US20160017357A1

US20160017357A1 - Polynucleotides and methods for improving plants

Info

Publication number: US20160017357A1
Application number: US14/706,801
Authority: US
Inventors: Sathish Puthigae; Jonathan Robert Phillips; Claudia Jeannette Smith-Espinoza; Catherine Jane Bryant; Kieran Michael Elborough; Margaret Biswas
Original assignee: Fonterra Cooperative Group Ltd
Current assignee: Fonterra Cooperative Group Ltd
Priority date: 2007-11-27
Filing date: 2015-05-07
Publication date: 2016-01-21
Also published as: AR069441A1; WO2009070038A2; CN101938897B; US20090229009A1; ZA201004974B; EP2219434A2; AU2008330292B2; BRPI0819610A2; WO2009070038A3; US20120260369A1; MX2010005716A; US8227665B2; NZ585431A; AU2008330292A1; MY157117A; CA2706184A1; CN101938897A; EP2219434A4

Abstract

The invention provides methods and compositions for producing plant with altered biomass, the methods comprising the step of altering the expression and/or activity of the polypeptide comprising the sequence of SEQ ID NO:1, or a variant thereof, in a plant cell or plant. The invention also provides a polypeptide comprising the sequence of SEQ ID NO:1, and fragments of variants thereof the sequence. The invention also provides polynucleotides encoding such polypetide sequences. The invention also provides constructs, cells and plants comprising such polynucleotides.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority as a continuation of U.S. application Ser. No. 13/528,558, filed Jun. 20, 2012, which itself is a continuation of U.S. application Ser. No. 12/324,664, filed Nov. 26, 2008, and claims priority to U.S. Provisional Application No. 60/990,590, filed Nov. 27, 2007. The priority applications are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled Sequence-Listing-JAMES161002C2.txt, created May 7, 2015, which is 76.8 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention
The present invention relates to compositions and methods for producing plants with increased biomass.
2. Description of the Related Art
As the population of the world increases, a major goal of agricultural research is to improve the biomass yield of crop and forage plant species.
Such improvements have until recently depended on selective breeding of plants for desirable characteristics. However for many plants the heterogeneous genetic complements produced in off-spring do not result in the same desirable traits as those of their parents, thus limiting the effectiveness of selective breeding approaches.
Advances in molecular biology now make it possible to genetically manipulate the germplasm of both plants and animals. Genetic engineering of plants involves the isolation and manipulation of genetic material and the subsequent introduction of such material into a plant. This technology has led to the development of plants that are capable of expressing pharmaceuticals and other chemicals, plants with increased pest resistance, increased stress tolerance, and plants that express other beneficial traits.
Whilst it is known in the art that certain growth factors may be applied to increase plant size, the application of such growth factors is both costly and time consuming Thus, there exists a need for plants with increased biomass relative to their cultivated counterparts.
It is an object of the invention to provide improved compositions and/or methods for developing plant varieties with altered biomass or at least to provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect the invention provides a method for producing a plant with altered biomass, the method comprising transformation of a plant with a:

- a) a polynucleotide including a sequence encoding of a polypeptide with the amino acid sequence of SEQ ID NO:1 or a variant of the polypeptide; or
- b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); or
- c) a polynucleotide comprising a compliment, of at least 15 nucleotides in length, of the polynucleotide of a); or d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions.

Preferably the polynucleotide is included as part of a genetic construct.
In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
Preferably the variant is capable of modulating biomass in a plant
In a further embodiment the polynucleotide of a) encodes a polypeptide with the amino acid sequence of SEQ ID NO: 1.
Preferably expression of the polynucleotide in the plant results in down-regulation of an endogenous polynucleotide/polypeptide capable of modulating biomass production in the plant.
Preferably the reduced expression is effected by antisense suppression, sense suppression or RNA interference.
Preferably the plant produced has increased biomass relative to a suitable control plant.
In a further aspect the invention provides a method for producing a plant with increased biomass, the method comprising transformation of a plant with a polynucleotide with sufficient sequence similarity to an endogenous nucleic acid encoding a polypeptide with the sequence of SEQ ID NO:1 or a variant thereof, such that expression of the polynucleotide results in inhibition of expression of the endogenous nucleic acid.
Preferably the polynucleotide is included as part of a genetic construct.
In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
Preferably the variant is capable of modulating biomass in a plant.
In a further embodiment the polypeptide has the sequence of SEQ ID NO:1
In a further aspect the invention provides a method of producing a plant with altered biomass, the method comprising transformation of a plant cell or plant with a:

- a) a polynucleotide comprising the nucleotide sequence of SEQ ID NO:10, or a variant thereof; or
- b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); or
- c) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of a); or
- d) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of a) under stringent conditions.

Preferably the polynucleotide is included as part of a genetic construct.
Preferably the variant encodes a polypeptide capable of modulating biomass in a plant
In one embodiment the polynucleotide of a) comprises the sequence of SEQ ID NO:10. Preferably expression of the polynucleotide in the plant results in down-regulation of an endogenous polynucleotide/polypeptide capable of modulating biomass production in the plant.
Preferably the down-regulation is effected by antisense suppression, sense suppression or RNA interference.
Preferably the plant produced by the method of the invention has increased biomass relative to a suitable control plant.
In a further aspect the invention provides a method for producing a plant with increased biomass the method comprising transformation of a plant with a polynucleotide with sufficient sequence similarity to an endogenous nucleic acid with the sequence of SEQ ID NO:10 or a variant thereof, such that in expression of the polynucleotide results in inhibition of expression of the endogenous nucleic acid.
Preferably the polynucleotide is included as part of a genetic construct.
In one embodiment the variant has at least 70% sequence identity with the full-length coding sequence of SEQ ID NO: 10.
In a further embodiment the variant encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a plant species and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a dicotyledonous plant species and encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 21.
Preferably the variant encodes a polypeptide capable of modulating biomass in a plant
In a further embodiment the endogenous nucleic acid comprises the full-length coding sequence of SEQ ID NO:10.
In a further aspect the invention provides a method for producing a plant cell or plant with altered biomass, the method comprising reducing the expression or activity of a polypeptide including the amino acid sequence of SEQ ID NO: 1 or variant thereof.
In one embodiment the variant has at least 70% sequence identity to a polypeptide with the amino acid sequence of SEQ ID NO: 1.
In a further embodiment the variant comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a plant species and comprises the amino acid sequence of SEQ ID NO: 20.
In a further embodiment the variant is derived from a dicotyledonous plant species and comprises the amino acid sequence of SEQ ID NO: 21.
In a further embodiment the polypeptide has the sequence of SEQ ID NO:1
In a further aspect the invention provides a method of producing a plant with altered biomass the method comprising the step of reducing the expression or activity in a plant cell or plant of a polypeptide comprising the sequence of SEQ ID NO: 20.
In one embodiment the polypeptide comprises the sequence of SEQ ID NO: 21.
In a further embodiment the the polypeptide comprises the sequence of with at least 70% identity to the sequence of SEQ ID NO: 1.
In a further embodiment the polypeptide comprises the sequence of SEQ ID NO: 1.
In a further embodiment the a polynucleotide capable of hybridising under stringent conditions to an endogenous nucleic acid encoding the polypeptide is introduced into the plant cell or plant to effect reduced expression of the polypeptide.
In a further embodiment the endogenous nucleic acid comprises a sequence with at least 70% identity to the full-length coding sequence of SEQ ID NO: 10.
In a further embodiment the endogenous nucleic acid comprises the full-length coding sequence of SEQ ID NO: 10.
In a further embodiment the polynucleotide comprises at least 15 contiguous nucleotides of a sequence with at least 70% identity to the sequence of SEQ ID NO: 10.
In a further embodiment the polynucleotide comprises at least 15 contiguous nucleotides of SEQ ID NO: 10.
In a further aspect the invention provides a plant cell or plant produced by a method of the invention.
Preferably the plant produced by the method of the invention has increased biomass production relative to a suitable control plant.
Preferably the plant produced by the method of the invention has an increased number of tillers relative to a suitable control plant.
In a further aspect the invention provides an isolated polynucleotide having at least 71% sequence identity to a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.
Preferably the polynucleotide encodes a polypeptide capable of modulating biomass in a plant
In one embodiment the polypeptide comprises the amino acid sequence of SEQ ID NO:1
In a further embodiment the nucleotide sequence comprises the sequence of SEQ ID NO:10.
In a further embodiment said nucleotide sequence comprises the full-length coding sequence of SEQ ID NO:10.
In a further aspect the invention provides an isolated polynucleotide that encodes a polypeptide comprising an amino acid sequence SEQ ID NO: 1.
In one embodiment the polynucleotide comprises the sequence of SEQ ID NO:10.
In a further embodiment the polynucleotide comprises the full-length coding sequence of SEQ ID NO:10.
In a further aspect the invention provides an isolated polynucleotide comprising the full-length coding sequence of SEQ ID NO: 10 or a variant thereof, wherein the variant is derived from ryegrass or fescue, and encodes a polypeptide capable of modulating biomass in a plant.
In one embodiment the variant has at least 70% sequence identity to the full-length coding sequence of SEQ ID NO:10.
In one embodiment the isolated polynucleotide comprises the sequence of SEQ ID NO:10.
In a further aspect the invention provides an isolated polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 1, wherein the polypeptide is capable of modulating biomass in a plant.
In one embodiment the isolated polypeptide the amino acid sequence of SEQ ID NO: 1.
In a further aspect the invention provides an isolated polynucleotide encoding a polypeptide of the invention.
In a further aspect the invention provides an isolated polynucleotide comprising:

- a) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of a polynucleotide of the invention; or
- b) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of the invention; or
- c) a polynucleotide comprising a sequence, of at least 15 nucleotides in length, capable of hybridising to the polynucleotide of the invention.

In a further aspect the invention provides a genetic construct which comprises a polynucleotide of the invention.
In one embodiment the genetic construct is an expression construct.
In a further aspect the invention provides a vector comprising an expression construct or genetic construct of the invention.
In a further aspect the invention provides a host cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
In a further aspect the invention provides a host cell comprising an expression construct or genetic construct of the invention.
In a further aspect the invention provides a plant cell genetically modified to express a polynucleotide of the invention, or a polypeptide of the invention.
In a further aspect the invention provides a plant cell which comprises an expression construct of the invention or the genetic construct of the invention.
Preferably the expression construct is capable of expressing the polynucleotide, resulting in inhibition of expression of an endogenous polynucleotide/polypeptide which is capable of modulating biomass production in the plant.
In a further aspect the invention provides a plant which comprises a plant cell of the invention.
In a further aspect the invention provides a method for selecting a plant with altered biomass, the method comprising testing of a plant for altered expression of a polynucleotide of the invention.
In a further aspect the invention provides a method for selecting a plant with altered biomass, the method comprising testing of a plant for altered expression of a polypeptide of the invention.
In a further aspect the invention provides a plant cell or plant produced by the method of the invention.
In a further aspect the invention provides a plant selected by the method of the invention.
In a further aspect the invention provides an antibody raised against a polypeptide of the invention.
The polynucleotides and polynucleotide variants, of the invention may be derived from any species and/or may be produced recombinantly or synthetically.
In one embodiment the polynucleotide or variant, is derived from a plant species.
In a further embodiment the polynucleotide or variant, is derived from a gymnosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from an angiosperm plant species.
In a further embodiment the polynucleotide or variant, is derived from a from dicotyledonous plant species.
In a further embodiment the polynucleotide or variant, is derived from a monocotyledonous plant species.
The polypeptide and polypeptide variants, of the invention may be derived from any species and/or may be produced recombinantly or synthetically.
In one embodiment the polypeptide or variant, is derived from a plant species.
In a further embodiment the polypeptide or variant, is derived from a gymnosperm plant species.
In a further embodiment the polypeptide or variant, is derived from an angiosperm plant species.
In a further embodiment the polypeptide or variant, is derived from a from dicotyledonous plant species.
In a further embodiment the polypeptide or variant, is derived from a monocotyledonous plant species.
The plant cell or plant may be derived from any plant species.
In a further embodiment the plant cell or plant, is derived from a gymnosperm plant species.
In a further embodiment the plant cell or plant, is derived from an angiosperm plant species.
In a further embodiment the plant cell or plant, is derived from a from dicotyledonous plant species.
In a further embodiment the plant cell or plant, is derived from a monocotyledonous plant 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, Mucana, Olea, Onobrychis, Ornithopus, papaver, Phaseolus, Phoenix, Pistacia, Pitum, Prunus, Pueraria, ribes, Richinus, 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, 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, Lupinus angustifolius, Lupinus luteus, Lupinus matabilis, Lespedeza sericea, Lespedeza striate, Lotus uliginosus, Luthyrus sativus, Lens culinaris, Lespedeza stipulacea, Linum usitatissimum, Lotus corniculatus, Lupinus albus, Medicago arborea, Medicago falcate, Medicago hispida, Medicago officinalis, medicago, sativa Alfalfa, medicago tribuloides, Macadamia integrifoniia, Medicago arabica, Melilotus albus, Mucuna prim:ens, Olea europaea, Onobrychis viciifolia, Ornithopus sativus, Phaseolus aureus, Prunus cerasifera, Prunus cerasus, Phaseolus coccineus, Prunus domestica, Phaseolus lumatus, Prunus maheleb, Phaseolus mango, Prunus persica, Prunus pseudocerasus, Phaseolus vulgaris, Papaver soinniferum, Phaseolus acutifolius, Phoenix dactylifera, Pistacia vera, Pisum sativum, Prunus amygdalus, Prunus armeniaca, Pueraria thunbergiana, Ribes nigrum, Ribes rubrum, Ribes grossularia, Ricinus communis, Sesamum indicum, Trifolium augustifolium, Trifolium diffusum, Trifolium hybridum, Trifolium incernatum, Trifolium ingrescens, Trifolium pratense, Trifolium repens, Trifolium resupinatum, Triolium subterraneum, Theobroma cacao, Trifolium alexandrinum, Trigonella foenumgraecum, Vicia angestifolia, Vicia atropurpurea, Vicia calcarata, Vicia dasycarpa, Vicia ervilia, Vaccinium oxycoccos, Vicia pannonica, Vigna sesquipedalis, Vigna sinensis, Vicia vollosa, Vicia faba, Vicia sative and Vigna angularis.
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, Album ascalonicum, Album cepa, Album chinense, Allium porrum, Album schoenoprasum, Album fistulosum, Album 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, 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.
Preferred plants are forage plant species from a group comprising but not limited to the following genera: Lolium, Festuca, Dactylis,Bromus, Trifolium, Medicago, Phleum, Phalaris, Holcus, Lotus, Plantago and Cichorium.
Particularly preferred plants are from the genera Lolium and Trifolium. Particularly preferred species are Lolium perenne and Trifolium repens.
Particularly preferred monocotyledonous plant species are: Lolium perenne and Oryza sativa.
The term “plant” is intended to include a whole plant, any part of a plant, propagules and progeny of a plant.
The term ‘propagule’ means any part of a plant that may be used in reproduction or propagation, either sexual or asexual, including seeds and cuttings.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

Polynucleotides and Fragments

The term “polynucleotide(s),” as used herein, means a single or double-stranded deoxyribonucleotide 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 sequence provided herein is a subsequence of contiguous nucleotides that is capable of specific hybridization to a target of interest, e.g., a sequence that is at least 15 nucleotides in length. The fragments of the invention comprise 15 nucleotides, preferably at least 20 nucleotides, more preferably at least 30 nucleotides, more preferably at least 50 nucleotides, more preferably at least 50 nucleotides and most preferably at least 60 nucleotides of contiguous nucleotides of a polynucleotide of the invention. A fragment of a polynucleotide sequence can be used in antisense, 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.
The term “primer” refers to a short polynucleotide, usually having a free 3′ H group, that is 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 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 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 polypeptide is a subsequence of the polypeptide that performs a function that is required for the biological activity and/or provides three dimensional structure of the polypeptide. 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 capable of performing the above enzymatic activity.
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. 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 removed from sequences that surround it in its natural context and/or is 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 and 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 which is derived from a 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 inventive polypeptides and polynucleotides possess biological activities that are the same or similar to those of the inventive polypeptides or polynucleotides. 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 specified polynucleotide sequence. 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 the specified polynucleotide sequence.
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/). The default parameters of bl2seq are utilized except that filtering of low complexity parts should be turned off.
The identity of polynucleotide sequences may be examined using the following unix command line parameters:
bl2seq -i nucleotideseq1-j nucleotideseq2 -F F -p blastn
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. The bl2seq program reports sequence identity as both the number and percentage of identical nucleotides in a line “Identities=”.
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.
Use of BLASTN as described above is preferred for use in the determination of sequence identity for polynucleotide variants according to the present invention.
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 [November 2002]) from NCBI (ftp://ftp.ncbi.nih gov/blast/).
The similarity of polynucleotide sequences may be examined using the following unix command line parameters:

- bl2seq -i nucleotideseq1-j nucleotideseq2-F F -p tblastx

The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. The size of this database is set by default in the bl2seq program.
For small E values, much less than one, the E value is approximately the probability of such a random match.
Variant polynucleotide sequences preferably exhibit an E value of less than 1×10⁻¹⁰more preferably less than 1×10⁻²⁰, more preferably less than 1×10⁻³⁰, more preferably less than 1×10⁴⁰, more preferably less than 1×10⁻⁵⁰, more preferably less than 1×10⁻⁶⁰, more preferably less than 1×10⁻⁷⁰, more preferably less than 1×10⁻⁸⁰, more preferably less than 1×10⁻⁹⁰and most preferably less than 1×10⁻¹⁰⁰when compared with any one of the specifically identified sequences.
Alternatively, variant polynucleotides of the present invention hybridize to a specified polynucleotide sequence, 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 30° C. (for example, 10° 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 6×SSC, 0.2% SDS; hybridizing at 65° C., 6×SSC, 0.2% SDS overnight; followed by two washes of 30 minutes each in 1×SSC, 0.1% SDS at 65° C. and two washes of 30 minutes each in 0.2×SSC, 0.1% SDS at 65° C.
With respect to polynucleotide molecules having a length less than 100 bases, exemplary stringent hybridization conditions are 5 to 10° C. below Tm. On average, the Tm of a polynucleotide molecule of length less than 100 by is reduced by approximately (500/oligonucleotide length)° 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 10° 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 [November 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 %, 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 [November 2002]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions 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.
Use of BLASTP as described above is preferred for use in the determination of polypeptide variants according to the present invention.
Polypeptide 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 [November 20021) from NCBI (ftp://ftp.ncbi.nih gov/blast/). The similarity of polypeptide sequences may be examined using the following unix command line parameters:

- bl2seq -i peptideseq1 -j peptideseq2 -F F -p blastp

Variant polypeptide sequences preferably exhibit an E value of less than 1×10⁻¹⁰more preferably less than 1×10⁻²⁰, more preferably less than 1×10⁻³⁰, more preferably less than 1×10⁴⁰, more preferably less than 1×10⁻⁵⁰, more preferably less than 1×10⁻⁶⁰, more preferably less than 1×10⁻⁷⁰, more preferably less than 1×10⁻⁸⁰, more preferably less than 1×10⁻⁹⁰and most preferably less than 1×10⁻¹⁰⁰when compared with any one of the specifically identified sequences.
The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an “E value” which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
Conservative substitutions of one or several amino acids of a described 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).
Constructs, Vectors and Components Thereof
The term “genetic construct” refers to a polynucleotide molecule, usually double-stranded DNA, which may have inserted into it another polynucleotide molecule (the insert polynucleotide molecule) such as, but not limited to, a cDNA molecule. A genetic construct may contain the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. The insert polynucleotide molecule may be derived from the host cell, or may be derived from a different cell or organism and/or may be a recombinant polynucleotide. Once inside the host cell the genetic construct may become integrated in the host chromosomal DNA. The genetic construct may be linked to a vector.
The term “vector” refers to a polynucleotide molecule, usually double stranded DNA, which is used to transport the genetic construct into a host cell. The vector may be capable of replication in at least one additional host system, such as E. coli.
The term “expression construct” refers to a genetic construct that includes the necessary elements that permit transcribing the insert polynucleotide molecule, and, optionally, translating the transcript into a polypeptide. An expression construct typically comprises in a 5′ to 3′ direction:

- a) a promoter functional in the host cell into which the construct will be transformed,
- b) the polynucleotide to be expressed, and
- c) a terminator functional in the host cell into which the construct will be transformed.

The term “coding region” or “open reading frame” (ORF) refers to the sense strand of a genomic DNA sequence or a cDNA sequence that is capable of producing a transcription product and/or a polypeptide under the control of appropriate regulatory sequences. The coding sequence is identified by the presence of a 5′ translation start codon and a 3′ translation stop codon. When inserted into a genetic construct, a “coding sequence” is capable of being expressed when it is operably linked to promoter and terminator sequences.
“Operably-linked” means that the sequenced to be expressed is placed under the control of regulatory elements that include promoters, tissue-specific regulatory elements, temporal regulatory elements, enhancers, repressors and terminators.
The term “noncoding region” refers to untranslated sequences that are upstream of the translational start site and downstream of the translational stop site. These sequences are also referred to respectively as the 5′ UTR and the 3′ UTR. These regions include elements required for transcription initiation and termination and for regulation of translation efficiency.
Terminators are sequences, which terminate transcription, and are found in the 3′ untranslated ends of genes downstream of the translated sequence. Terminators are important determinants of mRNA stability and in some cases have been found to have spatial regulatory functions.
The term “promoter” refers to nontranscribed cis-regulatory elements upstream of the coding region that regulate gene transcription. Promoters comprise cis-initiator elements which specify the transcription initiation site and conserved boxes such as the TATA box, and motifs that are bound by transcription factors.
A “transgene” is a polynucleotide that is taken from one organism and introduced into a different organism by transformation. The transgene may be derived from the same species or from a different species as the species of the organism into which the transgene is introduced.
An “inverted repeat” is a sequence that is repeated, where the second half of the repeat is in the complementary strand, e.g.,

	(5′)GATCTA . . . TAGATC(3′)
	(3′)CTAGAT . . . ATCTAG(5′)

