US20210261976A1

US20210261976A1 - Generation of biomass

Info

Publication number: US20210261976A1
Application number: US17/188,144
Authority: US
Inventors: Simon Turner; Laxmi Mishra; Manoj Kumar; Peter Etchells
Original assignee: Stora Enso Oyj
Current assignee: Stora Enso Oyj
Priority date: 2015-03-02
Filing date: 2021-03-01
Publication date: 2021-08-26
Also published as: EP3265569A1; EP3265569A4; WO2016139579A1; CN107849578A; PL3265569T3; EP3265569B1; US10968456B2; US20180037900A1; BR112017018900A2

Abstract

A method for manipulating the growth and/or structure of a plant, for increasing biomass by increasing the expression and/or activity of PXY and/or CLE in the vascular tissue of a plant. The method includes introducing into the plant a regulatory element which specifically directs expression of CLE in the vascular tissue of the plant, where expression and/or activity of CLE modified in the vascular tissue remains substantially unaltered in non-vascular tissue of the modified plant.

Description

RELATED APPLICATIONS

This application is divisional of U.S. patent application Ser. No. 15/555,129 filed on Sep. 1, 2017, which is a U.S. National Stage under 35 U.S.C. § 371 of International Application No. PCT/IB2016/051131, filed Mar. 1, 2016, which claims priority to U.S. Provisional Patent Application No. 62/126,965, filed Mar. 2, 2015, the entirety of which are hereby incorporated by reference.

BACKGROUND

The present invention relates to a method for manipulating the growth and/or structure of a plant. The invention also relates to an expression cassette for use in manipulating the growth and/or structure of a plant, and to a transgenic plant, plant part, plant cell or seed comprising the expression cassette. The present invention also relates to a method of producing a plant derived product, and the plant-derived product per se. The present invention also relates to the use of a vascular tissue specific regulatory element in a method of manipulating the growth and/or structure of a plant. The present invention also relates to a host cell or organism comprising the expression cassette of the present invention.
The woody tissue of trees is composed of xylem cells that arise from divisions of stem cells within the cambial meristem. The rate of xylem cell formation is dependent upon the rate of cell division within the cambium and is controlled by both genetic and environmental factors (Miyashima et al (2013) EMBO J. 32, 178-193; Ursache et al (2013) Physiol. Plant. 147, 36-45). In the annual plant Ambidopsis, signalling between a peptide ligand CLE41 and a receptor kinase PXY controls cambial cell divisions (Etchells and Turner (2010) Development 137, 767-774; Fisher and Turner (2007) Current Biology 17, 1061-1066; and Hirakawa et al (2008) PNAS, USA 105, 15208-15213).
International patent application number WO2010/029357 relates to methods for altering the growth and/or structure of a plant, in order to maximise its potential as a source of biomass, in particular as a source of feedstock for the paper industry. The patent application describes the overexpression of CLE41, CLE42 and/or PXY in order to achieve the desired increase in the growth and structure of the vascular tissue of the plant.
There is however still a need to be able to improve the manipulation of the growth of plants, in particular trees for example to increase the yield of biomass from trees.

