# Patent

## Title
Material and methods to increase plant growth and yield

## Abstract

The present invention relates to materials and methods for modulating growth rates, yield, and/or resistance to drought conditions in plants. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene or expression or activity of the protein encoded by an hc1 gene results in increased growth rate, yield, and/or drought resistance in the plant.

## Background
REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. provisional application No. 61/751,648, filed Jan. 11, 2013, and is herein incorporated by reference in its entirety.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under grant number DE-FG02-05ER64114 awarded by the U.S. Department of Energy. The government has certain rights in the invention.


INCORPORATION OF SEQUENCE LISTING
The sequence listing that is contained in the file named “UFFL042US_ST25.txt,” which is 3 kilobytes as measured in Microsoft Windows operating system and was created on Jan. 4, 2014, is filed electronically herewith and incorporated herein by reference.
FIELD OF THE INVENTION
The invention relates generally to the field of molecular biology and genetics. More particularly, the invention relates to methods of increasing growth and yield of plants.
BACKGROUND OF THE INVENTION
Water deficit and drought are the main factors that limit crop production and productivity, and are a major threat to food security worldwide. Plant varieties can be bred to be more productive per unit of water supplied, i.e., higher water-use efficiency (WUE). However, our lack of knowledge of the genetic mechanisms underlying WUE has hindered the improvement of this trait. Water-use efficiency may be impacted by a number of factors, including stomatal conductance, which is partially regulated by hydraulic conductivity. Hydraulic conductivity increases rapidly with greater xylem vessel diameter because flow is proportional to the fourth power of conduit diameter. Genes that regulate meristematic cell differentiation into vessels were unknown until recently, but their manipulation could increase hydraulic conductivity and, consequently, the photosynthetic rate and plant productivity.
BRIEF SUMMARY OF THE INVENTION
The subject invention concerns materials and methods for modulating growth rates, yield, and/or drought resistance in plants. In one embodiment, a method of the invention comprises upregulating expression of a hydraulic conductivity 1 (hc1) gene (or a homolog thereof that provides for substantially the same activity), or increasing expression or activity of the protein encoded by an hc1 gene thereof, in a plant, wherein increased expression of the hc1 gene or increased expression or activity of the protein encoded by an hc1 gene results in increased growth in the plant. In one embodiment, the hc1 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity. In a specific embodiment, the hc1 gene comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In one embodiment, a plant is transformed with a polynucleotide encoding an Hc1 protein, or a fragment or variant thereof having substantially the same activity, wherein the Hc1 protein is expressed in the plant.
In another embodiment, a method of the invention provides for increased expression of an hc1 gene of the invention (or a homolog thereof that provides for substantially the same activity), or increased expression or activity of a protein encoded by the hc1 gene (or a homolog thereof). In one embodiment, multiple copies of an hc1 gene of the invention, or a protein encoding portion thereof, are incorporated in a plant. In one embodiment, the hc1 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity. In a specific embodiment, the hc1 gene comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1—Shows a linear regression plot demonstrating the positive association (r2=0.4164, P=0.0012) between leaf-specific hydraulic conductivity (KL) and diameter increment (DINC). Points are progeny genotype means.
FIG. 2—Shows a linear regression plot demonstrating the positive association (r2=0.7196, P<0.0001) between hydraulic vessel diameter (Dh) and diameter increment (DINC). Points are progeny genotype means.
FIG. 3—Shows a graph depicting up to a 90-fold increase in expression of hc1 in comparison to wildtype (level of expression=1) using Delta Delta CT analysis. Actin was used as the control gene.
FIG. 4—Shows growth comparisons demonstrating visibly higher growth of transgenic lines overexpressing hc1 compared to wildtype.
FIG. 5—Shows light microscopy images of xylem cross-sections demonstrating higher vessel number and diameter in a transgenic line overexpressing hc1 (panel A) compared to wildtype (panel B).
FIG. 6—Shows bar graphs demonstrating vessel mean diameter (panel A) and number (panel B) in a transgenic line overexpressing POPTR—0001s33660, compared to wildtype.