Read-through transcription will produce a transcript that undergoes complementary base-pairing to form a hairpin structure provided that there is a 3-5 by spacer between the repeated regions.
A “transgenic plant” refers to a plant which contains new genetic material as a result of genetic manipulation or transformation. The new genetic material may be derived from a plant of the same species as the resulting transgenic plant or from a different species.
The terms “to alter expression of” and “altered expression” of a polynucleotide or polypeptide of the invention, are intended to encompass the situation where genomic DNA corresponding to a polynucleotide of the invention is modified thus leading to altered expression of a polynucleotide or polypeptide of the invention. Modification of the genomic DNA may be through genetic transformation or other methods known in the art for inducing mutations. The “altered expression” can be related to an increase or decrease in the amount of messenger RNA and/or polypeptide produced and may also result in altered activity of a polypeptide due to alterations in the sequence of a polynucleotide and polypeptide produced.
The term “biomass” refers to the size and/or mass and/or number of vegetative organs of the plant at a particular age or developmental stage. Thus a plant with increased biomass has increased size and/or mass and/or number of vegetative organs than a suitable control plant of the same age or at an equivalent developmental stage. Conversely, a plant with decreased biomass has decreased size and/or mass and/or number of vegetative organs than a suitable control. Altered biomass may also involve an alteration in rate of growth and/or rate of formation of vegetative organs during some or all periods of the life cycle of a plant relative to a suitable control. Thus altered biomass may result in an advance or delay in the time taken for such a plant to reach a certain developmental stage.
Suitable control plants may include non-transformed plants of the same species and variety, or plants of the same species or variety transformed with a control construct.
The invention provides methods for producing and selecting plants with altered biomass relative to suitable control plants, including plants with both increased and decreased biomass and plants produced by such methods.
The invention provides a polynucleotide (SEQ ID NO:10) encoding a polypeptide (SEQ ID NO:1) which modulates biomass in plants. The invention provides polynucleotide variants of SEQ ID NO:10 (SEQ ID NOs: 11 to 18) which encode polypeptide variants of SEQ ID NO:1 (SEQ ID NO:2 to 9). The applicants have also identified a consensus polypeptide sequence motif present in SEQ ID NO:1 and all of the polypeptide variants of SEQ ID NO:1, as shown in SEQ ID NO:20, and a further consensus motif (SEQ ID NO: 21) present in SEQ ID NO:1 (ORF54) and all polypeptide variants thereof that are derived from dicotyledonous plants.
Methods for Isolating Polynucleotides
The polynucleotide molecules of the invention can be isolated by using a variety of techniques known to those of ordinary skill in the art. By way of example, such polypeptides can be isolated through use of the polymerase chain reaction (PCR) described in Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser, incorporated herein by reference. The polypeptides of the invention can be amplified using primers, as defined herein, derived from the polynucleotide sequences of the invention.
Further methods for isolating polynucleotides of the invention, or polynucleotides useful in methods of the invention, include use of all, or portions of, the polynucleotides set forth herein as hybridization probes. The technique of hybridizing labelled polynucleotide probes to polynucleotides immobilized on solid supports such as nitrocellulose filters or nylon membranes, can be used to screen the genomic or cDNA libraries. Exemplary hybridization and wash conditions are: hybridization for 20 hours at 65° C. in 5.0×SSC, 0.5% sodium dodecyl sulfate, 1× Denhardt's solution; washing (three washes of twenty minutes each at 55° C.) in 1.0×SSC, 1% (w/v) sodium dodecyl sulfate, and optionally one wash (for twenty minutes) in 0.5×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C. An optional further wash (for twenty minutes) can be conducted under conditions of 0.1×SSC, 1% (w/v) sodium dodecyl sulfate, at 60° C.
The polynucleotide fragments of the invention may be produced by techniques well-known in the art such as restriction endonuclease digestion and oligonucleotide synthesis.
A partial polynucleotide sequence may be used, in methods well-known in the art to identify the corresponding full-length polynucleotide sequence. Such methods include PCR-based methods, 5′RACE (Frohman Mass., 1993, Methods Enzymol. 218: 340-56) and hybridization-based method, computer/database -based methods. Further, by way of example, inverse PCR permits acquisition of unknown sequences, flanking the polynucleotide sequences disclosed herein, starting with primers based on a known region (Triglia et al., 1998, Nucleic Acids Res 16, 8186, incorporated herein by reference). The method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template. Divergent primers are designed from the known region. In order to physically assemble full-length clones, standard molecular biology approaches can be utilized (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987).
It may be beneficial, when producing a transgenic plant from a particular species, to transform such a plant with a sequence or sequences derived from that species. The benefit may be to alleviate public concerns regarding cross-species transformation in generating transgenic organisms. Additionally when down-regulation of a gene is the desired result, it may be necessary to utilise a sequence identical (or at least highly similar) to that in the plant, for which reduced expression is desired. For these reasons among others, it is desirable to be able to identify and isolate orthologues of a particular gene in several different plant species. Variants (including orthologues) may be identified by the methods described.
Methods for Identifying Variants
Physical Methods
Variant polynucleotides may be identified using PCR-based methods (Mullis et al., Eds. 1994 The Polymerase Chain Reaction, Birkhauser). Typically, the polynucleotide sequence of a primer, useful to amplify variant polynucleotide molecules by PCR, may be based on a sequence encoding a conserved region of the corresponding amino acid sequence.
Alternatively library screening methods will be known to those skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) may be employed. When identifying variants of the probe sequence hybridisation and/or wash stringency conditions will typically be reduced relative to when exact sequence matches are sought.
Polypeptide variants of the invention may be identified by physical methods, for example by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) or by identifying polypeptides from natural sources with the aid of such antibodies.
Computer Based Methods
The variant sequences of the invention, including both polynucleotide and polypeptide variants, may also be identified by computer-based methods well-known to those skilled in the art, using public domain sequence alignment algorithms and sequence similarity search tools to search sequence databases (public domain databases include Genbank, EMBL, Swiss-Prot, PIR and others). See, e.g., Nucleic Acids Res. 29: 1-10 and 11-16, 2001 for examples of online resources. Similarity searches retrieve and align target sequences for comparison with a sequence to be analyzed (i.e., a query sequence). Sequence comparison algorithms use scoring matrices to assign an overall score to each of the alignments.
An exemplary family of programs useful for identifying variants in sequence databases is the BLAST suite of programs (version 2.2.5 [Nov 2002]) including BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX, which are publicly available from (ftp://ftp.ncbi.nih gov/blast/) or from the National Center for Biotechnology Information (NCBI), National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894 USA. The NCBI server also provides the facility to use the programs to screen a number of publicly available sequence databases. BLASTN compares a nucleotide query sequence against a nucleotide sequence database. BLASTP compares an amino acid query sequence against a protein sequence database. BLASTX compares a nucleotide query sequence translated in all reading frames against a protein sequence database. tBLASTN compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. tBLASTX compares the six-frame translations of a nucleotide query sequence against the six-frame translations of a nucleotide sequence database. The BLAST programs may be used with default parameters or the parameters may be altered as required to refine the screen.
The use of the BLAST family of algorithms, including BLASTN, BLASTP, and BLASTX, is described in the publication of Altschul et al., Nucleic Acids Res. 25: 3389-3402, 1997.
The “hits” to one or more database sequences by a queried sequence produced by BLASTN, BLASTP, BLASTX, tBLASTN, tBLASTX, or a similar algorithm, align and identify similar portions of sequences. The hits are arranged in order of the degree of similarity and the length of sequence overlap. Hits to a database sequence generally represent an overlap over only a fraction of the sequence length of the queried sequence.
The BLASTN, BLASTP, BLASTX, tBLASTN and tBLASTX algorithms also produce “Expect” values for alignments. The Expect value (E) indicates the number of hits one can “expect” to see by chance when searching a database of the same size containing random contiguous sequences. The Expect value is used as a significance threshold for determining whether the hit to a database indicates true similarity. For example, an E value of 0.1 assigned to a polynucleotide hit is interpreted as meaning that in a database of the size of the database screened, one might expect to see 0.1 matches over the aligned portion of the sequence with a similar score simply by chance. For sequences having an E value of 0.01 or less over aligned and matched portions, the probability of finding a match by chance in that database is 1% or less using the BLASTN, BLASTP, BLASTX, tBLASTN or tBLASTX algorithm.