SUMMARY OF THE INVENTION

In a first aspect of the invention, there is provided a method for manipulating the growth and/or structure of a plant comprising modifying the plant to specifically increase the expression and/or activity of PXY and/or CLE in the vascular tissue of the plant compared to the native expression and/or activity of the PXY and/or CLE in the vascular tissue of a wild type plant of the same species maintained under identical conditions.
Modifying the plant preferably comprises specifically increasing the expression and/or activity of PXY and/or CLE in the vascular tissue of the plant, wherein the expression and/or activity of the PXY and/or CLE in non-vascular tissue of the modified plant remains substantially unaltered. In other words, expression and/or activity in the non-vascular tissue of the modified plant may be substantially the same as in the non-vascular tissue of a wild type plant maintained under identical conditions.
CLE may be CLE41 and/or CLE42. Alternatively CLE may be one or more of CLE41, CLE42 and CLE44.
Modifying the plant preferably comprises specifically increasing the expression and/or activity of PXY in the cambium of the plant, such that the expression and/or activity of PXY in non-cambium tissue (including the phloem and xylem) of the modified plant remains substantially unaltered. In other words, expression and/or activity in the non-cambium tissue of the modified plant may be substantially the same as in the non-cambium tissue of a wild type plant of the same species maintained under identical conditions.
Modifying the plant preferably comprises specifically increasing the expression and/or activity of CLE in the vascular tissue of the plant, such that the expression and/or activity of CLE in non-vascular tissue of the modified plant remains substantially unaltered. In other words, expression and/or activity of CLE in the non-vascular tissue of the modified plant may be substantially the same as in the non-vascular tissue of a wild type plant of the same species maintained under identical conditions.
Modifying the plant preferably comprises specifically increasing the expression and/or activity of CLE in the phloem of the plant, such that the expression and/or activity of CLE in non-phloem tissue (including the cambium and xylem) of the modified plant remains substantially unaltered. In other words, expression and/or activity in the non-phloem tissue of the modified plant may be substantially the same as in the non-phloem tissue of a wild type plant of the same species maintained under identical conditions.
Modifying the plant to specifically increase vascular tissue specific expression and/or activity may comprise introducing into the plant a regulatory element which specifically directs expression of PXY in the vascular tissue of the plant, more preferably in the cambium of the plant.
Modifying the plant to specifically increase vascular tissue specific expression and/or activity may comprise introducing into the plant a regulatory element which specifically directs expression of CLE in the vascular tissue of the plant, more preferably in the phloem of the plant.
A method of manipulating the growth and/or structure of a plant, as defined herein, may comprise modifying the plant to specifically increase the expression and/or activity of PXY and CLE in the vascular tissue of the plant compared to the expression and/or activity of PXY and CLE in the vascular tissue of a wild type plant of the same species maintained under identical conditions. The expression and/or activity of both PXY and CLE is increased compared to native expression in the wild type plant.
Thus, the method of manipulating the growth and/or structure of a plant as defined herein may comprise: i) modifying the plant to specifically increase the expression and/or activity of PXY in the cambium of a plant compared to the expression and/or activity of PXY in the vascular tissue of a wild type plant of the same species maintained under identical conditions; and ii) modifying the plant to specifically increase the expression and/or activity of CLE in the phloem of the plant compared to the expression and/or activity of CLE in the vascular tissue of a wild type plant of the same species maintained under identical conditions.
The method of modifying the plant may comprise directing expression and/or activity of PXY to the vascular tissue of a plant by placing PXY under the control of a vascular-tissue specific promoter. The promoter may be phloem, xylem or cambium specific. Preferably, the promoter is cambium specific. The promoter may be the promoter of the plant ANTEGUMENTA (herein referred to as “ANT”) gene, or a functional fragment or variant thereof. The ANT promoter may preferably be derived from hybrid Aspen (PttANT).
The method of modifying the plant may comprise directing expression and/or activity of CLE to the vascular tissue of a plant by placing CLE under the control of a vascular-tissue specific promoter. The promoter may be phloem, xylem or cambium specific. Preferably, the promoter may be phloem specific. The promoter may be the promoter of the phloem specific lectin gene PHLOEM PROTEIN 2 (herein referred to as “PP2”), or a functional fragment or variant thereof. The PP2 promoter may preferably be derived from hybrid Aspen (PttPP2).
In a second aspect, the present invention also provides a method for increasing the growth rate of a plant, the method comprising manipulating the growth and/or structure of the plant as described herein.
In a third aspect, the present invention also provides a method for increasing the radial growth of a plant, the method comprising manipulating the growth and/or structure of the plant as described herein.
In a fourth aspect, the present invention also provides a method for increasing the amount of leaf tissue in a plant, the method comprising manipulating the growth and/or structure of the plant as described herein.
In a fifth aspect, the present invention provides a method for increasing the biomass of a plant, the method comprising manipulating the growth and/or structure of the plant as described herein.
In a sixth aspect, the present invention provides a method of producing a plant derived product; comprising manipulating the growth and/or structure of the plant as described herein, and harvesting a plant product from the plant.
In a seventh aspect, the present invention provides an expression cassette comprising a nucleic acid sequence encoding a regulatory element which specifically directs expression in the vascular tissue of a plant. The regulatory element may specifically direct expression in the phloem, xylem or cambium. Preferably, the regulatory element will specifically direct expression in the cambium. The regulatory element may be the promoter of the ANT gene, or a functional fragment or variant thereof. The regulatory element may be the promoter of the PP2 gene, or a functional fragment or variant thereof. A regulatory element of the expression cassette may be operably linked to a gene, such as PXY and/or CLE. An expression cassette may comprise a first regulatory element operably linked to a nucleic acid molecule encoding PXY, and a second regulatory element operably linked to a nucleic acid molecule encoding CLE.
In an eighth aspect, the present invention provides a transgenic plant cell comprising an expression cassette as defined herein. Thus, the plant cell is manipulated according to the method of the first aspect.
In a ninth aspect, the present invention provides a transgenic plant, plant part or transgenic seed comprising a plant cell as defined above. Thus, the plant, plant part or transgenic seed is manipulated according to the method of the first aspect.
In a tenth aspect, the present invention provides a plant derived product produced according to a method of the sixth aspect.
In an eleventh aspect, the present invention provides the use of a vascular tissue specific regulatory element, for use in the manipulation of the growth and/or structure of a plant. A regulatory element may be, for example a promoter of the ANT gene or a functional fragment or variant thereof, and/or a promoter of the PP2 gene or a functional fragment or variant thereof. A regulatory element may be provided in an expression cassette according to the seventh aspect of the invention. Preferably, the use is according to a method of any one of the first to sixth aspects of the invention.
In a twelfth aspect the present invention provides a host cell or organism comprising an expression construct according to the seventh aspect.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described further, by way of example, with reference to the accompanying drawings, in which:

FIG. 1 shows the phenotypes of hybrid aspen ectopically overexpressing PttCLE41 and/or PttPXY genes. (A): Sections from tissue culture grown plantlets 3 weeks post rooting. Where two images are shown in the upper panel, they were selected to show the range of phenotypes observed. Scale bars indicate 200 μM (upper panels) and 50 μM (lower panels). The xylem (x) and phloem (ph) are indicated. Asterisks show examples of organised files of cells. (B): Representative greenhouse grown plants 3 months after transfer to soil. (C): Phenotypes of hybrid aspen with targeted overexpression of PttCLE41 and PttPXY. Left hand panels show sections from tissue culture grown piantlets 3 weeks post rooting while greenhouse grown plants 3 months after transfer to soil are shown on the right. Scale bars indicate 200 μM. The xylem (x) and phloem (ph) are also indicated. Arrows highlight the disrupted xylem.

FIG. 2 shows the expression patterns derived from PttANT and PtPP2 promoters. GUS stained and cleared control (A), PttANT::GUS (B) and PtPP2::GUS (C) plants. Upper panels show leaves, lower panels are transverse stem sections. Scale bars indicate 200 μm (upper panels) 100 μm (lower panels).

FIG. 3 shows the growth characteristics of trees with targeted PttCLE41/PttPXY overexpression. Mean stem diameter (A) and plant height (B) measurements from hybrid aspen grown in soil are shown. Trees rooted in April were measured at 15 weeks (July), 26 weeks (August) and 33 weeks (October). Further analysis of 6 month old plants: number of internodes (C), length of 50th internode (D), leaf area calculated from measurements of 5 leaves front around the 50th internode (E) and xylem cell number in a stem cross sector with a central angle of 40° C. (F). (G) Graph showing dry weight of 10 cm pieces of sapling stem. Samples were taken from the base, middle (50th internode) and top, except for 35S::PttCLE41 which had less than 50 internodes and a section taken midway between the top and bottom was used instead. All p values were calculated with an ANOVA and a LSD post-hoc test, n=15 (A-E) or 8 (F, G).

FIG. 4 shows the growth of clonally propagated plants derived from independent transformants of PtPP2::PttCLE41-PttANT::PttPXY. Diameter (top) and height (bottom) of plants was measured at weekly intervals starting 4 weeks after transfer from tissue culture to soil. Asterisk indicates a p value of less than 0.05 compared to the controls. All p values were calculated with an ANOVA and a LSD post-hoc test, n=6 for the control; n=5 for PtPP2::PttCLE41-PttANT::

PttPXY lines

1, 3 and 9; n=4 for

lines

2 and 4.