BRIEF DESCRIPTION OF THE SEQUENCES
SEQ ID NO:1—Nucleotide sequence of the coding region of an hc1 gene that encodes the amino acid sequence of SEQ ID NO:2.
SEQ ID NO:2—Amino acid sequence of a protein encoded by an hc1 gene having the nucleotide sequence of SEQ ID NO:1.
SEQ ID NO:3—Genomic nucleotide sequence that comprises the coding region of an hc1 gene that encodes the amino acid sequence of SEQ ID NO:2.
DETAILED DESCRIPTION OF THE INVENTION
The subject invention concerns materials and methods for modulating plant biomass and yield. In one aspect, the invention concerns materials and methods for increasing growth rates and/or biomass in plants. The subject invention also provides for improved resistance to drought conditions in a plant. In one embodiment, a method of the invention comprises increasing expression of an hc1 gene (or a homolog thereof that provides for substantially the same activity), or the protein encoded by an hc1 gene thereof, in a plant, wherein expression of the hc1 gene results in increased biomass levels in the plant (relative to biomass levels of a plant having lower levels of hc1 expression). In one embodiment, the hc1 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity as a full-length sequence. In a specific embodiment, the hc1 gene comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In one embodiment, a plant is transformed with a polynucleotide encoding an Hc1 protein, or a fragment or variant thereof having substantially the same activity, wherein the Hc1 protein is expressed in the plant.
In another embodiment, a method of the invention provides for increased expression of an hc1 gene of the invention (or a homolog thereof that provides for substantially the same activity), or a protein encoding portion thereof. In one embodiment, multiple copies of an hc1 gene of the invention, or a protein encoding portion thereof, are incorporated in a plant. In one embodiment, the hc1 gene encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity as a full-length sequence. In a specific embodiment, the hc1 gene comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
In one embodiment, a heterologous hc1-encoding polynucleotide is incorporated into a plant and the polynucleotide expressed therein. In one embodiment, the Hc1 protein encoded by the polynucleotide comprises the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity as a full-length sequence. In a further embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. The polynucleotide can comprise regulatory elements such as promoters, etc. that provide for increased expression of the hc1 in the plant.
In another embodiment, a method of the invention comprises introducing a polynucleotide into a plant wherein the polynucleotide, or the expression product thereof, provides for increased expression of an hc1 gene or protein relative to a plant wherein the polynucleotide has not been introduced (e.g., a wildtype plant). In another embodiment, a polynucleotide can be introduced that encodes an Hc1 protein that exhibits increased activity. In a further embodiment, a polynucleotide can be introduced that encodes a protein having Hc1 activity, wherein the polynucleotide comprises regulatory elements that provide for increased expression of the polynucleotide and/or the protein encoded thereby. Plants containing the polynucleotide, or progeny thereof, optionally can be screened for increased expression of the hc1 gene and/or protein, or increased activity of the protein.
The subject invention also concerns isolated polynucleotides encoding the gene product of an hc1 gene of Populus, or a homolog thereof having substantially the same activity. In one embodiment, the polynucleotide encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity. In a specific embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3.
The subject invention also concerns plants, plant tissue, and plant cells of the invention that exhibit increased expression of an hc1 gene (or homolog thereof) or the protein encoded thereby. In one embodiment, the plant, plant tissue, or plant cell is a woody tree. Plants contemplated within the scope of the present invention include, but are not limited to, plants of the genus Abies, Acacia, Acer, Aesculus, Ailanthus, Alnus, Amelanchier, Arbutus, Arctostaphylos, Artemisia, Asiminia, Atriplex, Aucuba, Berberis, Betula, Buddleia, Buxus, Calocedrus, Camellia, Campsis, Carpinus, Carya, Castanea, Catalpa, Ceanothus, Cedrus, Celastrus, Celtis, Cephalanthus, Cercidium, Cercis, Chaenomeles, Chamaecyparis, Chilopsis, Chionanthus, Chrysothamnus, Cistus, Cladrastis, Clematis, Coleogynia, Cornus, Corylus, Cotinus, Cotoneaster, Cowania, Crataegus, Crataegus, Cupressus, Cytisus, Daphne, Deutzia, Diospyros, Elaeagnus, Ephedra, Escallonia, Eucalyptus, Euonymus, Fagus, Forsythia, Fraxinus, Gaultheria, Ginkgo, Gleditsia, Grevillea, Gymnocladus, Hamamelis, Hebe, Hibiscus, Hydrangea, Hypericum, Ilex, Juglans, Juniperus, Kalmia, Kerria, Koelreuteria, Lagerstroemia, Larix, Larrea, Libocedrus, Ligustrum, Liquidambar, Liriodendron, Lonicera, Maclura, Magnolia, Mahonia, Malus, Menispermum, Morus, Myrica, Nyssa, Osmanthus, Ostrya, Oxydendron, Parthenocissus, Philadelphus, Photinia, Physocarpus, Picea, Pinus, Pittosporum, Platanus, Populus, Prosopis, Prunus, Pseudotsuga, Ptelea, Purshia, Pyrus, Quercus, Rhamnus, Rhaphiolepis, Rhododendron, Rhus, Ribes, Robinia, Rosa, Rubus, Salix, Sambucus, Sassafras, Sequoia, Shepherdia, Smilax, Sophora, Sorbus, Spiraea, Staphylea, Stewartia, Symphoricarpos, Syringa, Taxodium, Taxus, Thuja, Tilia, Tsuga, Ulmus, Umbellularia, Vaccinium, Viburnum, Vitis, Zanthoxylum, and Zelkova. The plant, plant tissue, or plant cell of the invention can be a hybrid plant or from a hybrid plant. Plants of the invention can have increased growth rates, biomass, and/or improved resistance to drought conditions. In one embodiment, a plant of the invention exhibits increased expression of hc1. In one embodiment, a plant comprises one or more mutations introduced into an hc1 gene of a plant that results in increased transcription of the hc1 gene, or increased translation of hc1 mRNA, and/or that results in an Hc1 protein exhibiting increased activity or function. In one embodiment, a plant, plant tissue, or plant cell comprises a heterologous polynucleotide that encodes a protein comprising the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity. In a specific embodiment, the heterologous polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. In one embodiment, a plant, plant tissue, or plant cell of the invention is a transgenic plant, plant tissue, or plant cell that exhibits increased expression of hc1. In another embodiment, a plant, plant tissue, or plant cell of the invention is one that has been obtained through a breeding program.
Optionally, the plants of the present invention may also further exhibit one or more agronomic traits that primarily are of benefit to a seed company, a grower, or a grain processor, for example, herbicide resistance, virus resistance, bacterial pathogen resistance, insect resistance, nematode resistance, and fungal resistance. See, e.g., U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; and 6,337,431. Such a trait may also be one that increases plant vigor or yield (including traits that allow a plant to grow at different temperatures, soil conditions and levels of sunlight and precipitation), or one that allows identification of a plant exhibiting a trait of interest (e.g., selectable marker gene, seed coat color, etc.). Various traits of interest, as well as methods for introducing these traits into a plant, are described, for example, in U.S. Pat. Nos. 5,569,823; 5,304,730; 5,495,071; 6,329,504; 6,337,431; 5,767,366; 5,928,937; 4,761,373; 5,013,659; 4,975,374; 5,162,602; 4,940,835; 4,769,061; 5,554,798; 5,879,903, 5,276,268; 5,561,236; 4,810,648; and 6,084,155; in European application No. 0242246; in U.S. Patent Application Publication No. 2001/0016956; and on the worldwide web at lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/.
Polynucleotides useful in the present invention can be provided in an expression construct. Expression constructs of the invention generally include regulatory elements that are functional in the intended host cell in which the expression construct is to be expressed. Thus, a person of ordinary skill in the art can select regulatory elements for use in bacterial host cells, yeast host cells, plant host cells, insect host cells, mammalian host cells, and human host cells. Regulatory elements include promoters, transcription termination sequences, translation termination sequences, enhancers, and polyadenylation elements. As used herein, the term “expression construct” refers to a combination of nucleic acid sequences that provides for transcription of an operably linked nucleic acid sequence. As used herein, the term “operably linked” refers to a juxtaposition of the components described wherein the components are in a relationship that permits them to function in their intended manner. In general, operably linked components are in contiguous relation.
An expression construct of the invention can comprise a promoter sequence operably linked to a polynucleotide sequence encoding a polypeptide of the invention. Promoters can be incorporated into a polynucleotide using standard techniques known in the art. Multiple copies of promoters or multiple promoters can be used in an expression construct of the invention. In a preferred embodiment, a promoter can be positioned about the same distance from the transcription start site in the expression construct as it is from the transcription start site in its natural genetic environment. Some variation in this distance is permitted without substantial decrease in promoter activity. A transcription start site is typically included in the expression construct.