Multiple sequence alignments of a group of related sequences can be carried out with CLUSTALW (Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research, 22:4673-4680, http://www-igbmc.u-strasbg.fr/Biolnfo/ClustalW/Top.html) or T-COFFEE (Cedric Notredame, Desmond G. Higgins, Jaap Heringa, T-Coffee: A novel method for fast and accurate multiple sequence alignment, J. Mol. Biol. (2000) 302: 205-217))or PILEUP, which uses progressive, pairwise alignments. (Feng and Doolittle, 1987, J. Mol. Evol. 25, 351).
Pattern recognition software applications are available for finding motifs or signature sequences. For example, MEME (Multiple Em for Motif Elicitation) finds motifs and signature sequences in a set of sequences, and MAST (Motif Alignment and Search Tool) uses these motifs to identify similar or the same motifs in query sequences. The MAST results are provided as a series of alignments with appropriate statistical data and a visual overview of the motifs found. MEME and MAST were developed at the University of California, San Diego.
PROSITE (Bairoch and Bucher, 1994, Nucleic Acids Res. 22, 3583; Hofmann et al., 1999, Nucleic Acids Res. 27, 215) is a method of identifying the functions of uncharacterized proteins translated from genomic or cDNA sequences. The PROSITE database (www.expasy.org/prosite) contains biologically significant patterns and profiles and is designed so that it can be used with appropriate computational tools to assign a new sequence to a known family of proteins or to determine which known domain(s) are present in the sequence (Falquet et al., 2002, Nucleic Acids Res. 30, 235). Prosearch is a tool that can search SWISS-PROT and EMBL databases with a given sequence pattern or signature.
Methods for Isolating Polypeptides
The polypeptides of the invention, including variant polypeptides, may be prepared using peptide synthesis methods well known in the art such as direct peptide synthesis using solid phase techniques (e.g. Stewart et al., 1969, in Solid-Phase Peptide Synthesis, WH Freeman Co, San Francisco Calif., or automated synthesis, for example using an Applied Biosystems 431A Peptide Synthesizer (Foster City, Calif.). Mutated forms of the polypeptides may also be produced during such syntheses.
The polypeptides and variant polypeptides of the invention may also be purified from natural sources using a variety of techniques that are well known in the art (e.g. Deutscher, 1990, Ed, Methods in Enzymology, Vol. 182, Guide to Protein Purification).
Alternatively the polypeptides and variant polypeptides of the invention may be expressed recombinantly in suitable host cells and separated from the cells as discussed below.
Methods for producing constructs and vectors
The genetic constructs of the present invention comprise one or more polynucleotide sequences of the invention and/or polynycleotides encoding polypeptides of the invention, and may be useful for transforming, for example, bacterial, fungal, insect, mammalian or plant organisms. The genetic constructs of the invention are intended to include expression constructs as herein defined.
Methods for producing and using genetic constructs and vectors are well known in the art and are described generally in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987).
Methods for Producing Host Cells Comprising Constructs and Vectors
The invention provides a host cell which comprises a genetic construct or vector of the invention. Host cells may be derived from, for example, bacterial, fungal, insect, mammalian or plant organisms.
Host cells comprising genetic constructs, such as expression constructs, of the invention are useful in methods well known in the art (e.g. Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987 ; Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing, 1987) for recombinant production of polypeptides of the invention. Such methods may involve the culture of host cells in an appropriate medium in conditions suitable for or conducive to expression of a polypeptide of the invention. The expressed recombinant polypeptide, which may optionally be secreted into the culture, may then be separated from the medium, host cells or culture medium by methods well known in the art (e.g. Deutscher, Ed, 1990, Methods in Enzymology, Vol 182, Guide to Protein Purification).
Host cells of the invention may also be useful in methods for production of an enzymatic product generated by an expressed polypeptide of the invention. Such methods may involve culturing the host cells of the invention in a medium suitable for expression of a recombinant polypeptide of the invention, optionally in the presence of additional enzymatic substrate for the expressed polypeptide of the invention. The enzymatic product produced may then be separated from the host cells or medium by a variety of art standard methods.
Methods for Producing Plant Cells and Plants Comprising Constructs and Vectors
The invention further provides plant cells which comprise a genetic construct of the invention, and plant cells modified to alter expression of a polynucleotide or polypeptide of the invention. Plants comprising such cells also form an aspect of the invention.
Production of plants altered in biomass may be achieved through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide capable of modulating biomass production in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polypeptides or polypeptides capable of modulating biomass production in such plant cells and plants.
Methods for transforming plant cells, plants and portions thereof with polynucleotides are described in Draper et al., 1988, Plant Genetic Transformation and Gene Expression. A Laboratory Manual, Blackwell Sci. Pub. Oxford, p. 365; Potrykus and Spangenburg, 1995, Gene Transfer to Plants. Springer-Verlag, Berlin.; and Gelvin et al., 1993, Plant Molecular Biol. Manual. Kluwer Acad. Pub. Dordrecht. A review of transgenic plants, including transformation techniques, is provided in Galun and Breiman, 1997, Transgenic Plants. Imperial College Press, London.
Methods for Genetic Manipulation of Plants
A number of strategies for genetically manipulating plants are available (e.g. Birch, 1997, Ann Rev Plant Phys Plant Mol Biol, 48, 297). For example, strategies may be designed to increase expression of a polynucleotide/polypeptide in a plant cell, organ and/or at a particular developmental stage where/when it is normally expressed or to ectopically express a polynucleotide/polypeptide in a cell, tissue, organ and/or at a particular developmental stage which/when it is not normally expressed. The expressed polynucleotide/polypeptide may be derived from the plant species to be transformed or may be derived from a different plant species.
Transformation strategies may be designed to reduce expression of a polynucleotide/polypeptide in a plant cell, tissue, organ or at a particular developmental stage which/when it is normally expressed. Such strategies are known as gene silencing strategies.
Genetic constructs for expression of genes in transgenic plants typically include promoters for driving the expression of one or more cloned polynucleotide, terminators and selectable marker sequences to detest presence of the genetic construct in the transformed plant.
The promoters suitable for use in the constructs of this invention are functional in a cell, tissue or organ of a monocot or dicot plant and include cell-, tissue- and organ-specific promoters, cell cycle specific promoters, temporal promoters, inducible promoters, constitutive promoters that are active in most plant tissues, and recombinant promoters. Choice of promoter will depend upon the temporal and spatial expression of the cloned polynucleotide, so desired. The promoters may be those normally associated with a transgene of interest, or promoters which are derived from genes of other plants, viruses, and plant pathogenic bacteria and fungi. Those skilled in the art will, without undue experimentation, be able to select promoters that are suitable for use in modifying and modulating plant traits using genetic constructs comprising the polynucleotide sequences of the invention. Examples of constitutive plant promoters include the CaMV 35S promoter, the nopaline synthase promoter and the octopine synthase promoter, and the Ubi 1 promoter from maize. Plant promoters which are active in specific tissues, respond to internal developmental signals or external abiotic or biotic stresses are described in the scientific literature. Exemplary promoters are described, e.g., in WO 02/00894, which is herein incorporated by reference.
Exemplary terminators that are commonly used in plant transformation genetic construct include, e.g., the cauliflower mosaic virus (CaMV) 35S terminator, the Agrobacterium tumefaciens nopaline synthase or octopine synthase terminators, the Zea mays zein gene terminator, the Oryza sativa ADP-glucose pyrophosphorylase terminator and the Solanum tuberosum PI-II terminator.
Selectable markers commonly used in plant transformation include the neomycin phophotransferase II gene (NPT II) which confers kanamycin resistance, the aadA gene, which confers spectinomycin and streptomycin resistance, the phosphinothricin acetyl transferase (bar gene) for Ignite (AgrEvo) and Basta (Hoechst) resistance, and the hygromycin phosphotransferase gene (hpt) for hygromycin resistance.
Use of genetic constructs comprising reporter genes (coding sequences which express an activity that is foreign to the host, usually an enzymatic activity and/or a visible signal (e.g., luciferase, GUS, GFP) which may be used for promoter expression analysis in plants and plant tissues are also contemplated. The reporter gene literature is reviewed in Herrera-Estrella et al., 1993, Nature 303, 209, and Schrott, 1995, In: Gene Transfer to Plants (Potrykus, T., Spangenberg. Eds) Springer Verlag. Berline, pp. 325-336.
Gene silencing strategies may be focused on the gene itself or regulatory elements which effect expression of the encoded polypeptide. “Regulatory elements” is used here in the widest possible sense and includes other genes which interact with the gene of interest.
Genetic constructs designed to decrease or silence the expression of a polynucleotide/polypeptide of the invention may include an antisense copy of a polynucleotide of the invention. In such constructs the polynucleotide is placed in an antisense orientation with respect to the promoter and terminator.
An “antisense” polynucleotide is obtained by inverting a polynucleotide or a segment of the polynucleotide so that the transcript produced will be complementary to the mRNA transcript of the gene, e.g.,

