WO2016019423A1

WO2016019423A1 - Methods for modulating plant biomass and yield

Info

Publication number: WO2016019423A1
Application number: PCT/AU2015/000474
Authority: WO
Inventors: Sergiy Lopato; Peter Langridge; Yuri SHAVRUKOV
Original assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Current assignee: Australian Centre for Plant Functional Genomics Pty Ltd
Priority date: 2014-08-08
Filing date: 2015-08-07
Publication date: 2016-02-11
Anticipated expiration: 2017-02-08

Abstract

The present invention relates to methods for modulating the biomass of plants and to genetically modified plants having modulated biomass. Specifically, methods for increasing plant biomass, including increasing seed yield, are provided. The methods rely on modulation of expression of various subunits of the Nuclear Factor Gamma (NF-Y) gene family, including NF-YB and NF-YC subunits. More specifically, increasing expression of wheat NF-YB4 has been shown to increase plant biomass and yield, including under nutrient-poor conditions.

Description

METHODS FOR MODULATING PLANT BIOMASS AND YIELD PRIORITY CLAIM

[0001] This application claims priority from United States provisional patent application number 62/035,337 filed on 8 August 2014, the contents of which are to be taken as incorporated herein by this reference.

FIELD OF THE INVENTION

[0002] The present invention relates to methods for modulating the biomass of plants and to genetically modified plants having modulated biomass. Specifically, methods for increasing plant biomass, including increasing seed yield, are provided.

BACKGROUND OF THE INVENTION

[0003] With the increasing world population and reduced availability of arable land for farming, the production of agricultural goods such as food and feed production in sufficient quantity and quality presents a significant challenge. To address this issue, thereby meeting consumer demand, it is essential to improve crop productivity and yield.

[0004] To date, efforts to improve the intrinsic productivity and yield of various plant crops have mainly focussed on exploiting the genetic variability within crops. For example, conventional means for crop and horticultural improvements have utilised selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogeneous genetic components that may not always result in the desirable trait being passed on from parent plants.

[0005] Recent advances in molecular biology have enabled a more rapid expansion of the pool of variability by allowing the exchange of genetic material across species. For example, genetic engineering of plants involves the manipulation of genetic material within a plant or plant material, or the isolation and subsequent introduction of that genetic material into a plant, to impart a favourable phenotype. Using these biotechnology tools, it has been possible to specifically and rapidly modify plants by genetic engineering to improve economic, agronomic or horticultural traits. [0006] Accordingly, the identification of genes responsible for influencing favourable traits in domesticated plants has become an important focus of agricultural research. As indicated above, traits such as biomass production and yield are of particular economic value. Biomass and yield are directly dependent on several factors, for example, the number and size of the organs, plant architecture (for example, the number of branches), seed production, nutrient utilisation, root development, and stress tolerance. Therefore, methods which can optimise one or more of the abovementioned factors are of vital importance for the improvement of crop productivity and yield.

[0007] The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed before the priority date of each claim of this application.

SUMMARY OF THE INVENTION

[0008] The present invention is predicated, in part, on the identification of polypeptides, and their corresponding nucleic acids, that can increase plant biomass, yield, and nutrient utilisation. In particular, the inventors have identified various hetero-trimeric nuclear factor Y (NF-Y) subunits that play a role in influencing such traits.

[0009] Accordingly, in a first aspect, the present invention provides a method for modulating biomass of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0010] In one embodiment, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0011] In some embodiments of the first aspect of the invention, the method further comprises the step of identifying the plant as a plant with modulated biomass relative to a wild-type form of the plant, thereby confirming modulation of the biomass of the plant. [0012] In some embodiments, increasing expression of the NF-YB4 polypeptide increases biomass of the plant relative to a wild-type form of the plant. In some embodiments, expression of the NF-YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide.

[0013] In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0014] In some embodiments, increasing expression of the nucleic acid increases biomass of the plant relative to a wild-type form of the plant. In one embodiment, yield of the plant is increased relative to a wild-type form of the plant. In one embodiment, the yield is seed yield.

[0015] In some embodiments, the number of tillers of the plant is increased relative to a wild-type form of the plant. In some embodiments, the plant is a spike-bearing plant and the number of spikes is increased relative to a wild-type form of the plant.

[0016] In some embodiments, the plant is grown under non-drought conditions. In some embodiments, the plant is grown under nutrient-poor conditions.

[0017] In some embodiments, the plant is a monocotyledonous plant, including a cereal crop plant. In one embodiment, the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

[0018] In a second aspect, the present invention provides a method for modulating yield of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0019] In one embodiment, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0020] In some embodiments, the method further comprises the step of identifying the plant as a plant with modulated yield relative to a wild-type form of the plant, thereby confirming modulation of the yield of the plant.

[0021] In some embodiments of the second aspect of the invention, increasing expression of the NF-YB4 polypeptide increases yield of the plant relative to a wild-type form of the plant. In some embodiments, expression of the NF-YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide.

[0022] In some embodiments of the second aspect of the invention, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0023] In some embodiments, increasing expression of the nucleic acid increases yield of the plant relative to a wild-type form of the plant. In one embodiment, the yield is seed yield.

[0024] In some embodiments of the second aspect of the invention, the number of tillers of the plant is increased relative to a wild-type form of the plant. In some embodiments, the plant is a spike-bearing plant and the number of spikes is increased relative to a wild- type form of the plant.

[0025] In some embodiments of the second aspect of the invention, the plant is grown under non-drought conditions. In some embodiments, the plant is grown under nutrient- poor conditions. [0026] In some embodiments of the second aspect of the invention, the plant is a monocotyledonous plant, including a cereal crop plant. In one embodiment, the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

[0027] In a third aspect, the present invention provides a method for modulating biomass of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

[0028] In one embodiment, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0029] In some embodiments, the method further comprises the step of identifying the multicellular structure as a multicellular structure with modulated biomass relative to a wild-type form of the multicellular structure, thereby confirming modulation of the biomass of the multicellular structure.

[0030] In some embodiments of the third aspect of the invention, increasing expression of the NF-YB4 polypeptide increases biomass of the multicellular structure relative to a wild- type form of the multicellular structure. In some embodiments, expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more plant cells of the multicellular structure, a nucleic acid which encodes the NF-YB4 polypeptide.

[0031] In some embodiments of the third aspect of the invention, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4. [0032] In some embodiments of the third aspect of the invention, increasing expression of the nucleic acid increases biomass of the multicellular structure relative to a wild-type form of the multicellular structure. In one embodiment, yield of the multicellular structure is increased relative to a wild-type form of the multicellular structure. In one embodiment, yield is seed yield.

[0033] In some embodiments of the third aspect of the invention, the number of tillers of the multicellular structure is increased relative to a wild-type form of the multicellular structure. In some embodiments, the multicellular structure is a spike-bearing multicellular structure and the number of spikes is increased.

[0034] In some embodiments of the third aspect of the invention, the multicellular structure is grown under non-drought conditions. In some embodiments, the multicellular structure is grown under nutrient-poor conditions.

[0035] In some embodiments of the third aspect of the invention, the multicellular structure is a whole plant, plant tissue, a plant organ, a plant part, plant reproductive material, or cultured plant tissue.

[0036] In a fourth aspect, the present invention provides a method for modulating yield of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

[0037] In one embodiment, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0038] In some embodiments of the fourth aspect of the invention, the method further comprises the step of identifying the multicellular structure as a multicellular structure with modulated yield relative to a wild-type form of the multicellular structure, thereby confirming modulation of the yield of the multicellular structure. [0039] In some embodiments of the fourth aspect of the invention, increasing expression of the NF-YB4 polypeptide increases yield of the multicellular structure relative to a wild- type form of the multicellular structure. In some embodiments, expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more plant cells of the multicellular structure, a nucleic acid which encodes the NF-YB4 polypeptide.

[0040] In some embodiments of the fourth aspect of the invention, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0041] In some embodiments of the fourth aspect of the invention, increasing expression of the nucleic acid increases yield of the multicellular structure relative to a wild-type form of the multicellular structure. In one embodiment, yield is seed yield.

[0042] In some embodiments of the fourth aspect of the invention, the number of tillers of the multicellular structure is increased relative to a wild-type form of the multicellular structure. In some embodiments, the multicellular structure is a spike-bearing multicellular structure and the number of spikes is increased relative to a wild-type form of the multicellular structure.

[0043] In some embodiments of the fourth aspect of the invention, the multicellular structure is grown under non-drought conditions. In some embodiments, the multicellular structure is grown under nutrient-poor conditions.

[0044] In some embodiments of the fourth aspect of the invention, the multicellular structure is a whole plant, plant tissue, a plant organ, a plant part, plant reproductive material, or cultured plant tissue.

[0045] In a fifth aspect, the present invention provides a genetically modified plant which has modulated biomass, wherein modulation of the biomass is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant. [0046] In one embodiment, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0047] In some embodiments of the fifth aspect of the invention, the plant comprises an increased expression of the NF-YB4 polypeptide and an increased biomass relative to a wild-type form of the plant.

[0048] In some embodiments of the fifth aspect of the invention, expression of the NF- YB4 polypeptide is modulated by modulating expression of, in the one or more cells of the plant, a nucleic acid which encodes the NF-YB4 polypeptide. In one embodiment, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0049] In some embodiments of the fifth aspect of the invention, the plant comprises an increased expression of the nucleic acid and an increased biomass relative to a wild-type form of the plant. In one embodiment, the plant has an increased yield relative to a wild- type form of the plant. In one embodiment, the yield is seed yield.

[0050] In some embodiments of the fifth aspect of the invention, the plant has an increased number of tillers relative to a wild-type form of the plant. In some embodiments, the plant is a spike-bearing plant and the plant has an increased number of spikes relative to a wild-type form of the plant.

[0051] In some embodiments of the fifth aspect of the invention, the plant is a monocotyledonous plant, including a cereal crop plant. In one embodiment, the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant. [0052] In a sixth aspect, the present invention provides a genetically modified plant which has modulated yield, wherein modulation of the yield is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0053] In one embodiment of the sixth aspect of the invention, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto. In some embodiments, the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

[0054] In some embodiments of the sixth aspect of the invention, the plant comprises an increased expression of the NF-YB4 polypeptide and an increased yield relative to a wild- type form of the plant. In some embodiments, expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more cells of the plant, a nucleic acid which encodes the NF-YB4 polypeptide. In one embodiment, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3. In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4. In some embodiments, the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

[0055] In some embodiments of the sixth aspect of the invention, the plant comprises an increased expression of the nucleic acid and an increased yield relative to a wild-type form of the plant. In one embodiment, the yield is seed yield.

[0056] In some embodiments of the sixth aspect of the invention, the plant has an increased number of tillers relative to a wild-type form of the plant. In some embodiments, the plant is a spike-bearing plant and the plant has an increased number of spikes relative to a wild-type form of the plant.

[0057] In some embodiments of the sixth aspect of the invention, the plant is a monocotyledonous plant, including a cereal crop plant. In one embodiment, the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

BRIEF DESCRIPTION OF THE FIGURES

[0058] For a further understanding of the aspects and advantages of the present invention, reference should be made to the following detailed description, taken in conjunction with the accompanying drawings.

[0059] FIGURE 1 - shows the cloning strategy, confirmation of interactions, and identification of protein segments responsible for the interaction of wheat NF-YB and NF- YC subunits. (A) Schematic representation of the yeast 2-hybrid screens and cloned products. Empty arrows indicate cloned genes that are not discussed herein. (B) Confirmation of interaction of TaNF-YC15 with maize and wheat NF-YB proteins in a reciprocal direction using re-cloned coding regions (5' and 3' untranslated regions removed) of cloned wheat cDNAs. (C) Identification of the smallest possible TaNF-YB2 segment interacting with TaNF-YB15 by using a set of N-terminal and C-terminal truncations.

[0060] FIGURE 2 - depicts an unrooted radial phylogenetic tree of (A) NF-YB, and (B) NF-YC TFs from wheat, rice, maize and Arabidopsis. Sequences of 52 NF-YB and 32 NF- YC proteins were aligned using ProMals3D (Pei et a/., 2008, "PROMALS3D: a tool for multiple protein sequence and structure alignments", Nucleic Acids Research 36: 2295- 2300) with branch lengths drawn to scale. Two letter prefixes at sequence identifiers indicate species of origin. GenBank accession numbers of all proteins are listed in Table 2. Maize sequences that have no names in GenBank annotations where given the same number as wheat proteins grouped to the same clades (where it was possible). New wheat proteins with a high level of identity to previously published proteins were designated with the same name and an additional letter and number, e.g. L (like) 1 , L2, etc. at the end of the name. TFs isolated in this work (in bold) are marked with three additional letters (RAC is a shorthand for cultivar, cv RAC875).

[0061] FIGURE 3 - is a depiction of the molecular model of the wheat TaNF-YB2/Ta F- YC15 dimeric sub-structure and structural bioinformatics of wheat NF-YB and NF-YC factors. (A) A multiple sequence alignment of HFDs of 1 1 TaNF-YB factors from wheat. The sequence of a human NF-YB (PDB accession 1 NIJ:A also designated NF-YB) is also included. Protein sequences were aligned with ProMals3D (Pei et a/., 2008, supra). Predicted consensus a-helices (h) are marked. Conservation of residues on a scale of 9-6 is shown at the top of the diagram. Predicted residues involved in binding to HFD of NF- YC are lightly shaded. An additional nine residues at the C-termini of TaNF-YB (each residue marked with an asterisk) might also participate in binding of TaNF-YC proteins. Conserved Gin or His residues at C-terminal regions are darkly shaded. TaNF-YB2 and TaNF-YB4 isolated in this work are in bold. (B) A multiple sequence alignment of HFDs of 12 TaNF-YC factors from wheat. The sequence of a human NF-YC (PDB accession 1 NIJ:B; also designated NF-YC) is also included. Predicted residues involved in binding to HFD of NF-YB are lightly shaded. TaNF-YC15 isolated in this work is in bold. Protein sequences were aligned as described in panel A. Other annotations are as specified in panel A. (C) Molecular model of the wheat TaNF-YB2/Ta NF-YC 15 dimeric sub-structure (sequences highlighted in bold in panels A and B). Secondary structures are shown in cartoon representations (dark for TaNF-YB, light for TaNF-YC). Residues participating in binding (TaNF-YB to TaNF-YC: Lys57, Ala59, Asp61 , Glu68, Ser75, Lys91 , Ile93, Tyr1 10 and Arg122; TaNF-YC to TaNF-YB: Asp1 17, Asp1 19, Met122, Ser124, Glu126, Glu141 , Arg156, Leu158 and Lys160) involved in dimer formation are shown in sticks. Gln1 19 and Arg122 of TaNF-YB are in close proximity. Distances between interacting residues are shown in black dashed lines and vary between 2.7 A to 3.5 A.

[0062] FIGURE 4 - graphs showing expression of (A) TaNF-YB2 and TaNF-YB4, and (B) TaNF-YC15, genes in different wheat tissues (cv. RAC875) in the absence of stress. Levels of expression were detected by qRT-PCR and are shown as normalised transcription levels in arbitrary units.

[0063] FIGURE 5 - graphs showing expression of NF-Y genes under drought and rapid dehydration. Drought-inducible expression of the TaNF-YB2 and TaNF-YB4 (B) and TaNF-YC15 (C) genes in leaves of 4-week-old seedlings. Expression of TaNF-YB2 (E), TaNF-YB4 (F) and TaNF-YC15 (G) in detached leaves during dehydration at room temperature. The stress-inducible TaCOR39 gene was used as a positive control (Panels A and D). Levels of expression were detected by qRT-PCR and are shown as normalised transcription levels in arbitrary units.