If the expression construct is to be provided in or introduced into a plant cell, then plant viral promoters, such as, for example, a cauliflower mosaic virus (CaMV) 35S (including the enhanced CaMV 35S promoter (see, for example U.S. Pat. No. 5,106,739)) or a CaMV 19S promoter or a cassava vein mosaic can be used. Other promoters that can be used for expression constructs in plants include, for example, prolifera promoter, Ap3 promoter, heat shock promoters, T-DNA 1′- or 2′-promoter of A. tumefaciens, polygalacturonase promoter, chalcone synthase A (CHS-A) promoter from petunia, tobacco PR-1a promoter, ubiquitin promoter, actin promoter, alcA gene promoter, pin2 promoter (Xu et al., 1993), maize WipI promoter, maize trpA gene promoter (U.S. Pat. No. 5,625,136), maize CDPK gene promoter, and RUBISCO SSU promoter (U.S. Pat. No. 5,034,322) can also be used. Tissue-specific promoters, for example xylem-specific promoters, such as the promoter of Cald5H, SAD, XCP1, CAD, CesA1, CesA2, CesA3, tubulin gene (TUB) promoter, lipid transfer protein gene (LTP) promoter, or coumarate-4-hydroxylase gene (C4H) promoter (see, for example, Lu et al., 2008; Funk et al., 2002; Sibout et al., 2005; published U.S. Application No. 2008/0196125) can be used. Leaf-specific promoters that can be used in a nucleic acid construct of the invention include Cabl promoter (Brusslan and Tobin, 1992), Cab19 promoter (Bassett et al., 2007), PPDK promoter (Matsuoka et al., 1993), and ribulose biphosphate carboxylase (RBCS) promoter (Matsuoka et al., 1994 and U.S. Pat. No. 7,723,575). Other plant leaf-specific promoters that can be used with an expression construct of the invention include, but are not limited to, the Act1 promoter (U.S. Published Application No. 2009/0031441), AS-1 promoter (U.S. Pat. No. 5,256,558), RBC-3A promoter (U.S. Pat. No. 5,023,179), the CaMV 35S promoter (Odell et al., 1985), the enhanced CaMV 35S promoter, the Figwort Mosaic Virus (FMV) promoter (Richins et al., 1987), the mannopine synthase (mas) promoter, the octopine synthase (ocs) promoter, or others such as the promoters from CaMV 19S (Lawton et al., 1987), nos (Ebert et al., 1987), Adh (Walker et al., 1987), sucrose synthase (Yang et al., 1990), α-tubulin, ubiquitin, actin (Wang et al., 1992), cab (Sullivan et al., 1989), PEPCase (Hudspeth et al., 1989), or those associated with the R gene complex (Chandler et al., 1989). See also published U.S. Application No. 2007/006346 and Yamamoto et al. (1997); Kwon et al. (1994); and Yamamoto et al. (1994). Other promoters that direct expression in the xylem of plants include the 4-coumarate Co-enzyme A ligase (4CL) promoter of Populus described in U.S. Pat. No. 6,831,208. Seed-specific promoters such as the promoter from a β-phaseolin gene (for example, of kidney bean) or a glycinin gene (for example, of soybean), and others, can also be used. Endosperm-specific promoters include, but are not limited to, MEG1 (EPO Application No. EP1528104) and those described by Wu et al. (1998), Furtado et al. (2001), and Hwang et al. (2002). Root-specific promoters, such as any of the promoter sequences described in U.S. Pat. Nos. 6,455,760 or 6,696,623, or in published U.S. Patent Application Nos. 2004/0078841; 2004/0067506; 2004/0019934; 2003/0177536; 2003/0084486; or 2004/0123349, can be used with an expression construct of the invention. Constitutive promoters (such as the CaMV, ubiquitin, actin, or NOS promoter), developmentally-regulated promoters, and inducible promoters (such as those promoters than can be induced by heat, light, hormones, or chemicals) are also contemplated for use with polynucleotide expression constructs of the invention.
Methods for identifying and characterizing promoter regions in plant genomic DNA are known in the art and include, for example, those described in the following references: Jordano et al. (1989); Bustos et al. (1989); Green et al. (1988); Meier et al. (1991); and Zhang et al. (1996). Published U.S. Application 2009/0199307 also describes methods for identifying tissue-specific promoters using differential display (see, e.g., U.S. Pat. No. 5,599,672). In differential display, mRNAs are compared from different tissue types. By identifying mRNA species which are present in only a particular tissue type, or set of tissue types, corresponding genes can be identified which are expressed in a tissue specific manner. RNA can be transcribed by reverse transcriptase to produce a cDNA, and the cDNA can be used to isolate clones containing the full-length genes. The cDNA can also be used to isolate homeologous or homologous promoters, enhancers or terminators from the respective gene using, for example, suppression PCR. See also U.S. Pat. No. 5,723,763.
Expression constructs of the invention may optionally contain a transcription termination sequence, a translation termination sequence, a sequence encoding a signal peptide, and/or enhancer elements. Transcription termination regions can typically be obtained from the 3′ untranslated region of a eukaryotic or viral gene sequence. Transcription termination sequences can be positioned downstream of a coding sequence to provide for efficient termination. A signal peptide sequence is a short amino acid sequence typically present at the amino terminus of a protein that is responsible for the relocation of an operably linked mature polypeptide to a wide range of post-translational cellular destinations, ranging from a specific organelle compartment to sites of protein action and the extracellular environment. Targeting gene products to an intended cellular and/or extracellular destination through the use of an operably linked signal peptide sequence is contemplated for use with the polypeptides of the invention. Classical enhancers are cis-acting elements that increase gene transcription and can also be included in the expression construct. Classical enhancer elements are known in the art, and include, but are not limited to, the CaMV 35S enhancer element, cytomegalovirus (CMV) early promoter enhancer element, and the SV40 enhancer element. Intron-mediated enhancer elements that enhance gene expression are also known in the art. These elements must be present within the transcribed region and are orientation dependent. Examples include the maize shrunken-1 enhancer element (Clancy and Hannah, 2002).
DNA sequences which direct polyadenylation of mRNA transcribed from the expression construct can also be included in the expression construct, and include, but are not limited to, an octopine synthase or nopaline synthase signal. The expression constructs of the invention can also include a polynucleotide sequence that directs transposition of other genes, i.e., a transposon.
Polynucleotides of the present invention can be composed of either RNA or DNA. Preferably, the polynucleotides are composed of DNA. The subject invention also encompasses those polynucleotides that are complementary in sequence to the polynucleotides disclosed herein. Polynucleotides and polypeptides of the invention can be provided in purified or isolated form.
Because of the degeneracy of the genetic code, a variety of different polynucleotide sequences can encode polypeptides of the present invention. A table showing all possible triplet codons (and where U also stands for T) and the amino acid encoded by each codon is described in Lewin (1985). In addition, it is well within the skill of a person trained in the art to create alternative polynucleotide sequences encoding the same, or essentially the same, polypeptides of the subject invention. These variant or alternative polynucleotide sequences are within the scope of the subject invention. As used herein, references to “essentially the same” sequence refers to sequences which encode amino acid substitutions, deletions, additions, or insertions which do not materially alter the functional activity of the polypeptide encoded by the polynucleotides of the present invention. Allelic variants of the nucleotide sequences encoding an Hc1 protein of the invention are also encompassed within the scope of the invention.
Substitution of amino acids other than those specifically exemplified or naturally present in a polypeptide of the invention are also contemplated within the scope of the present invention. For example, non-natural amino acids can be substituted for the amino acids of an Hc1 polypeptide, so long as the polypeptide having the substituted amino acids retains substantially the same functional activity as the polypeptide in which amino acids have not been substituted. Examples of non-natural amino acids include, but are not limited to, ornithine, citrulline, hydroxyproline, homoserine, phenylglycine, taurine, iodotyrosine, 2,4-diaminobutyric acid, α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid, γ-amino butyric acid, ε-amino hexanoic acid, 6-amino hexanoic acid, 2-amino isobutyric acid, 3-amino propionic acid, norleucine, norvaline, sarcosine, homocitrulline, cysteic acid, τ-butylglycine, τ-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methyl amino acids, C-methyl amino acids, N-methyl amino acids, and amino acid analogues in general. Non-natural amino acids also include amino acids having derivatized side groups. Furthermore, any of the amino acids in the protein can be of the D (dextrorotary) form or L (levorotary) form. Allelic variants of a protein sequence of a polypeptide of the present invention are also encompassed within the scope of the invention.
Amino acids can be generally categorized in the following classes: non-polar, uncharged polar, basic, and acidic. Conservative substitutions whereby a polypeptide of the present invention having an amino acid of one class is replaced with another amino acid of the same class fall within the scope of the subject invention so long as the polypeptide having the substitution still retains substantially the same functional activity as the polypeptide that does not have the substitution. Polynucleotides encoding a polypeptide having one or more amino acid substitutions in the sequence are contemplated within the scope of the present invention. Table 1 below provides a listing of examples of amino acids belonging to each class.