5′GATCTA 3′ (coding	3′CTAGAT 5′ (antisense strand)
strand)

3′CUAGAU 5′ mRNA	5′GAUCUCG 3′ antisense RNA

Genetic constructs designed for gene silencing may also include an inverted repeat. An ‘inverted repeat’ is a sequence that is repeated where the second half of the repeat is in the complementary strand, e.g.,

	5′-GATCTA . . . TAGATC-3′

	3′-CTAGAT . . . ATCTAG-5′

The transcript formed may undergo complementary base pairing to form a hairpin structure. Usually a spacer of at least 3-5 by between the repeated region is required to allow hairpin formation.
Another silencing approach involves the use of a small antisense RNA targeted to the transcript equivalent to an miRNA (Llave et al., 2002, Science 297, 2053). Use of such small antisense RNA corresponding to polynucleotide of the invention is expressly contemplated.
The term genetic construct as used herein also includes small antisense RNAs and other such polynucleotides useful for effecting gene silencing.
Transformation with an expression construct, as herein defined, may also result in gene silencing through a process known as sense suppression (e.g. Napoli et al., 1990, Plant Cell 2, 279; de Carvalho Niebel et al., 1995, Plant Cell, 7, 347). In some cases sense suppression may involve over-expression of the whole or a partial coding sequence but may also involve expression of non-coding region of the gene, such as an intron or a 5′ or 3′ untranslated region (UTR). Chimeric partial sense constructs can be used to coordinately silence multiple genes (Abbott et al., 2002, Plant Physiol. 128(3): 844-53; Jones et al., 1998, Planta 204: 499-505). The use of such sense suppression strategies to silence the expression of a polynucleotide of the invention is also contemplated.
The polynucleotide inserts in genetic constructs designed for gene silencing may correspond to coding sequence and/or non-coding sequence, such as promoter and/or intron and/or 5′ or 3′ UTR sequence, or the corresponding gene.
Other gene silencing strategies include dominant negative approaches and the use of ribozyme constructs (McIntyre, 1996, Transgenic Res, 5, 257).
Pre-transcriptional silencing may be brought about through mutation of the gene itself or its regulatory elements. Such mutations may include point mutations, frameshifts, insertions, deletions and substitutions.
The following are representative publications disclosing genetic transformation protocols that can be used to genetically transform the following plant species: Rice (Alam et al., 1999, Plant Cell Rep. 18, 572); maize (U.S. Pat. Nos 5,177,010 and 5,981,840); wheat (Ortiz et al., 1996, Plant Cell Rep. 15, 1996, 877); tomato (U.S. Pat. No. 5,159,135); potato (Kumar et al., 1996 Plant J. 9, : 821); cassava (Li et al., 1996 Nat. Biotechnology 14, 736); lettuce (Michelmore et al., 1987, Plant Cell Rep. 6, 439); tobacco (Horsch et al., 1985, Science 227, 1229); cotton (U.S. Pat. Nos. 5,846,797 and 5,004,863); grasses (U.S. Pat. Nos. 5,187,073, 6,020,539); peppermint (Niu et al., 1998, Plant Cell Rep. 17, 165); citrus plants (Pena et al., 1995, Plant Sci.104, 183); caraway (Krens et al., 1997, Plant Cell Rep, 17, 39); banana (U.S. Pat. No. 5,792,935); soybean (U.S. Pat. Nos. 5,416,011; 5,569,834; 5,824,877; 5,563,04455 and 5,968,830); pineapple (U.S. Pat. No. 5,952,543); poplar (U.S. Pat. No. 4,795,855); monocots in general (U.S. Pat. Nos. 5,591,616 and 6,037,522); brassica (U.S. Pat. Nos. 5,188,958; 5,463,174 and 5,750,871); alfalfa (Weeks et al., (2008) Transgenic Research 17: 587-597; Samac et al., (2006) Methods Mol Biol, Vol 343, Agrobacterium Protocols. 2nd edition. Totowa, N.J.: Humana Press. p 301-311.); and cereals (U.S. Pat. No. 6,074,877). Other species are contemplated and suitable methods and protocols are available in the scientific literature for use by those skilled in the art.
Several further methods known in the art may be employed to alter expression of a nucleotide and/or alter expression or activity of a polypeptide of the invention, or used in a method of the invention. Such methods include but are not limited to Tilling (Till et al., 2003, Methods Mol Biol, 2%, 205), so called “Deletagene” technology (Li et al., 2001, Plant Journal 27(3), 235) and the use of artificial transcription factors such as synthetic zinc finger transcription factors. (e.g. Jouvenot et al., 2003, Gene Therapy 10, 513). Additionally antibodies or fragments thereof, targeted to a particular polypeptide may also be expressed in plants to modulate the activity of that polypeptide (Jobling et al., 2003, Nat. Biotechnol., 21(1), 35). Transposon tagging approaches may also be applied. Additionally peptides interacting with a polypeptide of the invention may be identified through technologies such as phage-display (Dyax Corporation). Such interacting peptides may be expressed in or applied to a plant to affect activity of a polypeptide of the invention. Plantibodies (Stoger et al., Current Opinion in Biotechnology Volume 13, Issue 2, 1 April 2002, Pages 161-166; Sudarshana et al., Methods Mol Biol. 2007;354:183-95.) may also be used to modulate expression or activity of a polypeptide in a plant. Use of each of the above approaches, including the silencing methods discussed, in alteration of expression of a nucleotide and/or expression or activity of a polypeptide of the invention is specifically contemplated.
Methods for Selecting Plants
Methods are also provided for selecting plants with altered biomass. Such methods involve testing of plants for altered for the expression of a polynucleotide or polypeptide of the invention. Such methods may be applied at a young age or early developmental stage when the altered biomass may not necessarily be visible, to accelerate breeding programs directed toward improving biomass.
The expression of a polynucleotide, such as a messenger RNA, is often used as an indicator of expression of a corresponding polypeptide. Exemplary methods for measuring the expression of a polynucleotide include but are not limited to Northern analysis, RT-PCR and dot-blot analysis (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987). Polynucleotides or portions of the polynucleotides of the invention are thus useful as probes or primers, as herein defined, in methods for the identification of plants with altered biomass. The polypeptides of the invention may be used as probes in hybridization experiments, or as primers in PCR based experiments, designed to identify such plants.
Alternatively antibodies may be raised against polypeptides of the invention. Methods for raising and using antibodies are standard in the art (see for example: Antibodies, A Laboratory Manual, Harlow A Lane, Eds, Cold Spring Harbour Laboratory, 1998). Such antibodies may be used in methods to detect altered expression of polypeptides which modulate biomass in plants. Such methods may include ELISA (Kemeny, 1991, A Practical Guide to ELISA, NY Pergamon Press) and Western analysis (Towbin & Gordon, 1994, J Immunol Methods, 72, 313).
These approaches for analysis of polynucleotide or polypeptide expression and the selection of plants with altered expression are useful in conventional breeding programs designed to produce varieties with altered biomass.
Plants
The plants of the invention may be grown and either self-ed or crossed with a different plant strain and the resulting hybrids, with the desired phenotypic characteristics, may be identified. Two or more generations may be grown to ensure that the subject phenotypic characteristics are stably maintained and inherited. Plants resulting from such standard breeding approaches also form an aspect of the present invention.
It may be desirable to either increase or decrease biomass in a particular plant species. Increased biomass would be advantageous for example in human food, forage and forestry crops as well as in ornamental plants. Decreased biomass may also be desirable in certain of the above cases, for example in the miniaturization of ornamental plants.
Biomass in a plant may also be altered through methods of the invention. Such methods may involve the transformation of plant cells and plants, with a construct of the invention designed to alter expression of a polynucleotide or polypeptide which modulates biomass in such plant cells and plants. Such methods also include the transformation of plant cells and plants with a combination of the construct of the invention and one or more other constructs designed to alter expression of one or more polynucleotides or polypeptides which modulates biomass in plants.
Exemplary methods for assessing growth rate and biomass in plants of the invention are provided in Boyes D C et al., 2001, Plant Cell. 13(7):1499-510; Lancashire P.D et al., 1991, Ann Appl. Biol. 119: 560-601, and in Example 1 below.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be better understood with reference to the accompanying drawings in which:

FIG. 1 shows the summary output of a BLAST-P search of the NR-PLANT database (release date 1 Jan. 2005) in which the ORF54 polypeptide (SEQ ID NO:1) was used as a seed sequence.

FIG. 2-1 through 2-4 shows PrettyPlot alignment of polypeptides (SEQ ID NO: 1 to 9), including ORF54 and variants thereof

FIG. 3-1 through 3-4 shows a “T-COFFEE” alignment of the seven repeat elements, termed RCC repeats, found in the polypeptide sequences (SEQ ID NO: 1 to 9) as used by the applicants to identify a consensus region (SEQ ID NO: 10) present in each repeat of all the sequences.

FIG. 4 shows a map of a vector, for plant transformation, comprising ORF54 cloned in anti-sense orientation relative to the CaMV 35S promoter. The sequence of the vector is represented in SEQ ID NO:20.

FIG. 5 shows a DNA gel-blot analysis on genomic DNA from ORF54 T0 transgenic plants digested with HindIII and probed with a fragment of ORF54 coding sequence to determine gene copy number and to identify independent transformation events.

FIG. 5 a-5 e show growth parameters observed for these ORF54 T1 plant lines compared to the best performing wild type control (Nipponbare) in two separate experiments. Where FIG. 5 a Plant height measurements from Experiment 1, FIG. 5 b Plant tiller number from Experiment 1, FIG. 5 c Tiller number in ORF54 lines that exceeded the tittering capacity in the best performing wild-type control (Nipponbare), FIG. 5 d Plant height measurements from Experiment 2 and FIG. 5 e Plant tiller number from Experiment 2.