FIG. 5 shows amino acid sequence alignment of PXY and CLE. (A) Alignment of PXY sequences from Arabidopsis (AtPXY, AT5G61480), P. trichocarpa (PtPXY, Potri.003G107600) and hybrid Aspen (PttPXY, this study). (B) Alignment of CLE41 sequences from Arabidopsis (AtCLE41, AT3G24770), P. trichocarpa (PtCLE41, Potri.012G019400) and hybrid Aspen (PttCLE41, this study). TDIF peptides within the CLE41 sequences are indicated by the black rectangle. Ptt sequences were obtained translating the ORF from plasmids containing PttPXY and Supplemental Data PttCLE41 genes cloned as part of this study. Other protein sequences were obtained from phytozome (http://www.phytozome.net/).

FIG. 6 shows Poplar CLE41 and PXY genes are functional in Arabidopsis. Sections from Arabidopsis hypocotyls (LHS) and inflorescence stem vascular bundles (RHS) from typical wild type (A), 35S::PttCLE41 (B), pxy mutant (C), and a pxy mutant complemented with 35S::PttPXY (D). Scales bars indicate 50 μM.

FIG. 7 shows growth characteristics of Arabidopsis lines overexpressing poplar PXY genes. Number of cells per vascular bundle (A) and plant height (B) of Arabidopsis 35S::PttCLE41 lines compared to wild type counterparts. Number of cells per vascular bundle (C), plant height (D) and dry weight (E) of Arabidopsis pxy, pxy35S::PttPXY, SUC2::AtCLE41, SUC2::AtCLE41-35S::PttPXY lines compared to wild type counterparts. P values were calculated with an ANOVA and LSD post-hoc test with N=10 (A,C) or 40 (B,D,E).

FIG. 8 shows growth characteristics of hybrid aspen lines overexpressing PttCLE41/PttPXY. Height (A) and diameter (B) measurements from hybrid aspen grown in soil. Trees rooted in April, were measured at 15 weeks (July), 26 weeks (August) and 33 weeks (October). N=15.

FIG. 9 shows xylem cell number and biomass of transgenic trees. (A) Graph showing number of vascular cells in control and 35S::PttCLE41, 35S::PttPXY, 35S::PttCLE41 rolD:PttPXY, PtPP2::PttCLE41, PttANT::PttPXY and PtPP2::PttCLE41 PttANT::PttPXY hybrid aspen lines in tissue culture 3 weeks postrooting. (B) Wet weight of 10 cm pieces of sapling stem taken from the base, middle (50th internode) and top, except for 35S::PttCLE41 that had less than 50 internodes and sections were taken midway between the top and bottom instead. Graph shows the wet weight of stem pieces in FIG. 3G. *Significantly larger than wild type p<0.05; ** Significantly larger than all other lines p<0.001; values were calculated with an ANOVA and LSD post-hoc test, N=7.

FIG. 10 shows phenotypic characterisation using Cellprofiler. (A): Transverse stem section from the 50th internode showing xylem in a sector with a central angle of 40° C. (top) and recognition of cell lumens by Cellprofiler (below). Cells were identified with greater than 95% accuracy, but cells with no clear lumen, such as ray cells or very small fiber cells, were not recognized (B): Measurement of cell size and cell wall area is based upon a rectangle (top) outlined in (a). Primary objects (cell lumens) were identified (upper middle) and propagated outwards to identify the secondary objects (lower middle). The tertiary objects (cell walls) were obtained by subtracting the primary objects from the secondary objects (bottom). (C): Identification of vessels based upon identifying primary objects (middle) and then filtering by size and shape (bottom).

FIG. 11 shows PttCLE41 and PttPXY expression analysis in PtPP2::PttCLE41-PttATT::PttPXY lines. (A): RT-PCR showing expression in 8 independent transgenic lines. Stem material was taken adjacent to the 50th internode. (B): Relative intensity of PCR product in (A), was determined using Image Lab 5.1 software (Bio-rad). (C): Relationship between cell number and PttPXY expression. (D): Relationship between cell number and PttCLE41 expression.

FIG. 12 is a pictorial representation of the effects of the present invention.

FIG. 13 shows an alignment of PXY with similar genes from Arabidopsis (PXL1 and PXL2), together with homologous genes from Rice (Os02g02140.1 and Os03g05140.1) and Poplar (PttPXY). The consensus sequence of PXY is also shown.

FIG. 14 shows the amino acid sequence of PttPXY and the nucleic acid which encodes the protein.

FIG. 15 shows the nucleic acid sequence encoding Arabidopsis PXY.