TABLE 1







Classes of amino acids










Class of Amino Acid
Examples of Amino Acids







Nonpolar
Ala, Val, Leu, Ile, Pro, Met, Phe, Trp



Uncharged Polar
Gly, Ser, Thr, Cys, Tyr, Asn, Gln



Acidic
Asp, Glu



Basic
Lys, Arg, His










The subject invention also concerns variants of the polynucleotides of the present invention that encode functional polypeptides of the invention. Variant sequences include those sequences wherein one or more nucleotides of the sequence have been substituted, deleted, and/or inserted. The nucleotides that can be substituted for natural nucleotides of DNA have a base moiety that can include, but is not limited to, inosine, 5-fluorouracil, 5-bromouracil, hypoxanthine, 1-methylguanine, 5-methylcytosine, and tritylated bases. The sugar moiety of the nucleotide in a sequence can also be modified and includes, but is not limited to, arabinose, xylulose, and hexose. In addition, the adenine, cytosine, guanine, thymine, and uracil bases of the nucleotides can be modified with acetyl, methyl, and/or thio groups. Sequences containing nucleotide substitutions, deletions, and/or insertions can be prepared and tested using standard techniques known in the art.
Fragments and variants of a polypeptide of the present invention can be generated as described herein and tested for the presence of function using standard techniques known in the art. Thus, an ordinarily skilled artisan can readily prepare and test fragments and variants of a polypeptide of the invention and determine whether the fragment or variant retains functional activity relative to full-length or a non-variant polypeptide.
Polynucleotides and polypeptides contemplated within the scope of the subject invention can also be defined in terms of more particular identity and/or similarity ranges with those sequences of the invention specifically exemplified herein. The sequence identity will typically be greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and can be greater than 95%. The identity and/or similarity of a sequence can be 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein. Unless otherwise specified, as used herein percent sequence identity and/or similarity of two sequences can be determined using the algorithm of Karlin and Altschul (1990), modified as in Karlin and Altschul (1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990). BLAST searches can be performed with the NBLAST program, score=100, word length=12, to obtain sequences with the desired percent sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be used as described in Altschul et al. (1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (NBLAST and XBLAST) can be used. See the NCBI/NIH website.
The subject invention also contemplates those polynucleotide molecules having sequences which are sufficiently homologous with the polynucleotide sequences exemplified herein so as to permit hybridization with that sequence under standard stringent conditions and standard methods (Maniatis et al., 1982). As used herein, “stringent” conditions for hybridization refers to conditions wherein hybridization is typically carried out overnight at 20-25° C. below the melting temperature (Tm) of the DNA hybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. The melting temperature, Tm, is described by the following formula (Beltz et al., 1983):

Tm=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.61(% formamide)−600/length of duplex in base pairs.