FIG. 6 a shows shoot biomass analysis between ORF54 plants and wild-type control (Nipponbare). Student's t-test: *1 Mean differences between sample and Nipponbare is highly significant (p-0.01). *2 Mean differences between sample and Nipponbare is very highly significant (p=0.001).

FIG. 6 b shows dry matter yield in ORF54 lines that exceeded the dry matter yielding capacity in the best performing wild-type control (Nipponbare).

FIG. 7-1 through 7-4 shows an alignment of the ORF54 polypeptide and variants thereof from monocotyledonous species. Highlighting shows the position a consensus sequence motif (SEQ ID NO: 20) completely conserved in all of the sequences.

FIG. 8-1 through 8-2 shows an alignment of the ORF54 polypeptide and variants thereof from dicotyledonous species. Highlighting shows the position a consensus sequence motif (SEQ ID NO: 21) completely conserved in all of the sequences.

FIG. 9 shows altered biomass (dry weight in grams) in the transgenic ryegrass lines (1V20, 1V5, 1V1, 1V8, 1V11, 1V3, 2V9, 2V8, 2V5, 2V7, 2V2, 3V5, 3V12, 3V11, 3V10 and 3V7) over the non-transgenic control lines (T40, T41 and T101) bars over the columns represent standard deviation.

EXAMPLES

The invention will now be illustrated with reference to the following non-limiting examples.

Example 1

Increased Biomass by Down Regulation of a Polynucleotide of the Invention in Transgenic Plants

ORF54
A polynucleotide sequence designated ORF54 (SEQ ID NO: 10), encoding the polypeptide of SEQ ID NO:1, was identified in a ViaLactia Biosciences Ltd proprietary ryegrass (Lolium perenne) GeneThresher (Orion Genomics) genomic library.
ORF54 Variants
The polypeptide sequence of ORF54 (SEQ ID NO:1) was used as a seed sequence to perform a BLASTP search against the NR-Plant database (release date 2005-01-01) to identify variants of ORF54. A cut-off value of less than or equal to le-140 was identified as distinguishing ORF54 variants based upon the applicant's assessment of the associated score value and annotations in the public data base. The BLASTP output summary is shown in FIG. 1.
The selected variant sequences were aligned using the EMBOSS tool EMMA (Thompson et al 1994), which is an interface to the popular multiple alignment program ClustalW. Aligned sequences were visualised using another EMBOSS tool called prettyplot as shown in FIG. 2.
The polypeptide sequences of ORF54 variants are listed as SEQ ID NO:2 to 9 in the sequence listing. The corresponding polynucleotide sequences are listed as SEQ ID NO:11 to 18 respectively.
Analysis of the polypeptide sequences revealed the prescence of seven repeats per polypeptide sequence and the repeat sequences are termed RCC repeats. The repeat sequences from all polypeptides (SEQ ID NO:1-9) above were aligned using the T-COFFEE alignment programme Version_—1.37 (Notredame et. al 2000, Higgins) and the outcome is presented in FIG. 3.
A polypeptide motif based on, and present in, all of the RCC repeats from ORF54 and each of the ORF54 variants was identified and is represented in FIG. 3.
A Vector Comprising ORF54
The ORF54 polynucleotide sequence (SEQ ID NO:10) was cloned in the anti-sense orientation upstream of the double 35S promoter in order to down-regulate expression of the ORF54 homologue in rice. The vector comprising ORF54 was produced by standard molecular biology techniques. A map of the binary vector is shown in FIG. 4. The sequence of the vector is represented in SEQ ID NO:19.
Plant Transformation—Rice
The purified binary vector (SEQ ID NO:19) was introduced into Agrobacterium strain EHA105 by electroporation (den Dulk-Ras A and Hooykaas P J.) and the suspension was incubated at 26° C. for 30 minutes. A small aliquot was plated on AB minimal medium (Schmidt-Eisenlohr et. al 1999) containing Kanamycin at 100 mg/L. Plates were incubated at 26° C. for 3 days and single colonies were tested for presence of the plasmid using construct specific primers and transformation confirmed.
Agrobacterium
cultures were grown in AG minimal medium containing 100 mg/L kanamycin at 26° C. with shaking (200 rpm). The Agrobacterium suspensions were pelleted at 5,000 rpm for 5 minutes, washed once in basal MS medium containing 1% glucose and 3% sucrose, pH 5.2, and re-suspended in same medium containing 200 μM acetosyringone to OD₆₀₀0.6-0.8.
A. tumefaciens containing the binary vector ORF54 were used to co-cultivate at least 1,000 immature rice (Oryza sativa) cv. Nipponbare embryos. Immature seeds from rice were washed in sterile water and then surface sterilized with sodium hypochlorite containing 1.25% active chlorine with 10 μL Tween 20 for 20 minutes. After sterilization, the seeds were washed several times with sterile water and blotted dry on sterile filter paper (3M). The seeds were de-husked manually using sterile pair of forceps and the embryo dissected out with sterile knife. The isolated embryos were immersed in Agrobacterium suspension for 30 minutes with continuous shaking at 100 rpm in a 10 mL culture tube. The excess liquid was drained off and the embryos blotted on to sterile filter paper before placing them on to co-cultivation medium containing MS medium (Murashige and Skoog, 1964) supplemented with 3% sucrose, 1% glucose, 2 mg/L 2,4-D, 0.1 mg/L BA, 400 μM acetosyringone, pH 5.2 for 4 days in dark. After co-cultivation, the calli forming embryos were sub-cultured once every two weeks on selection medium consisting of MS medium supplemented with 3% sucrose, 1% glucose, 2 mg/L 2,4-D (2,4-dichlorophenoxy acetic acid), 0.1 mg/L BA (benzyl adenine) and containing 50 mg/L hygromycin and 300 mg/L timentinTM (ticarcillin +clavulanic acid) till at-least 30 healthy calli showing green spots indicative of healthy shoot emergence was achieved. Calli containing the green spots were transferred to selection medium lacking 2,4-D to regenerate a minimum of 10 transformed plants. Regenerated plants were rooted and then transplanted to six inch pots containing soil and plants grown in greenhouse. DNA gel-blot analysis was carried out (FIG. 5) by digesting genomic DNA from transgenic plants with HindIll and probing with a fragment of ORF54 coding sequence to determine gene copy number and to identify five independent transformation events. T1 seeds were harvested from the transformed plants (T0).
T1 Plant Phenotyping
Thirty seeds from Southern positive T0 plants were sown in individual cups containing cocopeat and twenty healthy plants out of them were transplanted in the green house. These plants were arranged using a CRD using the random numbers from a random table.
T1 plant phenotyping was carried out in two separate experiments. The first experiment involved progeny lines from T0 events 1129503 and 123602 and Nipponbare (a wild-type control), and the second experiment involved progeny lines from T0 events 1164906, 1164914 and 1164922 and Nipponbare (a wild-type control.)
Phenotypic Analysis of T1 Lines
Plants height and tiller numbers were measured once every week post-transplanting until seed set was achieved. FIGS. 5 a, b, c, d and e depict the growth parameters observed for these plants in two separate experiments. Transgenic ORF54 plants (T1) do not appear to be too different from the wildtype control (Nipponbare) in terms of plant height (FIG. 5 a and d). However tillering capacity of ORF54 plants (T1) appear to be higher than in the wild-type control (Nipponbare) (FIG. 5 b and e). A closer analysis revealed that 12.5% of the ORF54 T1 plants had out-performed the best tillering wild-type control plant (FIG. 5 c) with tiller numbers more than doubling. As a result, the biomass as measured by dry matter production in ORF54 plants (T1) also increased (FIG. 6 a). On an average, the increase in biomass amounts to roughly 66% as compared to the wild-type control (Nipponbare). Once again 12.5% of the ORF54 T1 plant population was seen to produce more dry matter than the highest dry matter yielding wild-type control (Nipponbare) (FIG. 6 b). In conclusion down-regulation of ORF54 gene expression, or that of variants of ORF54, in planta by anti-sense or similar technology leads to an increase plant biomass.
Plant Transformation—Ryegrass
Perennial ryegrass (Lolium perenne L. cv Impact) was transformed essentially as described in Bajaj et. al. (Plant Cell Reports, 2006, 25: 651-659). Embryogenic callus derived from mersitematic regions of the tillers of selected ryegrass lines and Agrobacterium tumefaciens strain EHA101 carrying a modified binary vector (ORF54, FIG. 4) was used for transformation experiments. Embryogenic calli were immersed with overnight-grown Agrobacterium cultures for 30 minutes with continuous shaking. Calli resistant to hygromycin were selected after sub-culturing them on co-cultivation medium for 4 weeks. After selection, the resistant calli were sub-cultured on regeneration medium every 2 weeks until the plants regenerated. The regenerants that continued to grow after two or three rounds of selection proved to be stable transformants. Each regenerated plant was then multiplied on maintenance medium to produce clonal plantlets and subsequently rooted on MS medium without hormones. A rooted plant from each clone was transferred into contained glasshouse conditions while retaining a clonal counterpart in tissue culture as backup. Eighteen independent transgenic lines (1V1, 1V3, 1V5, 1V8, 1V10, 1V11, 1V20, 2V2, 2V4, 2V5, 2V7, 2V8, 2V9, 3V5, 3V7, 3V10, 3V11, 3V12) and their non-transgenic control plants (T40, T41 and T101, respectively) have been analyzed in a climate-controlled environmental laboratory, where they were assessed for biomass production under fully water condition.
Screening for Increased Biomass in Growth Chamber
A plant growth system was built using 500 mm long; 90 mm diameter plastic storm-water pipes. The pipes were placed on a mobile tray and supported at the sides by ropes and metal frame. The tubes were plugged at the bottom with rockwool and progressively filled with washed mortar sand using water to achieve uniform packing. At the center of the open end of each tube a clump of perennial ryegrass (5 tillers) was planted. Plants from each event were replicate-planted in three tubes. The plants were arranged at random, one in each of the three replicates, and grown at 70% relative humidity; 16/8 hours day/night cycle and under 650 μmol.m⁻².s⁻¹light intensity. The plants were irrigated daily once in the morning with 50 mL Hoagland' s solution (Hoagland and Arnon, 1938) and again in the afternoon with 50 mL plain water. The plants were acclimated initially for twenty days and then the plants were trimmed back to 15 cm height. All plants were allowed to recover from trimming for the next fourteen days. Plant tiller numbers were recorded after seven and 14 days, respectively from the timed day. After fourteen days, plants were trimmed down to 15 cm height. The harvested samples were dried down at 60° C. for three days and then dry weight recorded. The plants were allowed to grow under fully watered conditions for another fourteen days. Again, the plants were trimmed back to 15 cm height and then the dry weight of the trimmed sample recorded after drying the samples at 60° C. for three days. The plants were allowed to grow for another 13 days and the final data on biomass production (dry matter) recorded by harvesting the plants above 15 cm and drying the samples at 60° C. for three days.
More than one-fourth of the transgenic events tested produced more biomass than wild type plants in each of the harvest. When cumulative growth was determined over a period of three harvests, one of the transgenic line, 3V7, produced more than 470% biomass; while in four other lines, 2V2; 2V7; and 3V10 the biomass increase ranged from over 35% to 95% (see FIG. 9).
The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.