[0064] FIGURE 6 - graphs showing a comparison of phenotypes (panels A to C) and yields (biomass and grain (seed) yields)(panels D to F) of transgenic and control (WT) wheat plants grown in large containers under well-watered conditions and constantly increasing drought (see Table 2). From 16 to 28 WT and transgenic T₂ plants with confirmed transgene expression for each sub-line of 3 independent lines were used in the experiment (see Table 4). All sub-lines except line 6-2 were confirmed to be homozygous. Differences between transgenic lines and WT plants were tested in the unpaired Student's f-test (^* mean P-value <0.05; ^** mean P-value < 0.01 ).

[0065] FIGURE 7 - results of a comparison of yield (panel A) and phenotypes (panels B to F) of transgenic and control (WT) wheat plants grown in soil mixes either without added fertiliser, or with the addition of a complete, slow-release fertiliser (x g kg^"1 soil; see Table 5). One WT plant and one T₃ transgenic plant for each of three homozygous sub-lines with confirmed transgene expression were grown in the same pot. Six pots were used for the soil mix without added fertiliser, and 3 pots for the fertilised treatment. Differences between transgenic lines and WT plants were tested in the unpaired Student's f-test (^* mean P-value <0.05; ^** mean P-value < 0.01 ).

[0066] FIGURE 8 - a depiction of the large container systems used for plant growth. (A) The soil-water potential shown by two sensors situated near the bottom of the container and just under the soil surface, in well-watered growth conditions and under drought. (B) Outlook of containers.

DETAILED DESCRIPTION OF THE INVENTION

[0067] Nucleotide and polypeptide sequences are referred to herein by a sequence identifier number (SEQ ID NO:). A summary of the sequence identifiers is provided in Table 1. A sequence listing has also been provided at the time of filing this application.

TABLE 1

Summary of Sequence Identifiers

Sequence Identifier Sequence or Purpose

SEQ ID NO: 1 TaNF-YB4 amino acid sequence - truncated

SEQ ID NO: 2 TaNF-YB4 amino acid sequence - full length

SEQ ID NO: 3 TaNF-YB4 nucleotide coding sequence - truncated

SEQ ID NO: 4 TaNF-YB4 nucleotide coding sequence - full length

SEQ ID NO: 5 Cloning ZmNF-YB2 into Y2H vector - F primer

SEQ ID NO: 6 Cloning ZmNF-YB2 into Y2H vector - R primer

SEQ ID NO: 7 Cloning TaNF-YB2 into Y2H vector - F primer Sequence Identifier Sequence or Purpose

SEQ ID NO: 8 Cloning TaNF-YB2 into Y2H vector - R primer

SEQ ID NO: 9 Cloning TaNF-YB4 into Y2H vector - F primer

SEQ ID NO: 10 Cloning TaNF-YB4 into Y2H vector - R primer

SEQ ID NO: 1 1 Cloning TaNF-YC15(d1) into Y2H vector - F primer

SEQ ID NO: 12 Cloning TaNF-YC15(d1) into Y2H vector - R primer

SEQ ID NO: 13 Cloning TaNF-YB4 into pENTR-D-TOPO vector - F primer

SEQ ID NO: 14 Cloning TaNF-YB4 into pENTR-D-TOPO vector - R primer

SEQ ID NO: 15 TaNF- YC15 Q-PCR - gene expression - F primer

SEQ ID NO: 16 TaNF- YC15 Q-PCR - gene expression - R primer

SEQ ID NO: 17 TaNF-YB2 Q-PCR - gene expression - F primer

SEQ ID NO: 18 TaNF-YB2 Q-PCR - gene expression - R primer

SEQ ID NO: 19 TaNF-YB4 Q-PCR - gene expression - F primer

SEQ ID NO: 20 TaNF-YB4 Q-PCR - gene expression - R primer

SEQ ID NO: 21 TaActin Q-PCR - normalisation - F primer

SEQ ID NO: 22 TaActin Q-PCR - normalisation - R primer

SEQ ID NO: 23 TaCyclophilin Q-PCR - normalisation - F primer

SEQ ID NO: 24 TaCyclophilin Q-PCR - normalisation - R primer

SEQ ID NO: 25 TaGAPDH Q-PCR - normalisation - F primer

SEQ ID NO: 26 TaGAPDH Q-PCR - normalisation - R primer

SEQ ID NO: 27 TaEFa Q-PCR - normalisation - F primer

SEQ ID NO: 28 TaEFa Q-PCR - normalisation - R primer

SEQ ID NO: 29 TaNF-YB4 - transgene specific - F primer

SEQ ID NO: 30 TaNF-YB4 - transgene specific - R primer

SEQ ID NO: 31 Nos terminator Q-PCR - copy number - F primer

SEQ ID NO: 32 Nos terminator Q-PCR - copy number - R primer

SEQ ID NO: 33 TaNF-YB2 - Y2H - CDS - F primer

SEQ ID NO: 34 TaNF-YB2 - Y2H - CDS - R primer

SEQ ID NO: 35 TaNF-YB2D1 - Y2H - deletions - F primer

SEQ ID NO: 36 TaNF-YB2D1 - Y2H - deletions - R primer

SEQ ID NO: 37 TaNF-YB2D2 - Y2H - deletions - F primer

SEQ ID NO: 38 TaNF-YB2D2 - Y2H - deletions - R primer

SEQ ID NO: 39 TaNF-YB2D3 - Y2H - deletions - F primer

SEQ ID NO: 40 TaNF-YB2D3 - Y2H - deletions - R primer

SEQ ID NO: 41 TaNF-YB2D4 - Y2H - deletions - F primer

SEQ ID NO: 42 TaNF-YB2D4 - Y2H - deletions - R primer

SEQ ID NO: 43 TaNF-YB2D5 - Y2H - deletions - F primer

SEQ ID NO: 44 TaNF-YB2D5 - Y2H - deletions - R primer

SEQ ID NO: 45 TaNF-YB2D6 - Y2H - deletions - F primer

SEQ ID NO: 46 TaNF-YB2D6 - Y2H - deletions - R primer

SEQ ID NO: 47 TaNF-YB2D7 - Y2H - deletions - F primer

SEQ ID NO: 48 TaNF-YB2D7 - Y2H - deletions - R primer

SEQ ID NO: 49 TaNF-YB2D8 - Y2H - deletions - F primer

SEQ ID NO: 50 TaNF-YB2D8 - Y2H - deletions - R primer

SEQ ID NO: 51 TaNF-YB2D9 - Y2H - deletions - F primer

SEQ ID NO: 52 TaNF-YB2D9 - Y2H - deletions - R primer

SEQ ID NO: 53 TaNF-YB2D10 - Y2H - deletions - F primer

SEQ ID NO: 54 TaNF-YB2D10 - Y2H - deletions - R primer Sequence Identifier Sequence or Purpose

SEQ ID NO: 55 TaNF-YB2D11 - Y2H - deletions - F primer

SEQ ID NO: 56 TaNF-YB2D11 - Y2H - deletions - R primer

SEQ ID NO: 57 TaNF-YB2D12 - Y2H - deletions - F primer

SEQ ID NO: 58 TaNF-YB2D12 - Y2H - deletions - R primer

SEQ ID NO: 59 TaNF-YB2D13 - Y2H - deletions - F primer

SEQ ID NO: 60 TaNF-YB2D13 - Y2H - deletions - R primer

SEQ ID NO: 61 TaNF-YB2D14 - Y2H - deletions - F primer

SEQ ID NO: 62 TaNF-YB2D14 - Y2H - deletions - R primer

SEQ ID NO: 63 TaNF-YB2D15 - Y2H - deletions - F primer

SEQ ID NO: 64 TaNF-YB2D15 - Y2H - deletions - R primer

SEQ ID NO: 65 Pin-b - F primer

SEQ ID NO: 66 Pin-b - R primer

SEQ ID NO: 67 Pin-b dual labelled Taqman probe

SEQ ID NO: 68 TaNF-YC15 mRNA sequence

SEQ ID NO: 69 TaNF-YC15 nucleotide coding sequence

SEQ ID NO: 70 TaNF-YC15 amino acid sequence

SEQ ID NO: 71 7aNF-YB4 mRNA sequence

[0068] The present invention is predicated, in part, on the identification of Nuclear Factor Gamma (NF-Y) transcription factors, which are capable of imparting favourable trait characertistics in plants, including influencing biomass production, yield and nutrient utilisation.

[0069] Accordingly, in a first aspect, the present invention provides a method for modulating biomass of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0070] As used herein, the "biomass of a plant" can refer to the mass, such as dry weight, of the plant in toto. However, given that modulating expression of the NF-YB4 polypeptide can modulate specific parts or structures of the plant, the term "biomass of a plant" may also be a measure of the biomass of one or more of those specific plant parts or plant structures. For example, biomass of the plant may be measured in terms of just the harvestable parts of the plant, including seeds, tillers, spikes, leaves, shoots, fruit, stems, roots, flowers, trichome, sepals, hypocotyls, petals, stamen, pollen, style, stigma, embryo, ovule, endosperm, seed coat, nodule, cambium, fiber, aleurone, wood, parenchyma, erenchyma, phloem, sieve element, vascular tissue and the like. In any event, modulated biomass is measured relative to the biomass of a plant (or part thereof) in which expression of the NF-YB4 polypeptide has not been modulated, i.e. an unmodified or wild- type form of the plant.

[0071] As used herein, the word "seed" is taken to include a "grain", for example a wheat, rice or barley grain.

[0072] Biomass of a plant may be measured according to any number of parameters. When the biomass of the whole plant is being determined, weight of the plant is typically measured. This may include the complete dessicated weight of the plant, part-dessicated weight of the plant, or the weight upon harvesting (i.e. minus drying). When the biomass of part of the plant is being determined, the measurement unit or parameter will depend upon the plant part. For example, seed size, seed weight per plant, weight of an individual seed, or seed weight per hectare or acre may be measured. The number of flowers (florets) per panicle can be measured and expressed as a ratio of the number of filled seeds over the number of primary panicles. The harvest index may be measured, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass. In another example, thousand kernel weight (TKW) can be measured, which is extrapolated from the number of filled seeds counted and their total weight. The present invention contemplates the use of any number of measurement units or parameters to determine the biomass of the plant.

[0073] Modulating biomass or modulated biomass of a plant refers to an increase or decrease in the biomass of the plant, or as indicated above, an increase or decrease in a specific part or structure of the plant, relative to an unmodified or wild-type form of the plant (or part thereof).

[0074] An "increase" in biomass is intended, for example, to include a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100- fold or greater increase in biomass compared to the biomass of a wild-type form of the plant. Conversely, a "decrease" biomass is intended, for example, to include a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater reduction in biomass compared to the biomass of a wild-type form of the plant.

[0075] As set out above, the present invention contemplates modulating biomass of a plant by modulating expression of a NF-YB4 polypeptide in the plant. As referred to herein, a "NF-YB4 polypeptide" includes a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. The term "NF-YB4 polypeptide" should also be understood to extend to functional homologs of SEQ ID NO: 1 or SEQ ID NO: 2.

[0076] Functional homologs of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 should be understood to include polypeptides which modulate the biomass of a plant. In some embodiments, a functional homolog may comprise, for example, a polypeptide which has one or more amino acid insertions, deletions or substitutions relative to the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; a mutant form or allelic variant of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2; an ortholog of the polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 in another plant species and the like.

[0077] In some embodiments, a functional homolog of a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 comprises an amino acid sequence which has at least 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% amino acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 2.

[0078] When comparing amino acid sequences, the sequences should be compared over a comparison window of at least 30 amino acid residues, at least 50 amino acid residues, at least 80 amino acid residues, at least 1 10 amino acid residues, at least 140 amino acid residues, or over the full length of SEQ ID NO: 1 or SEQ ID NO: 2. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms such as the BLAST family of programs as, for example, disclosed by Altschul et al. 1997, Nucl. Acids Res. 25: 3389-3402. Global alignment programs may also be used to align similar sequences of roughly equal size. Examples of global alignment programs include NEEDLE (available at www.ebi.ac.uk/Tools/psa/emboss_needle/) which is part of the EMBOSS package (Rice P et al., 2000, Trends Genet, 16: 276-277), and the GGSEARCH program (available at fasta.bioch. Virginia. edu/fasta_www2/fasta_www.cgi?rm=compare&pgm=gnw) which is part of the FASTA package (Pearson W and Lipman D, 1988, Proc. Natl. Acad. Sci. USA, 85: 2444-2448). Both of these programs are based on the Needleman-Wunsch algorithm which is used to find the optimum alignment (including gaps) of two sequences along their entire length. Programs such as Clustal Omega (Sievers et al., 201 1 , Molecular Systems Biology 7: 539) may also be used, and alignments can be inspected using applications such as PROMALS3D (Pei et al., 2008, Nucleic Acids Research 36: 2295-2300) and the Alignment Annotator (Gille et al., 2014, Bioinformatics 30: 121-122). A detailed discussion of sequence analysis can also be found in Unit 19.3 of Ausubel et al ("Current Protocols in Molecular Biology" John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998).

[0079] As a result of inconsistent nomenclature of genes and proteins within the NF-Y family, it should be understood that orthologs of Triticum aestivum NF-YB4 (SEQ ID NO: 1 or SEQ ID NO: 2) may be classified into different NF-Y subfamilies. For example, homologs or orthologs of Triticum aestivum NF-YB4 (SEQ ID NO: 1 or SEQ ID NO: 2) may include TaNF-YB2 (BT009078), ZmNF-YB2 (NP_001 106052), ZmNF-YB2a (NP_001 105435), OsHAP3B/OsNF-YB3 (BAC76332), OsHAP3A (BAC76331 ), ZmNF- YB4 (NP_001 152278), AtN F-YB 1 /HAP3A (NP_030436), AtNF-YB8 (AEC06243), AtNF- YB10 (AEE79070), TaNF-YB1 1 (CJ856713), OsHAP3C (BAC76333), ZmNF-YB1 1 (CBW53694), ZmNF-YB13 (DAA53600), and ZmNF-YB12 (CBW53703).

[0080] As referred to herein, modulating "expression" of a NF-YB4 polypeptide in one or more cells of the plant includes modulating the level and/or activity of the polypeptide in the one or more cells. Modulating the "level" of the polypeptide should be understood to include an increase or decrease in the level or amount of a NF-YB4 polypeptide in one or more cells of the plant. Similarly, modulating the "activity" of a NF-YB4 polypeptide should be understood to include an increase or decrease in, for example, the total activity, specific activity, half-life and/or stability of a NF-YB4 polypeptide in the one or more cells of the plant.

[0081] By "increasing" is intended, for example, to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100-fold or greater increase in the level and/or activity of the NF-YB4 polypeptide in one or more cells of the plant compared to one or more cells from a wild-type plant, i.e. a plant in which the level and/or activity of the polypeptide has not been modulated. By "decreasing" is intended, for example, to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater reduction in the level and/or activity of the NF-YB4 polypeptide in one or more cells of the plant compared to one or more cells from a wild-type plant.

[0082] "Modulating" should also be understood to include introducing a NF-YB4 polypeptide into one or more cells of a plant which does not normally express the NF-YB4 polypeptide, or the substantially complete inhibition of NF-YB4 polypeptide level and/or activity in one or more cells of a plant that normally has such activity.

[0083] In some embodiments, expression of a particular NF-YB4 polypeptide contemplated by the present invention is increased in one or more cells of the plant. An "increased" expression of the NF-YB4 polypeptide should be understood to include an increase in the level and/or activity of the NF-YB4 polypeptide in one or more cells of the plant and/or introducing a particular NF-YB4 polypeptide into one or more cells of the plant which do not normally express the introduced polypeptide.

[0084] In some embodiments, increasing expression of the NF-YB4 polypeptide in one or more cells of the plant increases biomass of the plant relative to a wild-type form of the plant.

[0085] In another embodiment, expression of a particular NF-YB4 polypeptide contemplated by the present invention is decreased in one or more cells of the plant. A "decreased" expression of the NF-YB4 polypeptide should be understood to include a decrease in the level and/or activity of the NF-YB4 polypeptide in one or more cells of the plant and/or substantially complete inhibition of a particular NF-YB4 polypeptide in one or more cells of a plant which normally express the polypeptide.