Washes are typically carried out as follows:
(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (low stringency wash).
(2) Once at Tm-20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderate stringency wash).
As used herein, the terms “nucleic acid” and “polynucleotide” refer to a deoxyribonucleotide, ribonucleotide, or a mixed deoxyribonucleotide and ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, would encompass known analogs of natural nucleotides that can function in a similar manner as naturally-occurring nucleotides. The polynucleotide sequences include the DNA strand sequence that is transcribed into RNA and the strand sequence that is complementary to the DNA strand that is transcribed. The polynucleotide sequences also include both full-length sequences as well as shorter sequences derived from the full-length sequences. Allelic variations of the exemplified sequences also fall within the scope of the subject invention. The polynucleotide sequence includes both the sense and antisense strands either as individual strands or in the duplex.
Techniques for transforming plant cells with a gene are known in the art and include, for example, Agrobacterium infection, biolistic methods, electroporation, calcium chloride treatment, PEG-mediated transformation, etc. (see, for example, Nagel et al., 1990; Song et al., 2006; de la Pena et al., 1987; and Klein et al., 1993). U.S. Pat. No. 5,661,017 teaches methods and materials for transforming an algal cell with a heterologous polynucleotide. Transformed cells can be selected, redifferentiated, and grown into plants that contain and express a polynucleotide of the invention using standard methods known in the art. The seeds and other plant tissue and progeny of any transformed or transgenic plant cells or plants of the invention are also included within the scope of the present invention.
The subject invention also concerns methods for producing a plant that exhibits increased hc1 content and/or protein functional activity relative to a wildtype plant. In one embodiment, a polynucleotide encoding an Hc1 or a mutant Hc1 protein of the present invention is introduced into a plant cell and the polypeptide(s) encoded by the polynucleotide(s) is expressed. In one embodiment, the polynucleotide or polynucleotides is incorporated into the genome of the plant cell and a plant is grown from the plant cell. In a preferred embodiment, the plant grown from the plant cell stably expresses the incorporated polynucleotide or polynucleotides.
The subject invention also concerns methods and materials for selecting for plants having increased levels of plant growth, biomass, and/or resistance to drought conditions. In one embodiment, an hc1 gene or polynucleotide is utilized as a genetic marker. In a specific embodiment, the Hc1 protein comprises an amino acid sequence of SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity as the full-length sequence. In a specific embodiment, the hc1 gene or polynucleotide comprises a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or a fragment or variant thereof. Methods of the invention comprise determining whether a plant, plant tissue, or plant cell contains an hc1 gene or polynucleotide of the invention, and/or determining whether a plant, plant tissue, or plant cell comprises or expresses an Hc1 protein of the present invention. In one embodiment, the presence of an hc1 gene or polynucleotide is determined by screening nucleic acid from the plant, plant tissue, or plant cell for hybridization with a nucleic acid probe (e.g., an oligonucleotide of the invention) that hybridizes with an hc1 gene or polynucleotide of the invention. In another embodiment, the presence of an hc1 gene or polynucleotide is determined by restriction fragment length polymorphism (RFLP) analysis, by polymerase chain reaction (PCR) amplification of specific hc1 nucleic acid sequences, or by sequencing hc1-encoding nucleic acid from the plant, plant tissue, or plant cell and identifying whether the gene or polynucleotide comprises a sequence that provides for increased hc1 mRNA levels or increased hc1 activity.
The subject invention also concerns methods for marker assisted selection and breeding in plants using a gene or polynucleotide that provides for modulated expression (increased or decreased) of hc1 or the gene product thereof for selecting for plants, plant tissue, or plant cells that exhibit a phenotypic characteristic of interest, e.g., increased plant biomass and/or growth rates. Methods for marker assisted selection are known in the art.
The subject invention also concerns oligonucleotide probes and primers, such as polymerase chain reaction (PCR) primers, that can hybridize to a coding or non-coding sequence of a polynucleotide of the present invention. Oligonucleotide probes of the invention can be used in methods for detecting and quantitating nucleic acid sequences encoding a polypeptide of the invention. Oligonucleotide primers of the invention can be used in PCR methods and other methods involving nucleic acid amplification. In a preferred embodiment, a probe or primer of the invention can hybridize to a polynucleotide of the invention under stringent conditions. Probes and primers of the invention can optionally comprise a detectable label or reporter molecule, such as fluorescent molecules, enzymes, radioactive moiety (e.g., 3H, 35S, 125I, etc.), and the like. Probes and primers of the invention can be of any suitable length for the method or assay in which they are being employed. Typically, probes and primers of the invention will be 10 to 500 or more nucleotides in length. Probes and primers that are 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 to 60, 61 to 70, 71 to 80, 81 to 90, 91 to 100 or more nucleotides in length are contemplated within the scope of the invention. Probes and primers of the invention can have complete (100%) nucleotide sequence identity with the polynucleotide sequence, or the sequence identity can be less than 100%. For example, sequence identity between a probe or primer and a sequence can be 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, 75%, 70% or any other percentage sequence identity so long as the probe or primer can hybridize under stringent conditions to a nucleotide sequence of a polynucleotide of the invention. In one embodiment, a probe or primer of the invention has 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, or 95% to 100% sequence identity with a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3, or the complement thereof.
The subject invention also concerns isolated Hc1 polypeptides. In a specific embodiment, a polypeptide of the invention has an amino acid sequence as shown in SEQ ID NO:2, or functional fragment or variant thereof that exhibits substantially the same activity as a full-length amino acid sequence. A polypeptide of the invention can be purified using standard techniques known in the art. In one embodiment, a polynucleotide of the invention encoding an Hc1 polypeptide is incorporated into a microorganism, such as E. coli, and the polypeptide expressed in the microorganism and then isolated therefrom.
Polypeptides of the invention, and functional peptide fragments thereof, can be used to generate antibodies that bind specifically to a polypeptide of the invention, and such antibodies are contemplated within the scope of the invention. The antibodies of the invention can be polyclonal or monoclonal and can be produced and isolated using standard methods known in the art. In one embodiment, an antibody of the invention binds specifically to a polypeptide that comprises the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof. Antigen binding fragments (such as Fab or Fab2 or Fv fragments) of antibodies of the invention can be routinely prepared and are also contemplated within the scope of the invention.
Fragments of a polypeptide of the invention, as described herein, can be obtained by cleaving the polypeptides of the invention with a proteolytic enzyme (such as trypsin, chymotrypsin, or collagenase) or with a chemical reagent, such as cyanogen bromide (CNBr). Alternatively, polypeptide fragments can be generated in a highly acidic environment, for example at pH 2.5. Polypeptide fragments can also be prepared by chemical synthesis or using host cells transformed with an expression vector comprising a polynucleotide encoding a fragment of a polypeptide of the invention, for example, a polypeptide that is a fragment of the amino acid sequence shown in SEQ ID NO:2. Fragments of a polypeptide of the invention also contemplated herein include fragments of the polypeptides wherein all or a part of a transit or signal sequence of the polypeptide is removed.
The subject invention also concerns cells transformed with a polynucleotide of the present invention encoding an Hc1 polypeptide of the invention, or that exhibit increased expression of an Hc1 encoding polynucleotide or the protein encoded by the polynucleotide, or that expresses a mutant hc1 polynucleotide or a mutant Hc1 protein that is characterized by increased expression or activity or function, or a fragment or variant thereof. In one embodiment, the cell is transformed with a polynucleotide sequence comprising a sequence encoding the amino acid sequence shown in SEQ ID NO:2, or a functional fragment or variant thereof. In a specific embodiment, the cell is transformed with a polynucleotide sequence shown in SEQ ID NO:1 and/or SEQ ID NO:3, or a sequence encoding a functional fragment or variant of SEQ ID NO:2. In one embodiment, the polynucleotide sequence is provided in an expression construct of the invention. The transformed cell can be a prokaryotic cell, for example, a bacterial cell such as E. coli or B. subtilis, or the transformed cell can be a eukaryotic cell, for example, a plant cell, including protoplasts, or an animal cell. Plant cells include, but are not limited to, dicotyledonous, monocotyledonous, and gymnosperm cells, such as conifer cells. In one embodiment, the plant cell is a cell from a Populus plant. The plant cell can be a cell from a hybrid plant, e.g., a poplar hybrid. Animal cells include human cells, mammalian cells, avian cells, and insect cells. Mammalian cells include, but are not limited to, COS, 3T3, and CHO cells.
The subject invention also concerns plant tissue and plant parts, including, but not limited to, plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells that are intact in plants or parts of plants such as branches, kernels, ears, cobs, husks, root tips, anthers, seeds, roots, embryos, hypocotyls, cotyledons, pollen, ovules, anthers, shoots, stalks, stems, leaves, fruits, and flowers, derived from a plant of the invention. The subject invention also concerns cuttings produced from a plant of the invention. In one embodiment, the cutting is a rootstock or a scion. In one embodiment, the cutting is a stem or branch from a young plant of the invention. In a specific embodiment, the stem is from a poplar plant comprising an hc1 gene, or the protein encoding portion thereof. In one embodiment, the poplar plant stem or branch is from a hybrid poplar plant.
The subject invention also encompasses plants and plant tissue that are bred from or otherwise derived from a plant of the present invention comprising a polynucleotide encoding an Hc1 polypeptide of the invention, or a fragment or variant thereof that provides for substantially the same activity. Seeds encompassed within the scope of the invention include hybrid seeds produced from a cross of a plant of the invention with another plant, such as an inbred plant. In one embodiment, the plant of the invention and/or the other plant is a homozygous inbred line. In one embodiment, the other plant can be one that exhibits desirable agronomic traits and/or fruit quality. In a specific embodiment, the other plant is one that exhibits resistance to one or more plant pathogens, diseases, or herbicides. The subject invention also concerns hybrid plants grown from hybrid seed or cuttings of the invention. The subject invention also concerns plants on which plant tissue of the subject invention has been grafted. In one embodiment, the Hc1 protein encoded by the polynucleotide comprises the amino acid sequence shown in SEQ ID NO:2, or a fragment or variant thereof having substantially the same activity as a full-length sequence. In a further embodiment, the polynucleotide comprises the nucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:3. All patents, patent applications, provisional applications, and publications referred to or cited herein are incorporated by reference in their entirety, including all figures and tables, to the extent they are not inconsistent with the explicit teachings of this specification.
EXAMPLES
Following are examples that illustrate procedures for practicing the invention. These examples should not be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.
It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims. In addition, any elements or limitations of any invention or embodiment thereof disclosed herein can be combined with any and/or all other elements or limitations (individually or in any combination) or any other invention or embodiment thereof disclosed herein, and all such combinations are contemplated with the scope of the invention without limitation thereto.
Example 1
Plant Material, Propagation, and Growth Measurements
The pedigree used to identify hc1 was a pseudo-backcross of the hybrid female parent 52-225 (Populus trichocarpa 93-968×P. deltoides ILL-101, P. t×d) and male parent D124 (Populus deltoides), hereafter referred to as Family 52-124. The parental plants and 100 individuals from the segregating population were clonally propagated as root cuttings, transplanted to deep pots, and placed randomly in a checkerboard arrangement on a flood bench. The plants were grown for 60 days, during which time the flood benches were flooded twice daily for approximately 30 minutes with a nutrient solution. Temperatures in the greenhouse ranged between 22° C. and 38° C., and interior photosynthetically active radiation ranged up to 1200 μmol s−1 m−2 (over the waveband 400-700 nm) during the daily 14 hours of natural irradiance.
Example 2
Plant Harvest, Biomass, and Growth Measurements
Sixty days after up-potting, plants were harvested. At the time of the harvest, plant final diameters and heights were recorded. Shoots were cut at the root collar and immediately re-cut under water, removing at least 5 cm from the cut end. Stem segments at least 10-cm long were stored in 15-mL conical tubes with deionized, distilled water in a cooler with ice for up to 5 hours until hydraulic conductivity could be measured. All leaves, sylleptic (lateral) branches, stems, and woody roots were dried in paper envelopes at 65° C., and weighed for calculation of total plant biomass. Height (HINC) and diameter (DINC) growth increments, were calculated as final (60 days growth) minus initial (30 days growth) measurements.
Example 3
Hydraulic Conductivity and Xylem Vessel Measurements
Hydraulic conductivity was determined two ways: first by the low pressure flow method on a subset of individuals, and then by the theoretical calculation of hydraulic conductivity on all individuals. Prior to the low pressure flow measurements, the stems were allowed to equilibrate to room temperature (25° C.), and re-cut under water with fresh razor blades. Stem segments contained multiple nodes, which were wrapped with parafilm to prevent leaks during measurement. To control for ion effects on pit membrane conductivity, the perfusion solution contained 20 mM KCl in distilled, deionized water, deaerated by sparging with helium (to reduce embolism formation), filtered to 0.2 μm, and adjusted to pH 2 with HCl (to control microbial growth). Stems were connected under water to a hydraulic apparatus containing the perfusion solution, and measurements were not taken until the zero-pressure (background) flow was zero. Flow of the solution through the stems at low pressure (4 kPa), to prevent flushing native embolism, was recorded on a balance connected to a computer and converted to initial conductivity (Knative, kg s−1 m MPa−1). Embolisms were then flushed with a higher pressure (>100 kPa) produced by a syringe mounted in a caulk gun, and the flow was recorded again and converted to maximum conductivity (Kmax, kg m MPa−1 s−1). Percent loss of conductivity (PLC) was calculated as: PLC=100*(Kmax−Knative)/Kmax. After conductivity measurements were completed, all stems were placed in 15-mL conical tubes with 50% ethanol in deionized, distilled water, and stored at 4° C. until cross-sections were made. Cross-sections (approximately 50-μm thick) were made approximately 2 cm from the upstream end of the stem with a vibratome and mounted in deionized distilled water. Images of the xylem were captured by a digital camera attached to a light microscope at 3× magnification. In each cross-section, vessel area was measured by automated tracing and, when needed, manual drawing of the inner perimeter of the vessel lumen. The individual vessel areas were converted to diameters (d) and counted (n), and vessels per sapwood area (VSA, count per mm2) and mean hydraulic diameter (Dh, μm, ((Σd4)n−1)1/4) were calculated. To determine theoretical conductivity, d was used to calculate lumen resistivity for each vessel as follows:




  
    
      
        
          
            R
            L
          
          =
          
            
              128
              *
              η
            
            
              π
              *
              
                d
                4
              
            
          
        
        ,
      
    
    
      
        (
        3
        )
      
    
  




where η is the viscosity of water at 25° C. to agree with low pressure flow meter measurements (8.9×10−10 MPa s). Lumen conductivity for each vessel was calculated as the inverse of RL (Ohm's Law), and then summed (conductances in parallel are additive) to determine theoretical conductivity, Kt, in m4 MPa−1 s−1. The Kt values were converted to the same units as Kmax (kg m MPa−1 s−1) by multiplying by 1000 kg m3 H2O, Sapwood specific conductivity (KS=Kt/SA, kg m−1 MPa−1 s−1) and leaf specific conductivity (KL=Kt/LA, kg m−1 MPa−1 s−1, where LA is total leaf area distal to the stem segment) were also calculated.

