct comprises a promoter operatively linked to the polynucleotide or fragment thereof.

15. The method of claim 14 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide or fragment thereof, constitutively in all tissues of the plant.

16. The method of claim 14 in which the promoter is a tissue-preferred promoter.

17. The method of claim 14 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide, or fragment thereof, in the below ground tissues of the plant.

18. The method of claim 14 in which the promoter is a below ground tissues-preferred promoter.

19. The method of claim 14 in which the promoter is a light-repressed promoter.

20. The method of claim 14 in which the promoter is capable of driving, or drives, expression of the operatively linked polynucleotide, or fragment thereof, in the roots of the plant.

21. The method of claim 14 in which the promoter is a root-preferred promoter.

22. The method of claim 14 in which the promoter is a root-specific promoter.

23. A plant that has increased root biomass as a result of having increased expression a PEAPOD protein, or fragment thereof.

24. The plant of claim 23 wherein expression of the PEAPOD protein, or fragment thereof, is increased as a consequence of the plant, or its ancestor plant or plant cell, having been transformed with a polynucleotide encoding the PEAPOD protein, or fragment thereof.

25. The plant of claim 23 or 24 that is transgenic for a polynucleotide expressing the PEAPOD protein, or fragment thereof.

26. The plant of claim 24 or 25 in which the polynucleotide or fragment thereof is operatively linked polynucleotide to a tissue-preferred promoter.

27. The plant of claim 26 in which the promoter is a root- preferred promoter.

28. The plant of claim 26 in which the promoter is a root-specific promoter.

29. A cell, part, propagule or progeny of the plant of any one of claims 23 to 28 that is transgenic for at least one of:

a) the polynucleotide, and

b) the polynucleotide and operatively linked promoter.