[0086] Embodiments of the present invention contemplate any means by which the expression of a NF-YB4 polypeptide in a cell may be modulated. This includes, for example, methods such as the application of agents which modulate NF-YB4 polypeptide activity in a cell, including the application of a NF-YB4 agonist or antagonist; the application of agents which mimic NF-YB4 polypeptide activity in a cell; modulating the expression of a nucleic acid which encodes a NF-YB4 polypeptide in the cell; effecting the expression of an altered or mutated nucleic acid in a cell such that a NF-YB4 polypeptide with increased or decreased specific activity, half-life and/or stability is expressed by the cell; or modulating the expression level, pattern and/or targeting of a NF-YB4 polypeptide in a cell for example via modification of a transcriptional control sequence and/or signal polypeptide associated with the NF-YB4 polypeptide.

[0087] In some embodiments, the expression of the NF-YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide. A nucleic acid which encodes a NF-YB4 polypeptide (also referred to herein as a "NF-YB4 nucleic acid") includes any nucleic acid which encodes a NF-YB4 polypeptide as described above.

[0088] The NF-YB4 nucleic acid contemplated by the present invention may be derived from any source. For example, the NF-YB4 nucleic acid may be derived from an organism, such as a plant. Alternatively, the NF-YB4 nucleic acid may be a synthetic nucleic acid. The NF-YB4 nucleic acid contemplated by the present invention may also comprise one or more non-translated regions such as 3' and 5' untranslated regions and/or introns. The NF-YB4 nucleic acid contemplated by the present invention may comprise, for example, mRNA sequences, cDNA sequences or genomic nucleotide sequences.

[0089] The NF-YB4 nucleic acid may be composed of any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. For example, the NF-YB4 nucleic acid can be composed of single- and double- stranded DNA, DNA that is a mixture of single- and double-stranded regions, single- and double-stranded RNA, and RNA that is a mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, the NF-YB4 nucleic acid can be composed of triple-stranded regions comprising RNA or DNA or both RNA and DNA. The NF-YB4 nucleic acid may also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. "Modified" bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, "nucleic acid" embraces chemically, enzymatically, or metabolically modified forms. [0090] In some embodiments, the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence set forth in SEQ ID NO: 4. However, it would be understood by a person skilled in the art that any nucleotide sequence which encodes a NF-YB4 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (or functional homologs thereof) is contemplated by the present invention. For example, variants of SEQ ID NO: 3 or SEQ ID NO: 4 are contemplated which comprise one or more different nucleotides to these sequences but which still encode identical amino acid sequences. Because of the degeneracy of the genetic code, a large number of nucleotides can encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Therefore, at every position in SEQ ID NO: 3 or SEQ ID NO: 4 where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Accordingly, every nucleotide sequence herein which encodes a NF-YB4 polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2 (or functional homologs thereof) also describes every possible silent variation of the nucleotide sequence. One of skill will recognise that each codon in a nucleotide sequence (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleotide sequence that encodes a NF-YB4 polypeptide is implicit in each described sequence.

[0091] Accordingly, the present invention contemplates a variant of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4, wherein the variant encodes a functional NF-YB4 polypeptide. In some embodiments, the variant of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4 comprises a nucleotide sequence which is at least 50%, 52%, 54%, 56%, 58%, 60%, 62%, 64%, 66%, 68%, 70%, 72%, 74%, 76%, 78%, 80%, 82%, 84%, 86%, 88%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1 %, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% identical to SEQ ID NO:31 or SEQ ID NO: 4.

[0092] When comparing nucleotide sequences to SEQ ID NO: 3 or SEQ ID NO: 4 to calculate a percentage identity, the nucleotide sequences should be compared over a comparison window of at least 100 nucleotide residues, at least 200 nucleotide residues, at least 300 nucleotide residues, at least 400 nucleotide residues, at least 600 nucleotide residues, at least 700 nucleotide residues or over the full length of SEQ ID NO: 3 or SEQ ID NO: 4. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted using the methods referred to above with respect to comparing amino acid sequences.

[0093] The term "modulating" with regard to the expression of a NF-YB4 nucleic acid may include increasing or decreasing the transcription and/or translation of a NF-YB4 nucleic acid in one or more cells of the plant. By "increasing" is intended, for example, to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or greater increase in the transcription and/or translation of a NF- YB4 nucleic acid. By "decreasing" is intended, for example, to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20-fold, 50- fold, 100-fold or greater reduction in the transcription and/or translation of a CIPK16 nucleic acid. Modulating also comprises introducing expression of a NF-YB4 nucleic acid not normally found in a particular cell; or the substantially complete inhibition (e.g. knockout) of expression of a NF-YB4 nucleic acid in a cell that normally has such activity.

[0094] In some embodiments, expression of a NF-YB4 nucleic acid in one or more cells of the plant is increased. "Increased expression" should be understood to include an increase in the transcription and/or translation of a NF-YB4 nucleic acid in a cell and/or introducing transcription and/or translation of a particular NF-YB4 nucleic acid in a cell which does not normally express the introduced nucleic acid.

[0095] In some embodiments, expression of a NF-YB4 nucleic acid in one or more cells of the plant is decreased. "Decreased expression" should be understood to include a decrease in the transcription and/or translation of a NF-YB4 nucleic acid in a cell and/or substantially eliminating transcription and/or translation of a particular NF-YB4 nucleic acid in a cell which does not normally expresses the NF-YB4 nucleic acid.

[0096] The present invention contemplates any means by which expression of a NF-YB4 nucleic acid may be modulated. Methods for modulating expression of a NF-YB4 nucleic acid include, for example: genetic modification of the cell to upregulate or downregulate endogenous NF-YB4 nucleic acid expression; genetic modification by transformation with a NF-YB4 nucleic acid; genetic modification to increase the copy number of a NF-YB4 nucleic acid in the cell; administration of a nucleic acid molecule to the cell which modulates expression of an endogenous NF-YB4 nucleic acid in the cell; and the like.

[0097] In some embodiments, expression of a NF-YB4 nucleic acid is modulated by genetic modification of the cell. The term "genetically modified", as used herein, should be understood to include any genetic modification that effects an alteration in the expression of a NF-YB4 nucleic acid in the genetically modified cell relative to a non-genetically modified form of the cell. Exemplary types of genetic modification include: random mutagenesis such as transposon, chemical, UV and phage mutagenesis together with selection of mutants which overexpress or underexpress an endogenous NF-YB4 nucleic acid; transient or stable introduction of one or more nucleic acid molecules into a cell which direct the expression and/or overexpression of NF-YB4 nucleic acid in the cell; modulation of an endogenous NF-YB4 polypeptide by site-directed mutagenesis of an endogenous NF-YB4 nucleic acid; introduction of one or more nucleic acid molecules which inhibit the expression of an endogenous NF-YB4 nucleic acid in the cell, e.g. a cosuppression construct, an RNAi construct or a miRNA construct; and the like.

[0098] In some embodiments, the present invention contemplates increasing the level of a NF-YB4 polypeptide in one or more cells of a plant by introducing the expression of a NF- YB4 nucleic acid into the one or more cells, increasing expression of a NF-YB4 nucleic acid in theone or more cells and/or increasing the copy number of a NF-YB4 nucleic acid in the one or more cells.

[0099] Methods for transformation and expression of an introduced nucleic acid in plant cells are well known in the art, and the present invention contemplates the use of any suitable method. However, by way of example, reference is made to Zhao et al., 2006, Mol. Breeding DOI 10.1007/s1 1032-006-9005-6), Katsuhara et al., 2003, Plant Cell Physiol 44(12): 1378-1383), Ohta et al., 2002, FEBS Letters 532: 279-282) and Wu et ai., 2005, Plant Science 169: 65-73. Further suitable methods for introduction of a nucleic acid into plant cells include, for example: yAgrobacferium-mediated transformation, other bacterially-mediated transformation (see Broothaerts et al., 2005, Nature 433: 629-633), microprojectile bombardment based transformation methods and direct DNA uptake based methods. Roa-Rodriguez et al., 2003, Agrobacterium-med/^'afed transformation of plants, 3^rd Ed. CAMBIA Intellectual Property Resource, Canberra, Australia, review a wide array of suitable / grobacfer/um-mediated plant transformation methods for a wide range of plant species. Microprojectile bombardment may also be used to transform plant tissue and methods for the transformation of plants, particularly cereal plants, and such methods are reviewed by Casas et al., 1995, Plant Breeding Rev. 13: 235-264. Direct DNA uptake transformation protocols such as protoplast transformation and electroporation are described in detail in Galbraith et al., 1995, (eds.), Methods in Cell Biology Vol. 50, Academic Press, San Diego. In addition to the methods mentioned above, a range of other transformation protocols may also be used. These include infiltration, electroporation of cells and tissues, electroporation of embryos, microinjection, pollen-tube pathway, silicon carbide- and liposome mediated transformation. Methods such as these are reviewed by Rakoczy-Trojanowska, 2002, Cell. Mol. Biol. Lett. 7: 849-858. A range of other plant transformation methods may also be evident to those of skill in the art.

[0100] In further embodiments the present invention also provides methods for decreasing expression of a NF-YB4 nucleic acid in a cell. For example, with the identification of NF- YB4 nucleic acid sequences, the present invention also facilitates methods such as knockout, knockdown or downregulation of a NF-YB4 nucleic acid in a cell using methods including, for example:

(i) insertional mutagenesis including knockout or knockdown of a NF-YB4 nucleic acid in a cell by homologous recombination with a knockout construct (for an example of targeted gene disruption - see Terada et al., 2002, Nat. Biotechnol. 20: 1030- 1034);

(ii) post-transcriptional gene silencing (PTGS) or RNAi of a NF-YB4 nucleic acid in a cell (for review of PTGS and RNAi - see Sharp, 2001 , Genes Dev. 15(5): 485- 490; and Hannon, 2002, Nature 418: 244-51 );

(iii) transformation of a cell with an antisense construct directed against a NF- YB4 nucleic acid (for examples of antisense suppression see van der Krol et al., Nature 333: 866-869; van der Krol et al., BioTechniques 6: 958-967; and van der Krol et al., Gen. Genet. 220: 204-212);

(iv) transformation of a cell with a co-suppression construct directed against a NF-YB4 nucleic acid (for an example of co-suppression see van der Krol et al., Plant Cell 2(4): 291 -299);

(v) transformation of a cell with a construct encoding a double stranded RNA directed against a NF-YB4 nucleic acid (for an example of dsRNA mediated gene silencing see Waterhouse et al., 1998, Proc. Natl. Acad. Sci. USA 95: 13959-13964);

(vi) transformation of a cell with a construct encoding an siRNA or hairpin RNA directed against a NF-YB4 nucleic acid (for an example of siRNA or hairpin RNA mediated gene silencing see Lu et al., 2002, Nucl. Acids Res. 32(21 ): e171 ; doi:10.1093/nar/gnh170);

(vii) insertion of a miRNA target sequence such that it is in operable connection with a NF-YB4 nucleic acid (for an example of miRNA mediated gene silencing see Brown et al., 2002, Blood 1 10(13): 4144-4152);

(viii) mutageneis and gene knock-down using zinc-finger nucleases (see Carroll D, 201 1 , Genetics 188: 773-782; Sander JD et al., 201 1 , Nat. Methods 8: 67-69; and Miller JC et al., 2007, Nat. Biotechnol., 25: 778-785);

(ix) mutageneis and gene knock-down using transcription activator-like effector nuclease (TALEN) systems (see Bogdanove AJ and Voytas DF, 201 1 , Science 333: 1843-1846; Streubel J et al., 2012, Nat. Biotechnol., 30: 593-595; Cermak T et al., 201 1 , Nucl. Acids Res., 39: e82; Chen K and Gao C, 2013, J. Genet. Genomics 40: 271 -279; Voytas DF, 2013, Ann. Rev. Plant Biol., 64: 327-350; and Wang Y et al., 2014, Nat. Biotechnol., 32: 947-951 ); and

(x) mutageneis and gene knock-down using the CRISPR/Cas9 system (see Belhaj K et al., 2015, Current Opinion in Biotechnology, 32: 76-84; Shan Q et al., 2014, Nature Protocols, 9: 2395-2410; and Wang Y et al., 2014, Nat. Biotechnol., 32: 947-951 )

[0101] The present invention also facilitates decreasing expresion of a NF-YB4 nucleic acid in a cell via the use of synthetic oligonucleotides, for example, siRNAs or miRNAs directed against a NF-YB4 nucleic acid (for examples of synthetic siRNA mediated silencing see Caplen et al., 2001 , Proc. Natl. Acad. Sci. USA 98: 9742-9747; Elbashir et al., 2001 , Genes Dev. 15: 188-200; Elbashir et al., 2001 , Nature 41 1 : 494-498; Elbashir et al., 2001 , EMBO J. 20: 6877-6888; and Elbashir et al., 2002, Methods 26: 199-213).

[0102] In addition to the examples above, the introduced nucleic acid may also comprise a nucleotide sequence which is not directly related to a NF-YB4 nucleic acid but, nonetheless, may directly or indirectly modulate the expression of a NF-YB4 nucleic acid in a cell. Examples include nucleic acid molecules that encode transcription factors or other proteins which promote or suppress the expression of an endogenous NF-YB4 nucleic acid molecule in a cell; and other non-translated RNAs which directly or indirectly promote or suppress endogenous NF-YB4 polypeptide expression and the like. [0103] In order to effect expression of an introduced nucleic acid in a cell, where appropriate, the introduced nucleic acid may be operably connected to one or more transcriptional control sequences and/or promoters.

[0104] The term "transcriptional control sequence" as used herein should be understood to include any nucleic acid sequence which effects the transcription of an operably connected nucleic acid. A transcriptional control sequence may include, for example, a leader, polyadenylation sequence, promoter, enhancer or upstream activating sequence, and transcription terminator. Typically, a transcriptional control sequence at least includes a promoter. The term "promoter" as used herein, describes any nucleic acid which confers, activates or enhances expression of a nucleic acid molecule in a cell.

[0105] In some embodiments, at least one transcriptional control sequence is operably connected to a NF-YB4 nucleic acid. For the purposes of the present specification, a transcriptional control sequence is regarded as "operably connected" to a given gene or other nucleotide sequence when the transcriptional control sequence is able to promote, inhibit or otherwise modulate the transcription of the gene or other nucleotide sequence.

[0106] A promoter may regulate the expression of an operably connected nucleic acid constitutively, or differentially, with respect to the cell, tissue, organ or developmental stage at which expression occurs, in response to external stimuli such as physiological stresses, pathogens, or metal ions, amongst others, or in response to one or more transcriptional activators. As such, the promoter used in accordance with the methods of the present invention may include, for example, a constitutive promoter, an inducible promoter, a tissue-specific promoter or an activatable promoter.

[0107] Plant constitutive promoters typically direct expression in nearly all tissues of a plant and are largely independent of environmental and developmental factors. Examples of constitutive promoters that may be used in accordance with the present invention include the plant ubiquitin promoter (Pub/), plant viral derived promoters such as the Cauliflower Mosaic Virus 35S and 19S (CaMV 35S and CaMV 19S) promoters; bacterial plant pathogen derived promoters such as opine promoters derived from Agrobacterium spp., e.g. the Agrobacterium-der^'wed nopaline synthase (nos) promoter; and plant-derived promoters such as the rubisco small subunit gene (rbcS) promoter, and the rice actin promoter (Pact). [0108] "Inducible" promoters include, but are not limited to, chemically inducible promoters and physically inducible promoters. Chemically inducible promoters include promoters which have activity that is regulated by chemical compounds such as alcohols, antibiotics, steroids, metal ions or other compounds. Examples of chemically inducible promoters include: alcohol regulated promoters (e.g. see European Patent 637 339); tetracycline regulated promoters (e.g. see US Patent 5,851 ,796 and US Patent 5,464,758); steroid responsive promoters such as glucocorticoid receptor promoters (e.g. see US Patent 5,512,483), estrogen receptor promoters (e.g. see European Patent Application 1 232 273), ecdysone receptor promoters (e.g. see US Patent 6,379,945) and the like; metal- responsive promoters such as metallothionein promoters (e.g. see US Patent 4,940,661 , US Patent 4,579,821 and US 4,601 ,978); and pathogenesis related promoters such as chitinase or lysozyme promoters (e.g. see US Patent 5,654,414) or PR protein promoters (e.g. see US Patent 5,689,044, US Patent 5,789,214, Australian Patent 708850, US Patent 6,429,362).