Example 4
Measured and Theoretical Conductivity
Using 29 young poplar stems, Kt was established as a good predictor of Kmax (Kmax=1.46*Kt, non-significant intercept, r2=0.9266, P<0.0001). Overall, the strength of the relationship between Kmax and Kt, and the fact that the overestimate was consistent across the range of conductivity, support the conclusion that Kt is an excellent predictor of the more difficult-to-measure Kmax. In addition, with the low pressure flow method, PLC averaged <3%, and the maximum observed PLC was about 9%, suggesting that watering twice a day was sufficient to minimize embolism and that Kt would be similar to Kh in the experiments described herein. Thus, all hydraulic conductivity and specific conductivity results presented are based on Kt measurements.
Example 5
Statistical Analysis
Regression analysis was used to relate hydraulic traits to growth. Means of all ramets per genotype were used in the plots and regression analyses, which were performed with SigmaPlot version 10.0 (Systat Software, Inc., San Jose, Calif., USA).
Genetic and phenotypic correlations were determined among growth and hydraulic conductivity traits. Phenotypically, hydraulic conductivity was positively correlated with diameter and height increment. A significant positive genetic correlation was also detected between diameter and height increment, and vessel diameter and conductivity (Table 2), which indicated a pleiotropic contribution of hydraulic conductivity to plant growth. The strongest phenotypic correlation occurred between DINC and KL (FIG. 1). Both Dh and VSA also had strong phenotypic and genetic correlations with DINC, with large vessels correlated with greater diameter increment (FIG. 2). KL also had a very strong positive genetic correlation with Dh (0.82±0.11). The strong genetic correlation between hydraulic conductivity and productivity, particularly KL and diameter increment, indicates that genes that regulate hydraulic conductivity are candidates for the regulation of growth in Populus. The positive direction of this correlation also supports the hypothesis that high hydraulic conductivity is a prerequisite for fast growth in angiosperms.







TABLE 2







Within-family broad sense heritabilities (diagonal, bold type), genetic


correlations (above diagonal), and phenotypic correlations (below diagonal)


for progeny genotypes (standard errors in parentheses).













Trait
DINC
HINC
Dh
KS
KL
BIO





Diameter
0.45
0.88
0.89
0.45
0.71
0.92


increment
(0.13)
(0.08)
(0.09)
(0.28)
(0.18)
(0.06)


(DINC)


Height
0.89
0.31
0.68
0.24
0.27
0.79


increment
(0.03)
(0.13)
(0.19)
(0.35)
(0.33)
(0.14)


(HINC)


Vessel
0.81
0.77
0.29
0.73
0.82
0.64


diameter
(0.05)
(0.06)
(0.13)
(0.17)
(0.11)
(0.22)


(Dh)


Sapwood
0.51
0.48
0.87
0.29
—
0.19


hydraulic
(0.10)
(0.10)
(0.04)
(0.13)

(0.34)


conductivity


(KS)


Leaf
0.66
0.56
0.88
—
0.34
0.34


hydraulic
(0.08)
(0.10)
(0.03)

(0.13)
(0.30)


conductivity


(KL)


Total plant
0.86
0.79
0.66
0.40
0.48
0.41


biomass (BIO)
(0.03)
(0.05)
(0.08)
(0.12)
(0.11)
(0.14)





Correlations that are not biologically applicable (—).






Example 6
Transcriptome Analysis of Family 52-124
A set of 396 individuals from Family 52-124 were propagated and grown as described above. From a common set of 192 randomly selected individuals, 180 samples of differentiating xylem and 183 expanding leaves were collected for gene expression analysis. Collected tissues were immediately flash-frozen in liquid nitrogen and stored at −80° C. until lyophilization and RNA extraction. RNA was extracted from each lyophilized sample by a standard protocol, converted to double-stranded cDNA, labeled with cy3, and hybridized to microarrays. Hybridizations were carried out using a previously described four-plex NimbleGen (Madison, Wis.) microarray platform (Gene Expression Omnibus Accession #GPL7234) using probes designed to minimize the effects of sequence polymorphism on the estimates of gene expression. The microarray comprised one probe per gene for 55,793 previously described gene models derived from the annotation of the genome sequence of P. trichocarpa clone ‘Nisqually-1’ (version 1.1), and a set of non-annotated ESTs. Raw data from hybridizations were background subtracted, log2-transformed, and quantile-normalized separately on a tissue-by-tissue basis. Raw and normalized gene expression data is publically available (Gene Expression Omnibus Accession #GSE12623, GSE20117, and GSE20118).
Example 7
Quantitative Trait Analysis
Quantitative trait loci (QTL) for growth, hydraulic, and physiological traits were identified using composite interval mapping performed with QTL Cartographer V2.5 on a previously established, high quality single-tree map of the hybrid mother of family 52-124 using each quantile-normalized gene expression value. The standard model (model 6) was used, with a walk speed of 2 cM, and significance level of P<0.05, determined by performing 1000 permutation tests. The magnitude of the QTL effect was calculated as the percentage variance explained (PVE). The likelihood ratio (LR) was converted to an equivalent log of odds (LOD) score by multiplying LR by 0.2171.
Significance of eQTL Log of Odds (LOD) values was estimated for xylem, leaf, and root using a global permutation threshold. eQTL were declared on the basis of a strategy wherein eQTL composed of unimodal LOD curves are located by the peak position. Bimodal peaks were declared as separate eQTL if the trough between them exceeded 2 LOD. The eQTL were classified as cis- or trans-regulated based on co-localization of the eQTL LOD peak with the genetic map marker bin containing the gene model in the ‘Nisqually-1’ genome sequence.
Vessel diameter QTL were detected between genetic markers at positions 28.5-36.9 megabase pair (Mbp) of chromosome 1, where a QTL for total plant biomass growth was previously mapped (Novaes et al., New Phytol 182:878-90, 2009) in the same population. Of 827 genes within the interval, 53 were previously recorded as expressed primarily in tissues derived from the vascular cambium (Quesada et al., New Phytol 180:408-20, 2008), where meristematic cell differentiation into vessels takes place. Transcriptional variation of these genes, measured in differentiating xylem of the pseudo-backcross population (Drost et al., PNAS USA 107:8492-7, 2010), was analyzed as a quantitative phenotype and cis-regulated expression QTL were identified for four genes. Assuming that regulation of vessel properties and hydraulic conductivity occurs at the transcriptional level, further analysis was conducted on these four genes that are positioned within the trait QTL interval and that are cis-regulated. Among the possible candidates, hc1 was selected as the most likely regulator of vessel formation, hydraulic conductivity, and growth.
Multiple significant (P<0.05) QTL were identified for growth and hydraulic traits (FIG. 3). Of particular interest for this study were co-localized QTL detected in linkage group one, for DINC, HINC, Dh, Kt, KS and KL. The data suggests that the observed genetic and phenotypic correlation among these traits is driven by single or few common genetic elements. Therefore, a genetic/genomics approach was used to identify these elements, where information from expression QTL analysis was used to define a putative regulator of the growth, hydraulic, and physiological traits.
Example 8
Genetic/Genomic Analysis of Growth, Hydraulic, and Physiological Traits
In order to identify candidate genes affecting hydraulic conductivity and growth in Populus, growth and hydraulic conductivity traits were quantified, differentiating xylem and leaves were sampled, and gene expression profiles of segregating progeny of P. deltoides and P. trichocarpa were obtained. Candidate genes for regulating a trait transcriptionally were expected to be regulated in cis, and to contain an expression QTL that co-localizes with the trait QTLs. In this analysis, a gene of unknown function was identified, annotated in the P. trichocarpa genome as POPTR—0001s33660 (or estExt_Genewise1_v1.C_LG_I3118 in the first annotation of the genome). The gene was located within the interval of the QTL for DINC, HINC, Dh, Kt, KS and KL, and genetic regulation of its expression in leaves and xylem was regulated by the same locus.
Example 9
Modification of the Expression of hc1
To verify the role of hc1 (previously referred to as POPTR—0001s33660) in the regulation of growth and hydraulic conductivity traits, its expression was altered by developing transgenic lines using RNAi-mediated gene silencing and ectopic expression using 35S promoter to generate loss- and gain-of-function variants, respectively. The coding sequence of hc1, obtained from P. trichocarpa reference genotype Nisqually-1, was cloned into pCAPT Transitive for the RNAi and pCAPO for overexpression. Agrobacterium-mediated transformation using strain GUV3101 was performed in the Populus tremula×P. alba (717-1B4 genotype) background. RNAi (i.e., loss of function) was lethal and did not generate any transgenics. Overexpression independent transgenic lines were screened for hc1 expression using RT-PCR, and the three lines that showed different levels of up-regulation were clonally replicated and planted in a greenhouse, and in growth chambers at the University of Florida.
Example 10
Hydraulic Conductivity and Xylem Vessel Measurements of Transgenic Lines
Stem vessel properties were characterized in the transgenic line with the highest growth rate observed in greenhouse (FIG. 4), using the conditions and methods described previously. Measurements showed that the transgenic line contains a significantly larger number of vessels per sapwood area, as well as larger vessel element diameter and height (FIGS. 5 and 6 and Table 3). Consequently, the area occupied by vessels per sapwood area is also significantly higher.