REFERENCES

Adams et al. 1991, Science 252:1651-1656.
Chen H, Nelson R S, Sherwood J L. (1994) Biotechniques;16 (4): 664-8, 670.
Chen et al. 2002, Nucleic Acids Res. 31:101-105
den Dulk-Ras A, Hooykaas P J. (1995) Methods Mol Biol.; 55: 63-72.
Lee et al. 2003, PNAS 99:12257-12262
Lee and Lee, 2003 Plant Physiol. 132: 517-529
Murashige T, Skoog F (1962) Physiol Plant 15: 473-497
Notredame C., Higgins, D. and Heringa, J. (2000) J. Mol. Biol., 302, 205-217.
Richmond and Somerville 2000, Current Opinion in Plant Biology. 3:108-116
Ruan et al. 2004, Trends in Biotechnology 22: 23-30.
Schmidt-Eisenlohr H, Domke N, Angerer C, Wanner G, Zambryski P C, Baron C. (1999) J. Bacteriol.; 181 (24): 7485-92.
Sun et al. 2004, BMC Genomics 5: 1.1-1.4
Thompson, J. D., Higgins, D. G. and Gibson, T. J. (1994) CABIOS, 10, 19-29.
Velculescu et al. 1995, Science 270: 484-487

The above examples illustrate practice of the invention. It will be appreciated by those skilled in the art that numerous variations and modifications may be made without departing from the spirit and scope of the invention.

Summary of Sequences

				FULL-LENGTH
SEQ				CODING SEQUENCE
ID NO:	TYPE	SPECIES	REFERENCE	(NUCLEOTIDES)

1	Polypeptide	Lolium perenne	ORF54	N/A
2	Polypeptide	Oryza sativa	AK098904.1	N/A
3	Polypeptide	Oryza sativa	AK065041.1	N/A
4	Polypeptide	Oryza sativa	AK065992.1	N/A
5	Polypeptide	Oryza sativa	AK065747.1	N/A
6	Polypeptide	Oryza sativa	AK062069.1	N/A
7	Polypeptide	Oryza sativa	XP466543.1	N/A
8	Polypeptide	Arabidopsis thaliana	BAB01075.1	N/A
9	Polypeptide	Arabidopsis thaliana	AAL15211.1/	N/A
			AAK59536.1
10	Polynucleotide	Lolium perenne	ORF54	Start 302; End 1904
11	Polynucleotide	Oryza sativa	AK098904.1	Start 326; End 1928
12	Polynucleotide	Oryza sativa	AK065041.1	Start 326; End 1928
13	Polynucleotide	Oryza sativa	AK065992.1	Start 136; End 1738
14	Polynucleotide	Oryza sativa	AK065747.1	Start 486; End 2161
15	Polynucleotide	Oryza sativa	AK062069.1	Start 2; End 1004
16	Polynucleotide	Oryza sativa	XP466543.1	Start 184; End 1795
17	Polynucleotide	Arabidopsis thaliana	BAB01075.1	Start 1; End 1597
18	Polynucleotide	Arabidopsis thaliana	AAL15211.1/	Start 1; End 1297
			AAK59536.1
19	Polynucleotide	Vector		N/A
20	Polypeptide	Consensus, all plants		N/A
21	Polypeptide	Consensus,		N/A
		dicotyledonous plants

Claims

What is claimed is:

1. A method of producing a plant with increased biomass the method comprising the step of reducing the expression, in a plant cell or plant, of a polypeptide comprising the sequence of SEQ ID NO: 20, wherein the reducing of expression leads to the increased biomass.

2. The method of claim 1, wherein the expression is reduced by transforming a plant cell, or plant, with a polynucleotide that is complementary to an endogenous gene encoding the polypeptide, such that expression of the polynucleotide results in reduced expression of the polypeptide and leads to the increased biomass.

3. The method of claim 2, wherein the plant cell or plant is transformed with at least one of:

a) a polynucleotide including a sequence encoding of a polypeptide comprising the amino acid sequence of SEQ ID NO:20;

b) a polynucleotide comprising a fragment, of at least 15 nucleotides in length, of the polynucleotide of a); and

c) a polynucleotide comprising a complement, of at least 15 nucleotides in length, of the polynucleotide of a);

wherein the expression of the polynucleotide in the plant cell or plant results in reduced expression of the polypeptide and leads to the increased biomass.

4. A plant produced by the method of claim 1.

5. A plant produced by the method of claim 1, wherein the expression of an endogenous polypeptide comprising the amino acid sequence of SEQ ID NO:20 is down-regulated in the plant relative to a non-transformed control plant, and wherein the plant has increased biomass relative to a non-transformed control plant.

6. The plant of claim 5 that has an increased number of tillers relative to a non-transformed control plant.

7. A method for selecting a plant with altered biomass, the method comprising testing of a plant for reduced expression of a polynucleotide encoding a polypeptide comprising the amino acid sequence of SEQ ID NO:20, wherein the reduced expression is indicative of increased biomass.