[0109] An inducible promoter may also be a physically regulated promoter which is regulated by non-chemical environmental factors such as temperature (both heat and cold), light and the like. Examples of physically regulated promoters include heat shock promoters (e.g. see US Patent 5,447858, Australian Patent 732872, Canadian Patent Application 1324097); cold inducible promoters (e.g. see US Patent 6,479,260, US Patent 6,184,443 and US Patent 5,847,102); light inducible promoters (e.g. see US Patent 5,750,385 and Canadian Patent 132 1563); and light repressible promoters (e.g. see New Zealand Patent 508103 and US Patent 5,639,952).

[0110] "Tissue specific promoters" include promoters which are preferentially or specifically expressed in one or more specific cells, tissues or organs in an organism and/or one or more developmental stages of the organism. It should be understood that a tissue specific promoter also be constitutive or inducible.

[0111] Examples of plant tissue specific promoters include: root specific promoters such as those described in US Patent Application 2001047525; fruit specific promoters including ovary specific and receptacle tissue specific promoters such as those described in European Patent 316 441 , US Patent 5,753,475 and European Patent Application 973 922; and seed specific promoters such as those described in Australian Patent 612326 and European Patent application 0 781 849 and Australian Patent 746032. [0112] The promoter may also be a promoter that is activatable by one or more transcriptional activators, referred to herein as an "activatable promoter". For example, the activatable promoter may comprise a minimal promoter operably connected to an Upstream Activating Sequence (UAS), which comprises, inter alia, a DNA binding site for one or more transcriptional activators.

[0113] As referred to herein the term "minimal promoter" should be understood to include any promoter that incorporates at least an RNA polymerase binding site and, optionally a TATA box and transcription initiation site and/or one or more CAAT boxes. In some embodiments wherein the cell is a plant cell, the minimal promoter may be derived from the Cauliflower Mosaic Virus 35S (CaMV 35S) promoter. The CaMV 35S derived minimal promoter may comprise, for example, a sequence that substantially corresponds to positions -90 to +1 (the transcription initiation site) of the CaMV 35S promoter (also referred to as a -90 CaMV 35S minimal promoter), -60 to +1 of the CaMV 35S promoter (also referred to as a -60 CaMV 35S minimal promoter) or -45 to +1 of the CaMV 35S promoter (also referred to as a -45 CaMV 35S minimal promoter).

[0114] As set out above, the activatable promoter may comprise a minimal promoter fused to an Upstream Activating Sequence (UAS). The UAS may be any sequence that can bind a transcriptional activator to activate the minimal promoter. Exemplary transcriptional activators include, for example: yeast derived transcription activators such as Gal4, Pdr1 , Gcn4 and Ace1 ; the viral derived transcription activator, VP16; Hap1 (Hach et al., J Biol Chem 278: 248-254, 2000); Gaf1 (Hoe et al., Gene 215(2): 319-328, 1998); E2F (Albani et al., J Biol Chem 275: 19258-19267, 2000); HAND2 (Dai and Cserjesi, J Biol Chem 277: 12604-12612, 2002); NRF-1 and EWG (Herzig et al., J Cell Sci 1 13: 4263-4273, 2000); P/CAF (Itoh et al., NucI Acids Res 28: 4291 - 4298, 2000); MafA (Kataoka et al., J Biol Chem 277: 49903-49910, 2002); human activating transcription factor 4 (Liang and Hai, J Biol Chem 272: 24088 - 24095, 1997); BcH O (Liu ef al., Biochem Biophys Res Comm 320(1 ): 1 -6, 2004); CREB-H (Omori et al., NucI Acids Res 29: 2154 - 2162, 2001 ); ARR1 and ARR2 (Sakai et al., Plant J 24(6): 703-71 1 , 2000); Fos (Szuts and Bienz, Proc Natl Acad Sci USA 97: 5351 -5356, 2000); HSF4 (Tanabe et al., J Biol Chem 274: 27845 - 27856, 1999); MAML1 (Wu et al., Nat Genet 26: 484-489, 2000).

[0115] In some embodiments, the UAS comprises a nucleotide sequence that is able to bind to at least the DNA-binding domain of the GAL4 transcriptional activator. An example of an activatable promoter includes the enhancer trap system for Arabidopsis and rice as described by Johnson et al. (Plant J. 41 : 779-789, 2005).and Moller et al. (Plant Cell 21 : 2163-2178, 2009).

[0116] The transcriptional control sequence may also include a terminator. The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3'-non-translated DNA sequences generally containing a polyadenylation signal, which facilitate the addition of polyadenylate sequences to the 3'-end of a primary transcript. As with promoter sequences, the terminator may be any terminator sequence which is operable in the cells, tissues or organs in which it is intended to be used. Examples of suitable terminator sequences which may be useful in plant cells include: the nopaline synthase (nos) terminator, the CaMV 35S terminator, the octopine synthase (ocs) terminator, potato proteinase inhibitor gene (pin) terminators, such as the pinll and pinlll terminators and the like.

[0117] In some embodiments, increasing expression of the NF-YB4 nucleic acid increases biomass of the plant relative to a wild-type form of the plant. The meaning of an increase in biomass is explained in detail above.

[0118] In some embodiments, yield of the plant in increased relative to a wild-type form of the plant. The term "yield" as used herein generally means a measurable produce of economic value, typically related to a specified crop, to an area, and to a period of time. Individual plant parts directly contribute to yield based on their number, size and/or weight, or the actual yield is the yield per square meter for a crop and year, which is determined by dividing total production (includes both harvested and appraised production) by planted square meters. Yield can be assessed a number of ways. For example, yield may refer to biomass of the plant, seed yield per plant or per cultivated area, numbers of tillers, number of spikes, harvest index (i.e. the ratio of seed yield to aboveground dry weight), seed filling rate, number of filled seeds, number of seed capsules/pods, seed size, and growth or branching.

[0119] An "increased yield" as used herein is taken to mean a 1 %, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 2-fold, 5-fold, 10-fold, 20 fold, 50-fold, 100-fold or greater increase in yield compared to a wild-type form of the plant. [0120] A plant with an "increased yield" as used herein can therefore be a plant having any one or more of the following characteristics or phenotypes, each relative to corresponding wild type plants: (i) increased biomass (weight) of one or more parts of the plant; (ii) increased seed yield, which includes an increase in seed biomass (seed weight) and which may be an increase in the seed weight per plant or on an individual seed basis or an increase in seed weight per hectare or acre; (iii) increased number of flowers (florets) per panicle, which is expressed as a ratio of the number of filled seeds over the number of primary panicles; (iv) increased number of (filled) seeds; (v) increased fill rate of seeds (which is the number of filled seeds divided by the total number of seeds and multiplied by 100); (vi) increased seed size, which may also influence the composition of seeds; (vii) increased seed volume, which may also influence the composition of seeds (for example due to an increase in amount or a change in the composition of oil, protein or carbohydrate); (viii) increased seed area; (ix) increased seed length; (x) increased seed width; (xi) increased seed perimeter; (xii) increased harvest index, which is expressed as a ratio of the yield of harvestable parts, such as seeds, over the total biomass; and (xiii) increased thousand kernel weight (TKW), which is extrapolated from the number of filled seeds counted and their total weight. An increased TKW may result from an increased seed size and/or seed weight and may also result from an increase in embryo size and/or endosperm size.

[0121] Taking wheat for example, a yield increase may be manifested as one or more of an increase in seed (grain) yield, an increase in seed (grain) weight per plant, an increase in individual seed (grain) weight, an increase in the number of tillers, an increase in the number of spikes, an increase in the number of seeds (grains) per spike, and an increase in the number of seeds (grains) per plant, among others.

[0122] Taking corn as an example, a yield increase may be manifested as one or more of the following: increase in the number of plants per hectare or acre, an increase in the number of ears per plant, an increase in the number of rows, number of kernels per row, kernel weight, thousand kernel weight, ear length/diameter, among others.

[0123] Taking rice as an example, a yield increase may be manifested by an increase in one or more of the following: number of plants per hectare or acre, number of panicles per plant, number of spikes per panicle, number of flowers per panicle, increase in the seed filling rate, increase in thousand kernel weight, among others. [0124] As indicated above, when comparing biomass, NF-YB4 expression and yield, comparison is made relative to a wild-type or control plant. The wild-type plant is a plant that has not been modified according to the methods of the invention. The wild-type plant is typically of the same plant species, and preferably the same ecotype as the plant to be assessed.

[0125] With respect to the first aspect of the invention, the inventors have shown that biomass can be increased in a plant grown in non-drought conditions. When the plant is grown under conditions of drought, biomass of the plant is unchanged compared to a wild- type plant.

[0126] "Drought" as referred to herein should be understood to include any situation wherein the amount of water available to a plant, at a physiologically appropriate level of salinity, is less than the optimum level of water for that plant. In some embodiments, drought may include low volumetric water content (VWC) in a soil. In some embodiments, drought may include soil VWC of less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than 5%, less than about 4%, or less than about 3%.

[0127] In some embodiments, drought may also include other forms of osmotic stress such as wherein a relatively high volume of water is available, but the level of salinity in the water is sufficiently high to cause osmotic stress in the plant. As would be understood by a person skilled in the art, "salinity" as used herein generally refers to the level of salt in the growing environment of a plant. A salt in this regard typically includes sodium chloride, magnesium and calcium sulphates, and bicarbonates. However, the most relevant salt for a majority of cropping systems is sodium chloride.

[0128] The inventors have also shown that biomass can be increased in a plant grown in nutrient-poor conditions, such as nutrient-poor soil, relative to a control or wild-type plant. Nutrient-poor soil may result from a lack of nutrient such as nitrogen, phosphates and other phosphorous-containing compounds, potassium, calcium, magnesium, manganese, iron and boron, amongst others.

[0129] The plants contemplated by each embodiment of the present invention may include any plant including angiosperm or gymnosperm higher plant cells as well as lower plant cells such as bryophyte, fern and horsetail cells.

[0130] In some embodiments, the plant may be a monocotyledonous plant. In some embodiments, the monocotyledonous plant may be a cereal crop plant. As used herein, the term "cereal crop plant" includes members of the Poaceae (grass family) that produce edible seed (grain) for human or animal food. Examples of Poaceae cereal crop plants which in no way limit the present invention include wheat, rice, barley, maize, millets, sorghum, rye, triticale, oats, teff, wild rice, spelt, turf grass, Italian rye grass, switchgrass, Miscanthus, Festuca, and the like. However, the term cereal crop plant should also be understood to include a number of non- Poaceae species that also produce edible seed (grain) and are known as the pseudocereals, such as amaranth, buckwheat and quinoa.

[0131] In some embodiments, the plant may be a dicotyledonous plant. Exemplary dicots include, for example, Arabidopsis spp., Medicago spp., Nicotiana spp., soybean, canola, oil seed rape, sugar beet, mustard, sunflower, tomato, potato, safflower, cassava, yams, sweet potato, other Brassicaceae such as Thellungiella halophila, among others.

[0132] In a second aspect, the present invention provides a method for modulating yield of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0133] Modulating the yield of a plant refers to increasing or decreasing the yield of the plant compared to the yield of a control or wild-type plant. Increasing and decreasing are relative terms and workable parameters have been described in detail above, for example with respect to expression of a NF-YB4 polypeptide and a NF-YB4 nucleic acid. Mechanisms for modulating expression of a NF-YB4 polypeptide, the nature of the NF- YB4 polypeptide, and what is meant by "yield" have also been described above with respect to the first aspect of the invention and so are not reiterated here.

[0134] In some embodiments of the second aspect of the invention, increasing expression of the NF-YB4 polypeptide increases yield of the plant relative to a wild-type form of the plant. The terms "increasing yield" and "increasing expression" of a NF-YB4 polypeptide have been described above with respect to the first aspect of the invention. [0135] In some embodiments of the second aspect of the invention, expression of the NF- YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide. In some embodiments, increasing expression of the nucleic acid increases yield of the plant relative to a wild-type form of the plant. Mechanisms for modulating expression, including increasing expression, of a nucleic acid which encodes the NF-YB4 polypeptide (i.e. a NF-YB4 nucleic acid as referred to herein), and the nature of the NF-YB4 nucleic acid have also been described above with respect to the first aspect of the invention and so are not reiterated here.

[0136] In a third aspect, the present invention provides a method for modulating biomass of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

[0137] In a fourth aspect, the present invention provides a method for modulating yield of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

[0138] In some embodiments of the third and fourth aspects of the invention, increasing expression of the NF-YB4 polypeptide or increasing expression of a nucleic acid encoding the NF-YB4 polypeptide (i.e. a NF-YB4 nucleic acid) increases biomass and/or yield of the multicellular structure relative to a wild-type form of the multicellular structure.

[0139] As referred to herein, a "multicellular structure" includes any aggregation of one or more plant cells. As such, a multicellular structure specifically encompasses tissues, organs, whole organisms and parts thereof. Furthermore, a multicellular structure should also be understood to encompass multicellular aggregations of cultured cells such as colonies, plant calli, liquid or suspension cultures and the like.

[0140] In light of the above, the term "multicellular structure" should be understood to include a whole plant, plant tissue, plant organ, plant part, plant reproductive material or cultured plant tissue (e.g. callus or suspension culture).

[0141] With respect to the third and fourth aspects of the invention, the terms "biomass" and "yield" have been described above with respect to the first aspect of the invention.

[0142] In a fifth aspect, the present invention provides a genetically modified plant which has modulated biomass, wherein modulation of the biomass is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0143] In a sixth aspect, the present invention provides a genetically modified plant which has modulated yield, wherein modulation of the yield is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

[0144] As referred to herein, a "genetically modified plant" comprises one or more cells that have been genetically modified with respect to the wild-type form of the one or more cells. As such, a genetically modified plant may include one or more cells which themselves have been genetically modified, and/or the progeny of such cells.

[0145] The genetically modified plant of the fifth and sixth aspects of the invention may include a plant as hereinbefore described. For example, in some embodiments, the plant may be any of an angiosperm, gymnosperm or bryophyte cell. In some embodiments, the plant may be a monocotyledonous plant, including a cereal crop plant such as a wheat plant. In some embodiments, the plant may be a dicotyledonous plant.

[0146] As set out above, expression of a NF-YB4 polypeptide and/or a NF-YB4 nucleic acid is modulated in the genetically modified plant. Modulation of a NF-YB4 polypeptide and/or a NF-YB4 nucleic acid may be performed as described with respect to the first aspect of the invention. Similarly, the terms "biomass" and "yield" have been described above with respect to the first aspect of the invention.

[0147] The term "about" as used in the specification means approximately or nearly and in the context of a numerical value or range set forth herein is meant to encompass variations of +/- 10% or less, +/- 5% or less, +/- 1 % or less, or +/- 0.1 % or less of and from the numerical value or range recited or claimed.

[0148] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

[0149] Finally, reference is made to standard textbooks of molecular biology that contain methods for carrying out basic techniques encompassed by the present invention. See, for example, Green MR and Sambrook J, Molecular Cloning: A Laboratory Manual (4th edition), Cold Spring Harbor Laboratory Press, 2012.