TABLE 3







Vessel element area, mean diameter, and number of vessels measured


in the transgenic line 3.1 overexpressing hc1, and in the wildtype.


All measurements have been normalized to total sapwood area, and are


based on the analysis of three biological replicates of transgenic


and wildtype. Standard deviations are presented in parenthesis.









Trait
Transgenic 3.1
Wildtype





Vessel element area
0.1925 (0.0111)
0.0878 (0.0057)


per sapwood area


Vessel number
139.1264 (11.62)  
108.32 (3.70) 


per sapwood area


Vessel element
0.0390 (0.0019)
0.0297 (0.0008)


mean diameter (mm2)


Vessel element
0.4549 (0.1386)
0.3439 (0.0924)


height (mm2)









It was also observed that line 3.1, as well as other transgenic lines, grew significantly more in greenhouse, compared to wildtype (FIG. 4). Therefore, growth traits were subsequently measured under highly controlled growth chamber conditions. Over-expression of hc1 may not only lead to higher growth through higher hydraulic conductivity, but it has also been shown that poplar trees with higher vessel diameter have also higher drought tolerance, because they can support better control of the stomatal aperture under water-limiting conditions.
Hydraulic conductivity was also measured in the transgenic lines and wildtype, to evaluate if the change in vessel properties would imply higher conductivity. As expected, hydraulic conductivity was significantly higher in all three transgenic lines relative to wildtype (average 28% increase), reflecting that flow is proportional to the fourth power of vessel diameter (Tyree et al., Xylem Structure and the Ascent of Sap. New York: Springer-Verlag, 2002).
Example 11
Growth of Transgenic Lines Under Standard and Heat Stress Conditions
Five to six biological replicates of each of three transgenic lines with the highest level of hc1 expression, and six biological replicates of the wildtype, were grown for up to 16 weeks in growth chamber with a continuous water supply. The temperature of the growth chamber was maintained at 25° C., and the interior photosynthetically active radiation ranged up to 600 μmol s−1 m−2 (over the waveband 400-700 nm) during the daily 12 hours of irradiance. Plant heights were measured weekly. For the first eight weeks, height growth rates remained consistent among transgenic and wildtype lines. As the plants reached >90 cm, growth rates increased 10-18% in the three transgenic lines compared to wildtype. The acceleration of growth is presumed to occur because, as the plants became larger and had higher leaf area, transpiration demand increased sufficiently to the point that hydraulic conductivity became restrictive in the wildtype.
Under heat stress, plants may reduce or cease growth when the transpiration demand surpasses what can be supported by the plant's hydraulic conductivity. In order to address if transgenic plants with higher hydraulic conductivity would be more tolerant to heat stress and thus support growth longer, transgenic and wildtype plants were grown under the same conditions described above, except that temperature was maintained at 33° C. after an acclimation period. Height growth was measured weekly in six biological replicates of three transgenic lines and wildtype until the weekly growth increment ceased or was less than 1 cm. For the first 11 weeks, height growth rates exceeded 1 cm per day in both transgenic and wildtype lines. Between the 11th and 12th week, height growth increment decreased dramatically in the wildtype (0.6 cm/week), and ceased in the following week. Among the three transgenic lines, height growth rates remained above 1 cm/week for an additional two weeks, before ceasing growth in the 15th week of the growth chamber experiment. Thus, the higher hydraulic conductivity provided the capability for the transgenic plants to maintain active growth for 2-3 additional weeks compared to wildtype plants.
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## Claims

1. A method for modulating growth rate, yield, and/or resistance to drought conditions in a plant, comprising introducing into said plant a polynucleotide comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1 or 3, or that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:2.

2. The method according to claim 1, wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2, wherein said fragment has at least 95% sequence identity to SEQ ID NO:2, and wherein expression of the nucleotide sequence in said plant confers said modulating growth rate, yield, and/or resistance to drought conditions in the plant.

3. The method according to claim 1, wherein the nucleotide sequence comprises the sequence of SEQ ID NO:1 or SEQ ID NO:3.

4. The method according to claim 1, wherein the plant is of the genus Abies, Acacia, Acer, Aesculus, Ailanthus, Alnus, Amelanchier, Arbutus, Arctostaphylos, Artemisia, Asiminia, Atriplex, Aucuba, Berberis, Betula, Buddleia, Buxus, Calocedrus, Camellia, Campsis, Carpinus, Carya, Castanea, Catalpa, Ceanothus, Cedrus, Celastrus, Celtis, Cephalanthus, Cercidium, Cercis, Chaenomeles, Chamaecyparis, Chilopsis, Chionanthus, Chrysothamnus, Cistus, Cladrastis, Clematis, Coleogynia, Cornus, Corylus, Cotinus, Cotoneaster, Cowania, Crataegus, Crataegus, Cupressus, Cytisus, Daphne, Deutzia, Diospyros, Elaeagnus, Ephedra, Escallonia, Eucalyptus, Euonymus, Fagus, Forsythia, Fraxinus, Gaultheria, Ginkgo, Gleditsia, Grevillea, Gymnocladus, Hamamelis, Hebe, Hibiscus, Hydrangea, Hypericum, Ilex, Juglans, Juniperus, Kalmia, Kerria, Koelreuteria, Lagerstroemia, Larix, Larrea, Libocedrus, Ligustrum, Liquidambar, Liriodendron, Lonicera, Maclura, Magnolia, Mahonia, Malus, Menispermum, Morus, Myrica, Nyssa, Osmanthus, Ostrya, Oxydendron, Parthenocissus, Philadelphus, Photinia, Physocarpus, Picea, Pinus, Pittosporum, Platanus, Populus, Prosopis, Prunus, Pseudotsuga, Ptelea, Purshia, Pyrus, Quercus, Rhamnus, Rhaphiolepis, Rhododendron, Rhus, Ribes, Robinia, Rosa, Rubus, Salix, Sambucus, Sassafras, Sequoia, Shepherdia, Smilax, Sophora, Sorbus, Spiraea, Staphylea, Stewartia, Symphoricarpos, Syringa, Taxodium, Taxus, Thuja, Tilia, Tsuga, Ulmus, Umbellularia, Vaccinium, Viburnum, Vitis, Zanthoxylum, or Zelkova.