[0150] It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0151] The invention is further illustrated in the following example. The example is for the purpose of describing particular embodiments only and is not intended to be limiting with respect to the above description.

EXAMPLE 1

Identification and Characterisation of Nuclear Factor Gamma Subunits from Plants

[0152] Hetero-trimeric Nuclear Factors Gamma (NF-Y) are involved in regulation of various vital functions in all eukaryotic organisms. NF-Y transcription factors (TF), also known as histone- or haem-associated proteins (HAP) or CCAAT-binding factors (CBF), are heterotrimeric DNA-binding proteins, structurally conserved in all eukaryotes. They bind specifically to the CCAAT box which is found in about 25% of eukaryotic promoters. NF-Y TF are composed of three different subunits named NF-YA (also known as HAP-2 or CBF-B), NF-YB (HAP3 or CBF-A), and NF-YC (HAP5 or CBF-C). The NF-Y subunits B and C contain histone-fold domains (HFDs) structurally related to histones H2B and H2A, respectively. These domains mediate formation of a stable, histone-like heterodimer. The NF-Y subunit A subsequently binds to the B-C heterodimer to form a heterotrimeric complex, which can specifically recognise the CCAAT box.

[0153] Various members of the Nuclear Factor Gamma (NF-Y) gene family in plants have been shown to be involved in plant performance, development and stress regulation. Although a number of NF-Y subunits have been characterised in model plants, only a few have been functionally evaluated in crops. In this work we isolated a number of genes encoding NF-YB and NF-YC subunits from drought-tolerant wheat (Triticum aestivum L. cv. RAC875), and investigated the impact of the overexpression of TaNF-YB4 in the Australian wheat cultivar Gladius.

Material and Methods

Yeast 2-hybrid screen

[0154] The coding sequence (CDS) of ZmNF-YB2a (Acc. NP_001 105435) was cloned into the vector pGBKT7 (Invitrogen, Victoria, Australia) for yeast 2-hybrid (Y2H) screening. ZmNF-YB2a was used because we could not isolate maize ZmNF-YB2 cDNA based on the sequence submitted to public databases by Nelson et al., 2007, Proceedings of the National Academy of Sciences USA, 104: 16450-16455. The only sequence difference between the two proteins is an insert of 7 amino acid residues (GKTIPAN) in the histone fold domain of ZmNF-YB2, which is absent in ZmNF-YB2a and similar NF-YB subunits from wheat (see Figure 3A) and other grasses. ZmNF-YB2a was used as bait to screen WGL and WENDL (Lopato et al., 2006, Plant Methods 2: 3-17), WHSL (Eini et al., 2013, PloS One 8: e58713), WGD (developing grain at 0-6 DAP collected from the wheat cv. RAC875 subjected to drought at flowering) and WRDL (roots of wheat cv. RAC875 seedlings grown in soil and subjected to drought) cDNA libraries as previously described (Eini et al., 2013, supra). A large number of independent clones containing full-length or partial coding regions of the TaNF-YC15 cDNA were isolated. Because of self-activating properties of the full-length TaNF-YC15, a truncated version of the protein beginning at residue Ala97 was used to re-screen the same cDNA libraries. Clones containing inserts encoding two different full-length NF-YB transcription factors, designated TaNF-YB2 (18 independent clones) and TaNF-YB4 (22 independent clones), were isolated. The nucleotide sequence of the TaNF-YB4 mRNA is set forth in SEQ ID NO: 71.

Phylogenetic analysis of NF-Y subunits containing histone fold domains

[0155] The amino acid sequences of 53 NF-YB and 32 NF-YC subunit proteins were aligned with Clustal Omega (Sievers et al., 201 1 , supra) and alignments were further inspected by PROMALS3D (Pei ef al., 2008, supra) and the Alignment Annotator (Gille et al., 2014, supra). Unrooted phylogenetic trees, based on crude distance measures were visualised in TreeView (Page, 1996, CABIOS 12: 357-358) and are shown in Figure 2. GenBank accession numbers of protein sequences included in the phylogenetic trees are listed in Table 2 below. TABLE 2

NY-YB Subunit Proteins

TaNF-YB1 (BT009029, TaNF-YB2 (BT009078), TaNF-YB3 (BT009265), TaNF-YB4 (BT009393), TaNF-YB5 (CK203103), TaNF-YB6 (CV776390), TaNF-YB7 (CK213790), TaNF-YB8 (CJ724868), Ta F-YB1 1 (CJ856713), TaNF-YB12 (GH723061 ), OsHAP3A (BAC76331 ), OsHAP3B/OsNF-Y3B (BAC76332), OsHAP3C (BAC76333), OsHAP3E (BAF6443), OsHAP3F (BAF64445), OsHAP3J (FAA00426), OsHAP3l (BAF64448), OsHAP3H/DTH8 (BAF64447), OsHAP3G (BAF64446), OsHAP3K/LEC1 -like (AAL47204), OsNF-YB1 (CAC37695), AtNF-YB1/HAP3A (NP_030436), AtNF-YB2 (NP_199575), At F-YB3/HAP3C (AEE83458), AtNF-YB4 (AEE28385), AtNF-YB5 (AEC10890), AtNF- YB6/LIL (AED95548), AtNF-YB7 (AEC06243), AtNF-YB8 (AEC06243), AtNF-YB9/LEC1 (AEE30179), AtNF-YB10 (AEE79070), AtNF-YB13 (CBW53706), ZmNF-YB1

(CBW53688), ZmNF-YB2 (NP_001 106052), ZmNF-YB2a (NP_001 105435), ZmNF-YB3 (NP_001 147638), ZmNF-YB4 (NP_001 152278), ZmNF-YB5 (NP_001266909), ZmNF- YB6 (DAA53600), ZmNF-YB7 (CBW53701 ), ZmNF-YB8 (AFW63719), ZmNF-YB9 (CBW53696), ZmNF-YB10 (NP_001 152628), ZmNF-YB1 1 (CBW53694), ZmNF-YB12 (CBW53703), ZmNF-YB13 (DAA53600), ZmNF-YB14 (DAA59933), ZmNF-YB15

(CBW53702)

NY-YC Subunit Proteins

TaNF-YC1 (GH7314), TaNF-YC2 (BT008988), TaNF-YC3 (BT009224), TaNF-YC4 (DN829033), TaNF-YC5 (DR738968), TaNF-YC6 (CJ855361 ), TaNF-YC7 (CV762585), TaNF-YC8 (CD883696), TaNF-YC10 (BT008988), TaNF-YC1 1 (CD888515), TaNF-YC13 (BJ308764), OsHAP5A (BAF64449), OsHAP5B (BAF64450), OsHAP5C (BAF64451 ), OsHAP5D (BAF64452), OsHAP5E (BAF64453), OsHAP5F (BAF64454), OsHAP5G (BAF64455), AtNF-YC1 (AEE78434), AtNF-YB2 (AEE33354), AtNF-YC3 (AEE33153), AtNF-YC4 (AED97756), AtNF-YC5 (AED95951 ), AtNF-YC6 (AED959550), AtNF-YC7 (AED95949), AtNF-YC8 (AED93743), AtNF-YC9 (AEE28377), AtNF-YC10 (AEE28222), AtNF-YC1 1 (AEE75200), AtNF-YC12 (AED94272), AtNF-YC13 (AED94931 )

Construction of a three-dimensional (3D) model of the TaNF-YB2/TaNF-YC15 dimer

[0156] A 3D model was constructed by using mapping monomeric threading alignments to protein-protein interactions based on oligomeric entries in the Protein Data Bank (PDB) (Guerler et al., 2013, Journal of Chemical Information and Modeling 53: 717-725). The crystal structure of the NF-YB/NF-YC dimer from Homo sapiens (PDB accession 1 N1J, chains A/B, designated as 1 N1J:A/1 N1 J:B) (Romier et al., 2003, The Journal of Biological Chemistry 278: 1336-1345) was identified as a suitable quaternary assembly template for structural modelling. The full-length TaNF-YB2 and TaNF-YC15 sequences were analysed by SMART (Letunic et al., 2012, Nucleic Acids Research 40: D302-305), ProDom (Bru et al., 2005, Nucleic Acids Research 33: D212-215) and SBASE (Vlahovicek et al., 2002, Nucleic Acids Research 30: 273-275) to determine domain arrangements and the positions of HFDs. The TaNF-YB2 and TaNF-YC15 sequences were aligned with those of 1 N1 J:A and 1 N1 J:B, respectively, using LOMETS (Wu and Zhang, 2007, Nucleic Acids Research 35: 3375-3382), and the alignment quality was checked by the Alignment Annotator (Gille et al., 2014, supra) and PSIPRED (Buchan et al., 2013, Nucleic Acids Research 41 : W349-357) to confirm that secondary structures remained undisturbed. The aligned sequences were submitted to SPRING (Guerler et al., 2013, supra) and the most suitable model of TaNF-YB2/TaNF-YC15, evaluated by structural criteria, was selected from a library of six models. The model was minimised using AMBER99 force field to achieve optimal stereo-chemical parameters. A Ramachandran plot of the optimised model indicated that 100% residues were in the most favoured, additionally allowed and generously allowed regions, when excluding Gly and Pro residues. The overall G-factor values evaluated by PROCHECK (Laskowski et al., 1993, Journal of Molecular Biology 231 : 1049-1067) were 0.55 and 0.1 1 for 1 N1J:A/1 N1J:B and Ta F-YB2/Ta F-YC 15, respectively. The Z-score values, deduced from Prosa2003 (Sippl, 1993, Proteins 17: 355-362) and reflecting combined statistical potential energy, were -5.21 and -5.12 for 1 N1 J:A/1 N1J:B and TaNF-YB2/TaNF-YC15, respectively. The RMSD values between 1 N1 J:A/1 N1J:B (165 residues) and TaNF-YB2/TaNF-YC15 (169 residues) determined with a PyMol 'super' algorithm were 1.1 A for 164 residues in Ca positions. Images of structural models were generated in the PyMol Molecular Graphics System, Version 1.3 Schrodinger, LLC.

Plasmid construction and plant transformation

[0157] A 492 bp long fragment of the TaNF-YB4 CDS was cloned into the pENTR-D- TOPO vector and verified by sequencing using primers listed in Table 3 below, and subcloned into the vector pUbi (Eini et al., 2013, supra) by recombination. The nucleotide sequence of the open reading frame encompassed by this 492 bp fragment is set forth in SEQ ID NO: 4. The amino acid sequence of the TaNF-YB4 polypeptide encoded by this fragment is set forth in SEQ ID NO: 2. The resulting construct was designated pUbi-TaNF- YB4. pUbi-TaNF-YB4 was linearized using unique Pme\ restriction site and co- transformed with a selection marker cassette (pUbi-Hyg-nos) into the Australian elite wheat cultivar Gladius, using a biolistic bombardment method described by Kovalchuk et al., 2009, Plant Molecular Biology 71 : 81 -98. Transgene integration was confirmed by PCR using a forward primer derived from the 3' end of the transgene CDS and a reverse primer from the 5' end of the nos terminator (Table 3), for 17 independent transgenic events. Further validation of the cloned TaNF-YB4 CDS identified the introduction of a premature stop codon at nucleotide positions 313-315 due to the substitution of an adenine for a thymine nucleotide at position 313. The nucleotide sequence of the open reading frame encompassed by this truncated fragment is set forth in SEQ ID NO: 3. The amino acid sequence of the TaNF-YB4 polypeptide encoded by this truncated fragment is set forth in SEQ ID NO: 1 .

TABLE 3

Primer Sequences

Short

gene Purpose Forward primer Reverse primer name

ZmNF- Cloning in the GAAGAATTCATGGCGGAAGCTC GGAGGATTCCCATTAGTTTGAGATA YB2 Y2H vectors CGGCGAG - SEQ ID NO: 5 TCC - SEQ ID NO: 6

TaNF- Cloning in the GAAGAATTCATGTCGGACGAGG GGAGGATCCTCAGTTTGAGATGTC YB2 Y2H vectors CGGCGAG - SEQ ID NO: 7 CCCATTATGGTACT - SEQ ID NO: 8

TaNF- Cloning in the GAAGAATTCATGGCCGACGACG GGAGGATCCTCAGGTGTCCCCATT YB4 Y2H vectors ACAG - SEQ ID NO: 9 ATGGTAC - SEQ ID NO: 10

TaNF- Cloning in the GAAGAATTCTTCTGGGCCGAAC GAAGGATCCTCAGCCGCTTCCAGA YC15(d1) Y2H vectors GACTG - SEQ ID NO: 1 1 TTG - SEQ ID NO: 12

TaNF- Cloning in the CACCATGGCCGACGACGACAG - TCAGGTGTCCCCATT ATGGTAC - YB4 pENTR-D-TOPO SEQ ID NO: 13 SEQ ID NO: 14

TaNF- Q-PCR, gene GATTCAGCATCGTCAGCTTAG - TTGTAAGGGCACCACCACCAC - YC15 expression SEQ ID NO: 15 SEQ ID NO: 16

TaNF- Q-PCR, gene TCCTAGGTGGGTCATCATGTG - AGACAACAACAACAACCATGC - YB2 expression SEQ ID NO: 17 SEQ ID NO: 18

TaNF- Q-PCR, gene GGACACCTGAAACTGAAGATC - GCCATCACAACCAACTGTTTC - YB4 expression SEQ ID NO: 19 SEQ ID NO: 20

TaActin Q-PCR, GACAATGGAACCGGAATGGTC - GTGTGATGCCAGATTTTCTCCAT - normalisation SEQ ID NO: 21 SEQ ID NO: 22

Ta Cyclop Q-PCR, CAAGCCGCTGCACTACAAGG - AGGGGACGGTGCAGATGAA - SEQ hilin normalisation SEQ ID NO: 23 ID NO: 24

TaGAPdH Q-PCR, TTCAACATCATTCCAAGCAGCA - CGTAACCCAAAATGCCCTTG - SEQ normalisation SEQ ID NO: 25 ID NO: 26

TaEFa Q-PCR, CAGATTGGCAACGGCTACG - CGGACAGCAAAACGACCAAG - normalisation SEQ ID NO: 27 SEQ ID NO: 28

TaNF- Transgene GGCCAACGGCAAGATCGCCAAG TCTAGTAACATAGATGACACC - YB4 specific primers - SEQ ID NO: 29 SEQ ID NO: 30

Nos Q-PCR, copy CTTAAGATTGAATCCTGTTGCCG CGAATTCAGTAACATAGATGACACC terminator number GTC - SEQ ID NO: 31 GC - SEQ ID NO: 32 TaNF- Y2H, CDS GAAGAATTCATGGCGGAAGCTC GGAGGATTCCCATTAGTTTGAGATA YB2 CGGCGAG - SEQ ID NO: 33 TCC - SEQ ID NO: 34

TaNF- Y2H, deletions GAAGAATTCGGCGTCAGGGAGC GGAGGATTCCCATTAGTTTGAGATA YB2D1 AG G AC AG - SEQ ID NO: 35 TCC - SEQ ID NO: 36

TaNF- Y2H, deletions GAAGAATTCATGAAGAAGGCCA GGAGGATTCCCATTAGTTTGAGATA YB2D2 TCC - SEQ ID NO: 37 TCC - SEQ ID NO: 38

TaNF- Y2H, deletions GAAGAATTCCAGGAGTGCGTCT GGAGGATTCCCATTAGTTTGAGATA YB2D3 CCGAGT - SEQ ID NO: 39 TCC - SEQ ID NO: 40

TaNF- Y2H, deletions GAAGAATTCGACAAGTGCCAGG GGAGGATTCCCATTAGTTTGAGATA YB2D4 G - SEQ ID NO: 41 TCC - SEQ ID NO: 42

TaNF- Y2H, deletions GAAGAATTCGACGACCTGCTCT GGAGGATTCCCATTAGTTTGAGATA YB2D5 G - SEQ ID NO: 43 TCC - SEQ ID NO: 44

TaNF- Y2H, deletions GAAGAATTCCAGAAGTACAGAG GGAGGATTCCCATTAGTTTGAGATA YB2D6 AG A - SEQ ID NO: 45 TCC - SEQ ID NO: 46

TaNF- Y2H, deletions GAAGAATTCGATGCACTTGGTC GGAGGATTCCCATTAGTTTGAGATA YB2D7 CTC - SEQ ID NO: 47 TCC - SEQ ID NO: 48

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCTACTTGTTGGCCCATC YB2D8 CGGCGAG - SEQ ID NO: 49 - SEQ ID NO: 50

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCGAGGACCAAGTGCAT YB2D9 CGGCGAG - SEQ ID NO: 51 C - SEQ ID NO: 52

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCCTCTCTGTACTTCTG - YB2D10 CGGCGAG - SEQ ID NO: 53 SEQ ID NO: 54

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCGATGTACTCCTCGAA YB2D11 CGGCGAG - SEQ ID NO: 55 G - SEQ ID NO: 56

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCCCAGAGCAGGTCGTC YB2D12 CGGCGAG - SEQ ID NO: 57 - SEQ ID NO: 58

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCGGAGACGCACTCCTG YB2D13 CGGCGAG - SEQ ID NO: 59 CAC - SEQ ID NO: 60

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCGTCCTTGGCGATCTT YB2D14 CGGCGAG - SEQ ID NO: 61 GC - SEQ ID NO: 62

TaNF- Y2H, deletions GAAGAATTCATGGCGGAAGCTC GGAGGATTCGATGGCCTTCTTCAT YB2D15 CGGCGAG - SEQ ID NO: 63 G - SEQ ID NO: 64

Genotyping and transgene expression analyses

[0158] Plant DNA was extracted from leaf tissue using a freeze-drying method described by Shavrukov et al., 2010, Functional & Integrative Genomics 10: 277-291 . Individuals were genotyped for the presence of the transgene. Transgene copy number was also estimated by efficiency adjusted real-time quantitative PCR, using nos terminator specific primers (Table 3; Kovalchuk et al., 2013, Plant Biotechnology Journal 1 1 : 659-670). For template loading normalisation, primers and a probe complimentary to a portion of the single copy endogenous Puroindoline-b (Pin-b) gene were used (Li et al., 2004, Plant Molecular Biology Reports 22: 179-188). The oligonucleotide sequences were: forward 5'- ATTTTCCAGTCACCTGGCCC-3' (SEQ ID NO: 65); reverse 5 - TGCTATCTGGCTCAGCTGC-3' (SEQ ID NO: 66); and dual-labelled TaqMan probe 5'- CAL fluor Gold 540 - ATGGTGGAAGGGCGGCTGTGA-BHQ1 -3' (SEQ ID NO: 67). [0159] Total RNA was isolated from leaf tissue using the Direct-zol RNA MiniPrep (Zymo Research Corporation, ACT, Australia). Transgene expression was confirmed using RT- PCR and transgene specific primers (Table 3). For Northern analysis of transgene expression, RNA was electrophoretically separated on a 1.3% agarose gel containing 6% formaldehyde, transferred to nylon membrane and hybridized with ³²P-labeled DNA probes according to the protocol described by Church and Gilbert, 1984, Proceedings of the National Academy of Sciences USA 81 : 1991-1995.

Analysis of transgenic plants

[0160] Experiment 1. Three lines estimated to have a single copy of the transgene (L3, L4, L5) and one line with two copies (L6), were selected for preliminary characterisation of plant phenotypes and seed multiplication, Six Ti plants for each line were grown in 6 inch pots, one plant per pot, in coco-peat soil in a greenhouse (24/16^°C, day/night temperature; 16 h day) under well-watered conditions. Untransformed wild type (WT) plants were also grown for comparison. Plant height, number of tillers and spikes, plant biomass, flowering time, seed number and seed weight were recorded for each plant. Transgene copy number and transgene expression levels were also determined, and null segregants were excluded from the analyses.

[0161] Experiment 2. Six T₂ sub-lines (L3-5, L4-2, L4-4, L5-4, L6-2 and L6-3) derived from the four independent Ti lines were selected for a second experiment under a controlled water regime, conducted in two large containers (190 x 68 x 60 cm) filled with a 1 :1 :1 mix of coco-peat, river sand and clay soil collected near Adelaide (South Australia). Plants were grown in rows, eight plants per row for each line and WT with three randomised blocks in each container comprising in total 18 - 26 biological replicates for each line and WT. Experimental plants were flanked by a border row of WT plants on each short side of the container. The experimental design was identical for each container. No significant differences were found in plant growth between the three blocks in preliminary experiments (data not shown), and therefore all replicates for each line and WT were used to calculate means and standard errors for the measurements.

[0162] Containers were watered every second day. The watering was withdrawn in the draughted container when the majority of plants reached tillering, watering was continued in the well-watered container until the end of grain maturation. A soil water tension of minus 3 to minus 3.5 MPa was reached in the draughted container a short time before flowering, and plants were kept under slowly increasing drought until about 10 days after the end of flowering. Soil water content was monitored by the system, Magpie-3 (Measuring Engineering Australia, www.mea.com.au), where data for water content in the soil were regularly collected by sensors from each container and from three levels of depth in the soil: 15, 30 and 45 cm. Curve graphs of drought and well-watered bins were automatically recorded. When water potential in the draughted container reached minus 5.0 MPa, watering re-commenced and soil water content was restored to a level similar to the well-watered container.

[0163] Measurements of yield-related plant growth traits and yield components were taken at harvest, as for Experiment 1 . Leaf material sampled from all plants was used for genotyping and analysis of gene expression. Three of the 6 T₂ sub-lines (L4-2, L4-4, and L5-4) were identified as homozygous for the transgene. In segregating heterozygous sublines, if the number of null segregants was sufficient, these data were used as an additional control; otherwise they were excluded from the analyses. Progeny of line L3 was excluded from the analysis due to a large number of dead plants (see Table 4 below).

[0164] Experiment 3. T₃ wheat sub-lines were grown in 6 inch pots, four plants per pot, including one control and three transgenic plants. Transgenic plants included representatives of two confirmed and one possible homozygous sub-lines (L4-4-51 , L5-4- 52; L6-3-31 ). A coco-peat soil amended with five rates (0, 0.7, 1.4, 2.1 , 2.8 g/L of soil) of slow release complete fertiliser (Osmocote Exact Mini; Everris International B.V. The Netherlands) was used in this experiment (see Table 4). Three pots of plants (replicates) were grown for each fertiliser rate except for the unamended treatment, for which there were six pots. Analysis of yield components was performed as described above for Experiments 1 and 2.

Gene expression analysis of native NF-Y genes in wheat

[0165] Quantitative RT-PCR (qRT-PCR) analyses of gene expression in various tissues of non-transgenic wheat were performed on cDNA samples as described by Burton et al., 2008, Plant Physiology 146: 1821 -1833. Samples tested included a cDNA developmental tissue series prepared from different tissues of T. aestivum cv. Chinese Spring (Morran et al., 201 1 , Plant Biotechnology Journal 9: 230-249), a stress cDNA series prepared using the drought tolerant cv. RAC875 (Kovalchuk et al., 2013, supra), and a dehydration cDNA series prepared from detached leaves of four weak-old wheat (cv. RAC875) seedlings, which were incubated for 0, 0.5, 1 , 2, 4, 5, and 7 hours at room temperature (23°C) in opened 15 ml plastic tubes, before snap-freezing in liquid nitrogen for RNA extraction.

TABLE 4

Information about Ti progenies of TaNF-YB4 transgenic lines analysed in large

containers

Growth

Lines WT L3-5** L4-2* L4-4* L5-4* L6-2 L6-3* conditions

Trangene copy number 0 1 1 1 1 2 1

Well- Total No. of plants 21 10 21 28 28 28 28 watered

Well- No. of dead plants 3 10 0 4 3 1 1 watered

Well- No. of nulls 0 0 0 0 4 0 watered

No. of plants with

Well- 10 21 24 25 23 27

expressed

watered

transgene^***

Drought Total No. of plants 24 9 24 24 24 24 24

Drought No. of dead plants 2 9 2 1 2 0 2

Drought No. of nulls 0 0 0 0 4 0

No. of plants with

Drought 9 22 23 22 20 28

expressed transgene

^* Homozygous sub-lines

^** Progeny of Sub-line 3-5 demonstrated a poor growth and high mortality and therefore it was not shown in the Figure 5 and used in further experiments

^*** Only data for plants with expressed transgene were used for generation of graphs in the Figure 6

Results

Isolation of wheat genes encoding NF-YB subunits

[0166] In order to identify and isolate cDNA clones encoding wheat NF-YB proteins homologous to maize ZmNF-YB2, a two-step Y2H screen was performed. Full length ZmNF-YB2a protein (Acc. NP_001 105435) was used to screen cDNA libraries prepared from different wheat tissues of drought tolerant wheat cv. RAC875 subjected to drought and heat stresses.

[0167] The screen resulted in isolation of several transcription factors and transcription related proteins (data not shown). The most abundant of the isolated sequences was from an unreported wheat gene encoding the NF-Y subunit C, designated TaNF-YC15. The mRNA, coding nucleotide sequence and encoded amino acid sequence of the isolated TaNF-YC15 subunit are set forth in SEQ ID NOs: 68, 69 and 70, respectively. To prevent a self-activation during the Y2H screen, TaNF-YC15 was truncated at the N-terminal end. Truncation did not affect the fold of the histone fold domain, important for interactions with NF-YB proteins. Y2H screens with TaNF-YC15 resulted in isolation of large numbers of independent clones encoding two different NF-YB subunits identical to TaNF-YB2 and TaNF-YB4, which were previously described by Stephenson et al., 2007, Plant Molecular Biology 65: 77-92 (see Figure 1A). Full-length coding regions of isolated wheat NF-YB cDNAs were re-cloned into the bait vector, and interaction of TaNF-YB2 and TaNF-YB4 with TaNF-YC15 was demonstrated in a reciprocal way (see Figure 1 B). In addition, it was found that TaNF-YC15 is the most abundant among several NF-YC subunits isolated in Y2H screens using TaNF-YB2 and TaNF-YB4 as bait proteins (data not shown).

[0168] To reveal phylogenetic relationships between NF-YB and NF-YC protein sequences from wheat, maize, rice and Arabidopsis, unrooted trees were constructed using Clustal Omega (Blackshields et al., 2010, Algorithms for Molecular Biology: AMB 5: 21 ) (see Figure 2). The tree for the NF-YB sequences (Figure 2A) shows that both TaNF- YB2 and TaNF-YB4 belong to two different branches of the same protein clade, and that TaNF-YB2 and ZmNF-YB2 are the products of orthologous genes as they are grouped together. The TaNF-YB4 protein is grouped with a protein from maize, which is designated as ZmNF-YB4, while two rice proteins are grouped within these two branches. In contrast, the Arabidopsis AtNF-YB1 protein has a high level of identity to proteins from both branches containing TaNF-YB2-like and TaNF-YB4-like proteins, and two other proteins, AtNF-YB8 and AtNF-YB10, have much lower levels of identity to TaNF-YB2 (59% and 63%, respectively) or TaNF-YB4 (58% and 61 %, respectively), but still are grouped into the same clade (Figure 2A). The isolated TaNF-YB2 protein has a single amino acid residue difference from that submitted to the GenBank database (Acc. BT009078). This may reflect the fact that these two proteins originate from different wheat cultivars. No differences were found in the protein sequences for TaNF-YB4 isolated from cv. RAC875 in this study and the GenBank entry for TaNF-YB4 (Acc. BT009393).

[0169] TaNF-YC15 has a high level of protein sequence identity to TaNF-YC7 (Acc. AED95949). Both proteins belong to the same clade together with orthologous proteins from rice and more distant TaNF-YC1 (Acc. CD888515). No proteins from Arabidopsis were grouped in the same clade (see Figure 2B). The specificity of TaNF-YB2 and TaNF-YB4 interactions with TaNF-YC15

[0170] To explain why only two out of more than a dozen wheat NF-YB factors interacted with TaNF-YC15, the essential segment of the TaNF-YB2 protein was mapped in a Y2H assay using a series of truncated forms of TaNF-YB2 (Figure 1 C). The identified minimal protein segment, which interacted with the NF-YC subunit, contained a significant proportion of the histone fold domain (HFD), as well as additional amino acid residues adjacent to the C-terminal part of HFD.

[0171] The HFD portions of 1 1 NF-YB and 12 NF-YC protein sequences from wheat were aligned with those of human NF-YB and NF-YC, showing an exceptionally high level of conservation as seen in Figures 3A and 3B. Furthermore, a comparison of the modelled TaNF-YB2/Ta NF-YC 15 wheat structural dimer with the crystal structure of the human NF- YB/NF-YC (1 N1 J:A/1 N1J:B) sub-complex indicated that the two sub-structures were highly similar. Both complexes that interacted through HFDs showed features that were common to this class of proteins. There were high levels of sequence identity/similarity (76.9/98.9% and 76.9/93.6% for A/B and B/C chains, respectively) (Smith and Waterman, 1981 , Journal of Molecular Biology 147: 195-197) and structural correspondence (RMSD values 1 .1 A) between wheat and human NF-YB/NF-YC complexes, indicating that this structural fold has not undergone significant diversification during evolution. The residues involved in dimerisation of TaNF-YB2/Ta NF-YC 15 included nine residues each in chain B (Lys57, Ala59, Asp61 , Glu68, Ser75, Lys91 , Ile93, Tyr1 10 and Arg122) and chain C (Asp1 17, Asp1 19, Met122, Ser124, Glu126, Glu141 , Arg156, Leu158 and Lys160) (Figure 3C). All nine residues in TaNF-YB2 and eight out of the nine residues in TaNF-YC15 were conserved compared to human NF-YB/NF-YC, except of Lys160 that was substituted with Arg75 in human NF-YC (Figures 3B and 3C).

[0172] All residues of TaNF-YB2 HFD (Figure 3) were present in the minimal segment of TaNF-YB2 (residues 50 to 46 of Figure 1 C) that were required for interaction with TaNF- YC15 in a Y2H assay. A truncated variant of TaNF-YB2 with C-terminal Glu123 (underlined in the motif QKYRE; Figure 3B) lost the ability to bind TaNF-YC15. This suggested a minimal length for TaNF-YB2 protein to ensure correct folding of TaNF-YB2 (Figure 3B), and allowing Arg122 (that precedes Glu123) to redeem its proper protonation state and function during formation of the TaNF-YB2/TaNF-YC15 complex. Gln1 19 of wheat TaNF-YB2 was located in a close proximity of Arg122 in TaNF-YB2 and was also present in TaNF-YB4 (and human NF-YB; Figure 3B), but was substituted in all other TaNF-YBs by a His residue.

[0173] Further indirect evidence that wheat TaNF-YC15 acts in concert with either TaNF- YB2 or TaNF-YB4 came from gene expression analyses of plant tissues treated under a variety of conditions. Expression levels of TaNF-YC15 in these wheat tissues were in a good correlation with levels of NF-YB transcripts, particularly with TaNF-YB4 (see Figure 4).

Expression levels of isolated NF-Y transcription factors in different tissues, during slow developing drought and rapid dehydration

[0174] Transcript levels of TaNF-YB2, TaNF-YB4 and TaNF-YC15 genes were analysed by quantitative RT-PCR (qRT-PCR) in different tissues of unstressed wheat plants and in leaves from plants subjected to ABA treatment, drought or rapid dehydration. Expression of both NF-YB factors was detected in all tested tissues of unstressed wheat plants. Higher levels of expression were observed in reproductive tissues and particularly in anthers, where levels of expression of both genes were 5-6 fold higher than in other tissues (Figure 4A). Correlation between TaNF-YB2 and TaNF-YB4 levels of expression was observed across most tissue types. The most significant difference between expression levels of NF-YB genes was found in mature endosperm, where the number of TaNF-YB2 transcripts was about nine fold higher than the number of TaNF-YB4 transcripts (Figure 4A). Expression levels of TaNF-YC15 in different wheat tissues were in good correlation with levels of NF-YB transcripts, particularly with TaNF-YB4, with one exception (Figure 4B). In contrast to NF-YB genes, level of TaNF-YC15 expression in anthers was lower than in bracts and pistil. Overall, the TaNF-YC15 gene was less highly expressed than TaNF-YB2/YB4.