5. The method according to claim 1, wherein the polynucleotide is stably incorporated into the genome of the plant.

6. A transformed or transgenic plant, plant tissue, or plant cell transformed with a polynucleotide that is stably incorporated into the genome of the plant, plant tissue, or plant cell, wherein the polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1 or 3, or wherein the polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:2, wherein the growth rate, yield, and/or resistance to drought conditions of the plant, plant tissue, or plant cell is increased relative to a plant, plant tissue, or plant cell that is not transformed with the polynucleotide.

7. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2, wherein expression of the nucleotide sequence in said plant, plant tissue, or plant cell confers said increased growth rate, yield, and/or resistance to drought conditions.

8. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the nucleotide sequence comprises the sequence of SEQ ID NO:1 or SEQ ID NO:3.

9. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the plant is of the genus Abies, Acacia, Acer, Aesculus, Ailanthus, Alnus, Amelanchier, Arbutus, Arctostaphylos, Artemisia, Asiminia, Atriplex, Aucuba, Berberis, Betula, Buddleia, Buxus, Calocedrus, Camellia, Campsis, Carpinus, Carya, Castanea, Catalpa, Ceanothus, Cedrus, Celastrus, Celtis, Cephalanthus, Cercidium, Cercis, Chaenomeles, Chamaecyparis, Chilopsis, Chionanthus, Chrysothamnus, Cistus, Cladrastis, Clematis, Coleogynia, Cornus, Corylus, Cotinus, Cotoneaster, Cowania, Crataegus, Crataegus, Cupressus, Cytisus, Daphne, Deutzia, Diospyros, Elaeagnus, Ephedra, Escallonia, Eucalyptus, Euonymus, Fagus, Forsythia, Fraxinus, Gaultheria, Ginkgo, Gleditsia, Grevillea, Gymnocladus, Hamamelis, Hebe, Hibiscus, Hydrangea, Hypericum, Ilex, Juglans, Juniperus, Kalmia, Kerria, Koelreuteria, Lagerstroemia, Larix, Larrea, Libocedrus, Ligustrum, Liquidambar, Liriodendron, Lonicera, Maclura, Magnolia, Mahonia, Malus, Menispermum, Morus, Myrica, Nyssa, Osmanthus, Ostrya, Oxydendron, Parthenocissus, Philadelphus, Photinia, Physocarpus, Picea, Pinus, Pittosporum, Platanus, Populus, Prosopis, Prunus, Pseudotsuga, Ptelea, Purshia, Pyrus, Quercus, Rhamnus, Rhaphiolepis, Rhododendron, Rhus, Ribes, Robinia, Rosa, Rubus, Salix, Sambucus, Sassafras, Sequoia, Shepherdia, Smilax, Sophora, Sorbus, Spiraea, Staphylea, Stewartia, Symphoricarpos, Syringa, Taxodium, Taxus, Thuja, Tilia, Tsuga, Ulmus, Umbellularia, Vaccinium, Viburnum, Vitis, Zanthoxylum, or Zelkova.

10. A method for preparing a transformed or transgenic plant, plant tissue, or plant cell having increased growth rate, yield, and/or resistance to drought conditions comprising incorporating a polynucleotide in a cell of the plant, wherein the polynucleotide comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:1 or 3, or wherein the polynucleotide comprises a nucleotide sequence that encodes a polypeptide having at least 95% sequence identity to SEQ ID NO:2.

11. The method according to claim 10, wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2, or a fragment of SEQ ID NO:2, wherein expression of the nucleotide sequence in said plant, plant tissue, or plant cell confers said increased growth rate, yield, and/or resistance to drought conditions.

12. The method according to claim 10, wherein the nucleotide sequence comprises the sequence of SEQ ID NO:1 or SEQ ID NO:3.

13. The method according to claim 10, wherein the plant is of the genus Abies, Acacia, Acer, Aesculus, Ailanthus, Alnus, Amelanchier, Arbutus, Arctostaphylos, Artemisia, Asiminia, Atriplex, Aucuba, Berberis, Betula, Buddleia, Buxus, Calocedrus, Camellia, Campsis, Carpinus, Carya, Castanea, Catalpa, Ceanothus, Cedrus, Celastrus, Celtis, Cephalanthus, Cercidium, Cercis, Chaenomeles, Chamaecyparis, Chilopsis, Chionanthus, Chrysothamnus, Cistus, Cladrastis, Clematis, Coleogynia, Cornus, Corylus, Cotinus, Cotoneaster, Cowania, Crataegus, Crataegus, Cupressus, Cytisus, Daphne, Deutzia, Diospyros, Elaeagnus, Ephedra, Escallonia, Eucalyptus, Euonymus, Fagus, Forsythia, Fraxinus, Gaultheria, Ginkgo, Gleditsia, Grevillea, Gymnocladus, Hamamelis, Hebe, Hibiscus, Hydrangea, Hypericum, Ilex, Juglans, Juniperus, Kalmia, Kerria, Koelreuteria, Lagerstroemia, Larix, Larrea, Libocedrus, Ligustrum, Liquidambar, Liriodendron, Lonicera, Maclura, Magnolia, Mahonia, Malus, Menispermum, Morus, Myrica, Nyssa, Osmanthus, Ostrya, Oxydendron, Parthenocissus, Philadelphus, Photinia, Physocarpus, Picea, Pinus, Pittosporum, Platanus, Populus, Prosopis, Prunus, Pseudotsuga, Ptelea, Purshia, Pyrus, Quercus, Rhamnus, Rhaphiolepis, Rhododendron, Rhus, Ribes, Robinia, Rosa, Rubus, Salix, Sambucus, Sassafras, Sequoia, Shepherdia, Smilax, Sophora, Sorbus, Spiraea, Staphylea, Stewartia, Symphoricarpos, Syringa, Taxodium, Taxus, Thuja, Tilia, Tsuga, Ulmus, Umbellularia, Vaccinium, Viburnum, Vitis, Zanthoxylum, or Zelkova.

14. The method according to claim 10, wherein the polynucleotide is stably incorporated into the genome of the plant.

15. The method according to claim 1, wherein the nucleotide sequence encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2.

16. The method according to claim 1, wherein the nucleotide sequence comprises at least 95% identity to SEQ ID NO:1 or 3.

17. The method according to claim 1, wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

18. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the nucleotide sequence encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2.

19. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

20. The transformed or transgenic plant, plant tissue, or plant cell according to claim 6, wherein the nucleotide sequence comprises at least 95% identity to SEQ ID NO:1 or 3.

21. The method according to claim 10, wherein the nucleotide sequence encodes a polypeptide comprising an amino acid sequence with at least 95% sequence identity to SEQ ID NO:2.

22. The method according to claim 10, wherein the nucleotide sequences encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:2.

23. The method according to claim 10, wherein the nucleotide sequence comprises at least 95% identity to SEQ ID NO:1 or 3.