[0175] Neither TaNF-YC15 nor TaNF-YB genes were induced by ABA treatment (data not shown). In a drought cDNA tissue series, the three NF-Y genes showed two periods of induction. The first induction point occurred on the 17^th day of drought treatment, when plants had just started to sense drought, and the second induction point happened at wilting point, 30 days after watering was withheld (Figure 5A). A control gene known to be drought stress-inducible, TaCOR39 (Kovalchuk et al, 2013, supra), showed a single increase in expression across the same tissue series, at 30 days after cessation of watering (Figure 5A). Following moderate re-watering, expression levels of all tested genes decreased, but began to increase again after a further 4 days as the soil profile dried out (day 35).

[0176] In contrast to slowly developing drought, the rapid dehydration of detached leaves at room temperature led to different patterns of TaNF-YB2 and TaNF-YB4 expression. While TaNF-YB2 was induced by three-fold, TaNF-YB4 levels decreased with dehydration. Interestingly, the patterns of expression of TaNF-YB4 and TaNF-YC15 in dehydrating leaves were nearly identical (Figure 5B).

Evaluation of transgenic wheat plants overexpressing TaNF-YB4 in well-watered, droughted and nutrient-depleted conditions

[0177] Transgenic wheat plants (cv. Gladius) were generated with constitutive overexpression of TaNF-YB4 driven by the maize ubiquitin promoter. In a preliminary evaluation of plant performance (Ti generation), three from four transgenic lines with one or two copies of the transgene produced more grain compared to control plants, without substantial changes in other yield components (data not shown).

[0178] In a second experiment, two large containers were used for phenotyping of a large number of transgenic plants (18-28 T₂ individuals from each of six T₂ sub-lines, L3-5, L4- 2, L4-4, L5-4, L6-2 and L6-3; see Table 4). All of these except L6-2 were determined to be homozygous for the transgene. Progeny of the sub-line L3-5 grew more slowly than other plants and showed a high level of mortality (Table 4), possibly due to a very high level of transgene expression (more than 10-fold higher than in other lines, data not shown), or as a result of effects due to the position of transgene integration. This sub-line was not considered for further analysis.

[0179] Supporting the our initial observations, all five analysed transgenic lines showed significant increases in the number of spikes, vegetative biomass and grain weight per plant compared to control plants (Figure 7). Under well-watered conditions the transgenic T₂ plants yielded up to 30% more mass of grain per plant (Figure 6). There was a slight reduction in single grain weight for four of the lines. Thus, the increased grain yield was attributable to an increased number of spikes per plant (Figure 7). No yield advantage was observed for transgenics compared to control plants under mild drought. [0180] Our preliminary observations of plant performance in nutrient-depleted media indicated that NF-Y factors may impart the greatest yield advantage in poor soils. Thus, a third growth experiment was conducted using TaNF-YB4 homozygous transgenic lines (T₃), to investigate this hypothesis. Plants were grown in a coco-peat soil with either no addition or with four different rates of a slow-release complete fertiliser (Osmocote Exact Mini, Everris International B.V. The Netherlands; see Table 5 below). Increases in grain yield of the transgenic plants compared to control plants were evident across all fertiliser treatments (Figure 7; Table 5). However, there was also an interaction between fertiliser rate and the yield advantage of transgenic lines. Control plants showed a 25% increase in yield in response to fertiliser addition. By contrast, no positive effect of additional fertiliser on the grain yield of transgenic lines was observed (Figure 7).

TABLE 5

Influence of slow release fertiliser on plant yield

Additional Osmocote (g / L of soil)

Line

0 0.7 1 .4 2.1 2.8

Spike number per plant

WT^* 5.7 ± 0.7 6.1 ± 0.8 6.3 ± 0.7 6.5 ± 0.5 6.9 ± 0.4

L4-4-51 9.2 ± 0.5 10.7 ± 0.4 10.3 ± 1 .2 1 1 .0 ± 0.7 10.7 ± 0.5

L5-4-52 9.5 ± 0.9 9.7 ± 1 .5 9.7 ± 0.4 10.3 ± 1 .1 9.7 ± 1 .0

L6-3-31 9.0 ± 1 .0 10.0 ± 1 .4 1 1 .0 ± 0.9 1 1 .0 ± 0.7 1 1 .0 ± 1 .2

Seed number per plant

WT^* 162.3 ± 14.6 169.0 ± 31 .3 189.7 ± 20.2 206.3 ± 26.0 215.7 ± 32.2

L4-4-51 301 .3 ± 12.3 291 .3 ± 1 1 .7 340.7 ± 28.0 341 .0 ± 13.7 336.3 ± 30.1

L5-4-52 339.8 ± 24.0 286.7 ± 32.3 269.3 ± 30.6 298.0 ± 33.4 333.3 ± 36.6

L6-3-31 290.7 ± 12.3 287.7 ± 21 .8 332.7 ± 29.5 324.3 ± 28.8 348.0 ± 35.8

Seed weight per plant (g)

WT^* 4.33 ± 0.34 4.29 ± 0.64 4.70 ± 0.49 5.53 ± 0.65 5.83 ± 0.64

L4-4-51 8.69 ± 0.31 8.28 ± 0.62 8.61 ± 0.70 8.28 ± 0.21 7.73 ± 0.45

L5-4-52 8.57 ± 0.48 8.02 ± 0.81 7.88 ± 0.87 7.76 ± 0.35 7.65 ± 1 .10

L6-3-31 7.85 ± 0.38 7.82 ± 0.88 8.29 ± 0.62 8.29 ± 0.88 8.22 ± 0.75

Influence of different amounts of the slow release fertiliser Osmocote Exact Mini on yield components of WT (wheat cv. Gladius) and three homozygous TaNF-YB4 transgenic lines. Data presented as means (n = 3) ± standard error. WT plants (^*) were statistically different from each of three lines at least for p<0.05 for each treatment and each trait.

[0181] Figure 8 shoes the large container systems used for plant growth in the aforementioned experiments. Discussion

ZmNF-YB2, TaNF-YB2 and TaNF-YB4 specifically bind to Ta NF-YC 15

[0182] Plant genomes contain more than ten genes for each of three subunits of NF-Y proteins (Riechmann et al., 2000, Science 290: 2105-21 10), making it difficult to identify the specific subunits that may come together to form hetero-trimeric complexes. However, Y2H screening methods such as employed in this study allow the direct identification of interacting proteins from libraries of expressed genes. We selected ZmNF-YB2a to use as starting bait in sequential Y2H screens, to find related wheat NF-Y subunits and other proteins. Aiming to identify related genes possibly implicated in drought stress tolerance in wheat, we screened Y2H cDNA libraries prepared from different tissues of the Australian drought-tolerant wheat cultivar RAC875, including tissues from plants subjected to drought or to combined drought and heat stresses.

[0183] In the first round of the Y2H screening we identified a novel wheat protein, TaNF- YC15, encoded by DNA inserts of most clones isolated with ZmNF-YB2a as bait. We then used a truncated N-terminal version of TaNF-YC15, containing the full length HFD that is important for protein-protein interactions (Figures 3B and 3C), to screen for interacting proteins from the same wheat tissue libraries. In these Y2H screens we found only two types of NF-YB subunits, TaNF-YB2 and TaNF-YB4, full length cDNAs of which were identified in the majority of isolated independent clones. Both proteins had high levels of sequence identities (83.6% for TaNF-YB2 and 74.9% for TaNF-YB4) to the starting bait ZmNF-YB2a.

[0184] It was previously reported that most NF-YB subunits of Arabidopsis can interact in Y2H assays either with the majority of or all NF-YC subunits, suggesting a low level of selectivity between NF-YB and NF-YC (Calvenzani et al., 2012, PloS One 7: e42902; Hackenberg et al., 2012, Molecular Plant 5: 876-888). In this study, however, we observed significant selectivity in recognition of TaNF-YC15 by wheat NF-YB subunits. At least three explanations can be proposed: (i) high enrichment with particular NF-Y cDNAs in particular cDNA libraries, e.g. up-regulation of both isolated NF-YB genes by drought (Figure 5B); (ii) high specificity of recognition between TaNF-YC15 and TaNF-YB2 or TaNF-YB4, as a result of differences in protein sequences that guide protein-protein interactions; or (iii) differences in post-translational modifications of NF-YB subunits. Identification of the same NF-YB genes in several different cDNA libraries and isolation of the same NF-YC subunit in reciprocal screens using TaNF-YB2 and TaNF-YB4 proteins as bait (data not shown), suggested that specificity of the NF-YB/NF-YC complex formation could be guided by a protein sequence rather than by abundance of cDNAs in libraries. We were able to isolate a number of other TaNF-YB gene family members from our cDNA libraries (data not shown), but these were not identified with TaNF-YC15 in Y2H screens.

[0185] Aiming to identify possible determinants of specificity in interactions between various NF-YBs and TaNF-YC15, we mapped a TaNF-YB2 segment that may be responsible for the interaction with TaNF-YC15 (Figure 1 C). A minimal size segment of TaNF-YB2, which preserves a strong ability to bind TaNF-YC15, started at residue 50 (inside the N-terminal end of the HFD) and was finished at residue 146 (downstream of the C-terminal end of the HFD). Further truncations from either side led to disruption of protein-protein interactions, either because of the deletion of key residues responsible for these interaction and/or disruption of HFD folds (Figure 3). The minimal interacting segment of TaNF-YB2 was compared with similar segments of TaNF-YB4 and other known wheat NF-YB subunits (Figure 3A). It was found that similarly to Gln90 of human NF-YB, Gln1 19 of TaNF-YB2 and respective Gin of TaNF-YB4 were situated in a close proximity to Arg93, which was involved in TaNF-YB2/ TaNF-YC15 complex formation (Figure 3C). In all other wheat NF-YB sequences this Gin residue was substituted by a His residue. Since His imidazole side chains play critical roles in electrostatic interactions of proteins and thus in proteins' stability and function, the His to Gin substitution in TaNF- YB2 and TaNF-YB4 may directly influence the strength of interaction with TaNF-YC15 (Hansen and Kay, 2014, Proceedings of the National Academy of Sciences USA 1 1 1 : E1705-1712). We suggest that the His/Gin substitution may play a key role in the specificity of the interaction between TaNF-YC15 and TaNF-YB2 or TaNF-YB4.

Expression of TaNF-YB2, TaNF-YB4 and TaNF-YC15 is regulated in a similar way in most tested tissues and under drought stress, but differs under rapid dehydration

[0186] Analysis of expression levels of TaNF-YB2, TaNF-YB4 and TaNF-YC15 revealed that all three genes are expressed in all tested tissues, indicating that their products are in close physical proximity to each other for the formation of protein complexes. However, significant differences in levels of TaNF-YB2 and TaNF-YB4 expression in particular tissues (e.g. mature endosperm) might suggest possible importance of some specific features of protein sequences for the formation of particular transcriptional complexes, and hence differences in function of TaNF-YB2 and TaNF-YB4. Our observations of NF- YB2/4 gene expression in all tested wheat tissues confirm the results of wheat NF-Y expression analysis reported by (Stephenson et al., 2007, supra). Furthermore, the presence of transcripts of other NF-YB and NF-YC subunits in the same tissues (Stephenson et al., 2007, supra) and detection of these in our cDNA libraries (data not shown), together with results of our Y2H screens, advocate the existence of specific interactions between particular NF-YB and NF-YC subunits.

[0187] All three isolated NF-Y genes were up-regulated by drought. In contrast to the stress-inducible control gene TaCor39 (Guo et al., 1992, Plant Physiology 100: 915-922; Kovalchuk et al., 2013, supra), which is progressively induced by slowly developing drought, both TaNF-YB and TaNF-YC15 genes were activated at two points, initially when plants began to sense insufficiency of water, and at a second time at wilting point, when water deficit became severe (Figure 5). Maximal levels of TaNF-YB2 activation in our experiments were considerably lower than the level of activation of the same gene by dehydration reported by Stephenson et al., 2007, supra. This may be explained by different stress conditions used in these experiments, and possibly an insufficient number of data points in our experiment for detecting the peak time of expression. By comparison, TaNF-YB2 transcript levels were increased by three-fold in detached leaves subjected to rapid dehydration, a similar level of induction to the stress-inducible reference gene TaCOR39 (Figure 5). TaNF-YB4 and TaNF-YC15 showed very similar patterns of expression under dehydration. Both genes were initially down-regulated, then after two hours of leaf dehydration they partially returned to initial levels of expression and later again decreased by as much as 2.5-fold. These results suggest three interesting conclusions: (i) TaNF-YB2 and TaNF-YB4 may be induced by different components of drought stress, in the case of TaNF-YB2 by dehydration, and therefore may play different roles in drought stress responses; (ii) Significant differences in levels of expression of TaNF-YB2 and TaNF-YB4 in mature endosperm may be a result of TaNF-YB2 activation by natural desiccation of grain at this stage of development; (iii) TaNF-YB4 and TaNF- YC15 are more likely to be constituents of the same NF-Y protein complex than TaNF- YB2 and TaNF-YC15.

[0188] The activation of NF-Y genes by drought was ABA independent, because no significant changes in expression of all three genes were induced by treatment with 200 μΜ ABA. Constitutive over-expression of TaNF-YB4 improves performance of transgenic wheat

[0189] While constitutive over-expression of stress-related TFs generally confers improved plant reactions to corresponding stresses, in many cases it does not increase yield (seed weight per plant) (Hsieh et al., 2002, Plant Physiology 129: 1086-1094; Kobayashi et al., 2008, Transgenic Research 17: 755-767; Kobayashi et al., 2008, Journal of Experimental Botany 59: 891 -905; Oh et al., 2007, Plant Biotechnology Journal 5: 646- 656; Oh et al., 2005, Plant Physiology 138: 341 -351 ; Takumi et al., 2008, Plant Physiology and Biochemistry: PPB / Societe Francaise de Pysiologie Vegetale 46: 205-21 1 ). On the contrary, strong constitutive over-expression of TFs often results in development of undesirable pleiotropic phenotypes, and consequently reduces grain yields (Hsieh et al., 2002, supra; Ito et al., 2006, Plant & Cell Physiology 47: 141-153; Jaglo-Ottosen et al., 1998, Science 280: 104-106; Kasuga et al., 1999, Nature Biotechnology 17: 287-291 ; Kovalchuk et al., 2013, supra; Liu and Zhu, 1998, Science 280: 1943-1945; Morran et al., 201 1 , supra). Reports of the over-expression of some NF-Y subunits in maize and rice, however, are promising exceptions to this rule (Nelson et al., 2007, supra; Wei et al., 2010, Plant Physiology 153: 1747-1758). The main purpose of this study was to identify and clone wheat NF-Y gene(s) responsible for yield increase and explore the possibility to improve performance of an elite wheat cultivar by over-expression of such gene(s).

[0190] Preliminary phenotyping revealed no negative influence of the constitutively expressed TaNF-YB4 transgene on plant phenotype and yield for three independent transgenic lines showing moderate transgene expression. By contrast, Line 3 showed slower growth, lower grain number and a 10% lower grain yield compared to control plants. Levels of transgene expression in this line were around 10-fold higher than in the other lines, suggesting that limiting transgene expression to more moderate levels may be critical for avoiding deleterious phenotypes.

[0191] A replicated experiment was conducted in large containers in which soil moisture could be controlled, to simulate seasonal drought stress for one of the treatments. In this experiment, spike number, biomass and grain yield of transgenic plants were significantly higher than for wild type plants under well-watered conditions. Yield increase was observed for all five analysed sub-lines (three independent events), of up to 20-30% (Figure 6). Yield increase in crop plants as a result of overexpression of NF-YB genes has been reported previously (Nelson et al., 2007, supra; Wei et al., 2010, supra). In the first report, transgenic maize plants with enhanced ZmNF-YB2 expression demonstrated increased tolerance to drought based on improvement of parameters such as chlorophyll content, stomatal conductance, leaf temperature, reduced wilting, and maintenance of photosynthesis. These stress adaptations contributed to a grain yield advantage for transgenic maize lines under water-limited conditions (Nelson et a/., 2007, supra). Although TaNF-YB4 is closely related to ZmNF-YB2, under limited water conditions we did not observe either reduced wilting or increased grain yield in transgenic wheat plants compared to untransformed control plants. This may be explained by differences in protein sequences between ZmNF-YB2/TaNF-YB2 and TaNF-YB4, leading to formation of different complexes with other transcription factors and/or different roles of these proteins. The differences in expression patterns of TaNF-YB2 and TaNF-YB4 under dehydration stress also suggest diverse roles of their products under stress and during grain desiccation. We plan to investigate the drought stress adaptation of transgenic wheat with constitutive over-expression of TaNF-YB2.

[0192] In another study, a rice HAP3H {NF-YB) gene, DTH8 (QTL for days to heading on chromosome 8), was overexpressed in rice (Wei et a/., 2010, supra). It was shown that DTH8 suppresses rice flowering under long day conditions and plays an important role in the regulation of plant height and yield potential. Although the product of DTH8 gene belongs to another clade of NF-YB TFs (Figure 2A), the phenotype described for DTH8 transgenic rice plants to some extent resembles the phenotype of TaNF-YB4 transgenic wheat plants. Both transgenic events led to increased biomass production. TaNF-YB4 transgenic wheat plants in T₀ and T generations, similarly to DTH8 rice plants, were slightly higher than control plants, although this difference in height was not so pronounced as in DTH8 transgenic rice, and was not observed in T₂ and subsequent generations. There are also some differences in development between TaNF-YB4 wheat lines and DTH8 transgenic rice lines. DTH8 delayed flowering of transgenic rice by negatively influencing the expression of Ehd1 and Hd3a genes under long-day conditions.

[0193] No significant differences in flowering time of transgenic TaNF-YB4 and control wheat plants, or changes in the dependence of flowering time on day length, were observed (data not shown). The TaNF-YB4 gene in transgenic wheat appears to influence tiller number rather than plant height. The increased number of tillers is responsible for higher biomass and a higher number of grains per plant. Both transgenic events produce more grain per plant with little or no loss in grain weight, and therefore numbers of grain per plant was a crucial yield component responsible for yield improvement in both cases. [0194] Under water limited conditions TaNF-YB4 over-expressing wheat lines maintained parity in yield performance, but did not increase yield under drought conditions (Figure 6). One of the reasons for this may be due to higher demands of the transgenic lines for water as a consequence of larger numbers of tillers and greater vegetative biomass. This would reduce drought tolerance and yield under limiting water conditions. However, the drought conditions applied in this experiment did not reduce grain yield of transgenic lines to levels below wild type. Further experiments both in controlled conditions and multi- environment field trials are planned to more thoroughly investigate the effects of drought on TaNF-B4 over-expressing wheat.

[0195] The yield improvement observed for TaNF-YB4 transgenic lines under sufficient watering may have been a consequence of an improved ability of the transgenic plants to grow on nutritionally poor soil better than control plants. To investigate this hypothesis, an experiment was conducted where transgenic and control plants were grown in soil with different concentrations of a complete, slow-release fertilizer. Elevated concentrations of fertilizer significantly increased the yield of wild type plants, although yields still remained lower than those of the transgenic lines. By contrast, the yield of transgenic plants was not boosted by fertilizer, and in fact the highest concentration of fertilizer used in the experiment negatively influenced single grain weight (Figure 7). Further research with these transgenic lines is planned to investigate the possible mechanisms by which TaNF- YB4 over-expressing wheat plants overcome nutrient deficiency in soil.

Claims

1. A method for modulating biomass of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

2. The method of claim 1 , wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

3. The method of claim 1 or claim 2, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

4. The method of any one of claims 1 to 3, wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

5. The method of any one of claims 1 to 4, further comprising the step of identifying the plant as a plant with modulated biomass relative to a wild-type form of the plant, thereby confirming modulation of the biomass of the plant.

6. The method of any one of claims 1 to 5, wherein increasing expression of the NF- YB4 polypeptide increases biomass of the plant relative to a wild-type form of the plant.

7. The method of any one of claims 1 to 6, wherein expression of the NF-YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide.

8. The method of claim 7, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

9. The method of claim 7 or claim 8, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

10. The method of any one of claims 7 to 9, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

1 1 . The method of any one of claims 7 to 10, wherein increasing expression of the nucleic acid increases biomass of the plant relative to a wild-type form of the plant.

12. The method of claim 6 or claim 1 1 , wherein yield of the plant is increased relative to a wild-type form of the plant.

13. The method of claim 12, wherein the yield is seed yield.

14. The method of any one of claims 1 1 to 13, wherein the number of tillers of the plant is increased relative to a wild-type form of the plant.

15. The method of any one of claims 1 1 to 14, wherein the plant is a spike-bearing plant and the number of spikes is increased relative to a wild-type form of the plant.

16. The method of any one of claims 1 to 15, wherein the plant is grown under non- drought conditions.

17. The method of any one of claims 1 to 16, wherein the plant is grown under nutrient-poor conditions.

18. The method of any one of claims 1 to 17, wherein the plant is a monocotyledonous plant, including a cereal crop plant.

19. The method of claim 18, wherein the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

20. A method for modulating yield of a plant, the method including the step of modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

21 . The method of claim 20, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

22. The method of claim 20 or claim 21 , wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

23. The method of any one of claims 20 to 22, wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

24. The method of any one of claims 20 to 23, further comprising the step of identifying the plant as a plant with modulated yield relative to a wild-type form of the plant, thereby confirming modulation of the yield of the plant.

25. The method of any one of claims 20 to 24, wherein increasing expression of the NF-YB4 polypeptide increases yield of the plant relative to a wild-type form of the plant.

26. The method of any one of claims 20 to 25, wherein expression of the NF-YB4 polypeptide is modulated by modulating expression, in the one or more cells of the plant, of a nucleic acid which encodes the NF-YB4 polypeptide.

27. The method of claim 26, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

28. The method of claim 26 or claim 27, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

29. The method of any one of claims 26 to 28, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

30. The method of any one of claims 26 to 29, wherein increasing expression of the nucleic acid increases yield of the plant relative to a wild-type form of the plant.

31 . The method of claim 25 or claim 30, wherein the yield is seed yield.

32. The method of claim 30 or claim 31 , wherein the number of tillers of the plant is increased relative to a wild-type form of the plant.

33. The method of any one of claims 30 to 32, wherein the plant is a spike-bearing plant and the number of spikes is increased relative to a wild-type form of the plant.

34. The method of any one of claims 20 to 33, wherein the plant is grown under non- drought conditions.

35. The method of any one of claims 20 to 34, wherein the plant is grown under nutrient-poor conditions.

36. The method of any one of claims 20 to 35, wherein the plant is a monocotyledonous plant, including a cereal crop plant.

37. The method of claim 36, wherein the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

38. A method for modulating biomass of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

39. The method of claim 38, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

40. The method of claim 38 or claim 39, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

41 . The method of any one of claims 38 to 40, wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

42. The method of any one of claims 38 to 41 , further comprising the step of identifying the multicellular structure as a multicellular structure with modulated biomass relative to a wild-type form of the multicellular structure, thereby confirming modulation of the biomass of the multicellular structure.

43. The method of any one of claims 38 to 42, wherein increasing expression of the NF-YB4 polypeptide increases biomass of the multicellular structure relative to a wild-type form of the multicellular structure.

44. The method of any one of claims 38 to 43, wherein expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more plant cells of the multicellular structure, a nucleic acid which encodes the NF-YB4 polypeptide.

45. The method of claim 44, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

46. The method of claim 44 or claim 45, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

47. The method of any one of claims 44 to 46, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

48. The method of any one of claims 44 to 47, wherein increasing expression of the nucleic acid increases biomass of the multicellular structure relative to a wild-type form of the multicellular structure.

49. The method of claim 43 or claim 48, wherein yield of the multicellular structure is increased relative to a wild-type form of the multicellular structure.

50. The method of claim 49, wherein yield is seed yield.

51 . The method of any one of claims 48 to 50, wherein the number of tillers of the multicellular structure is increased relative to a wild-type form of the multicellular structure.

52. The method of any one of claims 48 to 51 , wherein the multicellular structure is a spike-bearing multicellular structure and the number of spikes is increased.

53. The method of any one of claims 38 to 52, wherein the multicellular structure is grown under non-drought conditions.

54. The method of any one of claims 38 to 53, wherein the multicellular structure is grown under nutrient-poor conditions.

55. The method of any one of claims 38 to 54, wherein the multicellular structure is a whole plant, plant tissue, a plant organ, a plant part, plant reproductive material, or cultured plant tissue.

56. A method for modulating yield of a multicellular structure comprising a plurality of plant cells, the method including modulating expression of a NF-YB4 polypeptide in one or more of the plant cells of the multicellular structure.

57. The method of claim 56, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

58. The method of claim 56 or claim 57, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

59. The method of any one of claims 56 to 58, wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

60. The method of any one of claims 56 to 59, further comprising the step of identifying the multicellular structure as a multicellular structure with modulated yield relative to a wild-type form of the multicellular structure, thereby confirming modulation of the yield of the multicellular structure.

61 . The method of any one of claims 56 to 60, wherein increasing expression of the NF-YB4 polypeptide increases yield of the multicellular structure relative to a wild-type form of the multicellular structure.

62. The method of any one of claims 56 to 61 , wherein expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more plant cells of the multicellular structure, a nucleic acid which encodes the NF-YB4 polypeptide.

63. The method of claim 62, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

64. The method of claim 62 or claim 63, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

65. The method of any one of claims 62 to 64, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

66. The method of any one of claims 62 to 65, wherein increasing expression of the nucleic acid increases yield of the multicellular structure relative to a wild-type form of the multicellular structure.

67. The method of claim 61 or claim 66, wherein yield is seed yield.

68. The method of claim 66 or claim 67, wherein the number of tillers of the multicellular structure is increased relative to a wild-type form of the multicellular structure.

69. The method of any one of claims 66 to 68, wherein the multicellular structure is a spike-bearing multicellular structure and the number of spikes is increased relative to a wild-type form of the multicellular structure.

70. The method of any one of claims 56 to 69, wherein the multicellular structure is grown under non-drought conditions.

71 . The method of any one of claims 56 to 70, wherein the multicellular structure is grown under nutrient-poor conditions.

72. The method of any one of claims 56 to 71 , wherein the multicellular structure is a whole plant, plant tissue, a plant organ, a plant part, plant reproductive material, or cultured plant tissue.

73. A genetically modified plant which has modulated biomass, wherein modulation of the biomass is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

74. The genetically modified plant of claim 73, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

75. The genetically modified plant of claim 73 or claim 74, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

76. The method of any one of claims 73 to 75, wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

77. The genetically modified plant of any one of claims 73 to 76, wherein the plant comprises an increased expression of the NF-YB4 polypeptide and an increased biomass relative to a wild-type form of the plant.

78. The genetically modified plant of any one of claims 73 to 77, wherein expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more cells of the plant, a nucleic acid which encodes the NF-YB4 polypeptide.

79. The genetically modified plant of claim 78, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

80. The genetically modified plant of claim 78 or claim 79, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

81 . The genetically modified plant of any one of claims 78 to 80, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

82. The genetically modified plant of any one of claims 78 to 81 , wherein the plant comprises an increased expression of the nucleic acid and an increased biomass relative to a wild-type form of the plant.

83. The genetically modified plant of claim 77 or claim 82, wherein the plant has an increased yield relative to a wild-type form of the plant.

84. The genetically modified plant of claim 83, wherein the yield is seed yield.

85. The genetically modified plant of any one of claims 82 to 84, wherein the plant has an increased number of tillers relative to a wild-type form of the plant.

86. The genetically modified plant of any one of claims 82 to 85, wherein the plant is a spike-bearing plant and the plant has an increased number of spikes relative to a wild- type form of the plant.

87. The genetically modified plant of any one of claims 73 to 86, wherein the plant is a monocotyledonous plant, including a cereal crop plant.

88. The genetically modified plant of claim 87, wherein the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

89. A genetically modified plant which has modulated yield, wherein modulation of the yield is effected by modulating expression of a NF-YB4 polypeptide in one or more cells of the plant.

90. The genetically modified plant of claim 89, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 1 , or comprises an amino acid sequence which is at least 50% identical thereto.

91 . The genetically modified plant of claim 89 or claim 90, wherein the NF-YB4 polypeptide comprises the amino acid sequence set forth in SEQ ID NO: 2, or comprises an amino acid sequence which is at least 50% identical thereto.

92. The genetically modified plant of any one of claims 89 to 91 , wherein the NF-YB4 polypeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2.

93. The genetically modified plant of any one of claims 89 to 92, wherein the plant comprises an increased expression of the NF-YB4 polypeptide and an increased yield relative to a wild-type form of the plant.

94. The genetically modified plant of any one of claims 89 to 93, wherein expression of the NF-YB4 polypeptide is modulated by modulating expression of, in the one or more cells of the plant, a nucleic acid which encodes the NF-YB4 polypeptide.

95. The genetically modified plant of claim 94, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 3, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 3.

96. The genetically modified plant of claim 94 or claim 95, wherein the nucleic acid comprises a nucleotide sequence set forth in SEQ ID NO: 4, or comprises a nucleotide sequence at least 50% identical to SEQ ID NO: 4.

97. The genetically modified plant of any one of claims 94 to 96, wherein the nucleic acid consists of the nucleotide sequence set forth in SEQ ID NO: 3 or SEQ ID NO: 4.

98. The genetically modified plant of any one of claims 94 to 97, wherein the plant comprises an increased expression of the nucleic acid and an increased yield relative to a wild-type form of the plant.

99. The genetically modified plant of claim 93 or claim 98, wherein the yield is seed yield.

100. The genetically modified plant of claim 98 or claim 99, wherein the plant has an increased number of tillers relative to a wild-type form of the plant.

101. The genetically modified plant of any one of claims 98 to 100, wherein the plant is a spike-bearing plant and the plant has an increased number of spikes relative to a wild- type form of the plant.

102. The genetically modified plant of any one of claims 89 to 101 , wherein the plant is a monocotyledonous plant, including a cereal crop plant.

103. The genetically modified plant of claim 102, wherein the plant is a wheat, rice, barley, maize, rye, millet, sorghum, triticale, oat, teff, wild rice, spelt, buckwheat, turf grass, Italian rye grass, switchgrass, Miscanthus, or Festuca plant.

104. A method for modulating an agronomic characteristic of a plant, the method including the step of modulating expression of a truncated NF-YB4 polypeptide in one or more cells of the plant.

105. The method of claim 104, wherein the NF-YB4 polypeptide comprises an amino acid sequence of SEQ ID NO: 1 or a sequence that is at least about 70% to about 95% identical to SEQ ID NO: 1.

106. The method of claim 104 or claim 105, wherein the plant is selected from the group consisting of maize, wheat, rice, sorghum, soybean, canola, sorghum, and sugarcane.

107. The method of any one of claims 104 to 106, wherein the agronomic characteristic of the plant is selected from the group consisting of increased biomass, abiotic stress tolerance, increased seed yield, increased grain filling, and improved nutrient uptake efficiency.