FIG. 16 shows an alignment of CLE41 and CLE42 and homologous sequences from other plant species, including the CLE41 consensus sequence.

FIG. 17 shows Arabidopsis amino acid and nucleic acid sequence.

FIG. 18 shows the nucleic acid sequence of PttCLE41.

FIG. 19 shows the amino acid sequence of the CLE42 proteins.

FIG. 20 and nucleotide sequence of the CLE42 gene (B).

DETAILED DESCRIPTION

The present inventors have shown that PttPXY and its peptide ligand PttCLE41 are functional orthologues and act to control a multifunctional pathway that regulates both the rate of cambial cell division and woody tissue organization in trees. The present invention is based upon the finding that vascular tissue-specific overexpression of PXY generated plants that exhibited an increase in the rate of wood formation, were taller and possessed larger leaves compared to wild type control plants. The results demonstrate that the PXY-CLE pathway has evolved to regulate secondary growth and manipulating this pathway can result in dramatically increased tree growth and productivity.

Definitions

As used herein, the term “nucleic acid molecule” includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., a niRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs. A nucleic acid molecule may be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid molecule may be recombinant.
A “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). A naturally occurring nucleic acid molecule may also be referred to as native.
The terms protein and polypeptide refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The terms protein and polypeptide may be used interchangeably herein.
“Foreign” referring to a nucleic acid molecule or polypeptide, with respect to a plant is used to indicate that the nucleic acid sequence or polypeptide is not naturally found in that plant, or is not naturally found in that genetic locus in that plant.
With regards to genomic DNA, the term “isolated” includes nucleic acid molecules that are separated from the chromosome with which the genomic DNA is naturally associated. Preferably, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′- and/or 3′-ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
By the term “recombinant nucleic acid molecule” is meant a nucleic acid molecule that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination may be accomplished by chemical synthesis or by the artificial manipulation of nucleic acid molecules, e.g., by genetic engineering techniques, such as by the manipulation of at least one nucleic acid by a restriction enzyme, ligase, recombinase, and/or a polymerase.
As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding protein, and can further include non-coding regulatory sequences and introns.
The term “complement of a nucleic acid sequence” is the nucleotide sequence which would be capable of forming a double stranded DNA molecule with the represented nucleotide sequence, and which can be derived from the represented nucleotide sequence by replacing the nucleotides by their complementary nucleotide according to Chargaff's rules (A< >T; G< >C) and reading in the 5′ to 3′ direction, i.e., in opposite direction of the represented nucleotide sequence.
A “regulatory element” is a non-coding region of a gene which regulates its transcription.
An “expression cassette” is a genetic vehicle comprising a regulatory element for expression of a gene or coding sequence, and optionally a coding sequence operably linked thereto. An expression cassette may facilitate expression of a gene in a cell into which it is introduced.
“Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is “under transcriptional initiation regulation” of the promoter.
A cell into which a foreign nucleic acid molecule has been introduced may be referred to herein as a recombinant cell, or a transgenic or transformed cell. Introduction may be defined as the insertion of a nucleic acid molecule into a cell. Once introduced into a host cell, a recombinant nucleic acid is replicated by the host cell, however, the recombinant nucleic acid once replicated in the cell remains a recombinant nucleic acid for purposes of this invention.
By “recombinant protein” herein is meant a protein produced by a method employing a recombinant nucleic acid. The term protein may be used interchangeably with the term polypeptide. As outlined above “recombinant nucleic acid molecules” and “recombinant proteins” also are “isolated” as described above. The cell into which the recombinant nucleic acid molecule may be introduced may be described as a recombinant cell, or a transformed or transgenic cell.
A transgenic plant, plant part or seed may comprise one or more transgenic plant cells, i.e. cells which comprise recombinant genetic material which is not normally found in a plant or tree of this type and which has been introduced into the plant in question (or into progenitors of the plant) by human manipulation.
Herein, “expression” refers to the biosynthesis of a gene product, i.e. in the case of a structural gene such as PXY or CLE, expression involves the transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
“Increased expression” means an increase in the level of transcription and translation compared to the level for the same gene in a wild type plant of the same species maintained under identical conditions.
“Activity” refers to a phenotypic property of the protein, for example its ability to bind to a binding partner, its ability to generate a signal within the pathway, and mediate downstream effects on growth and development.
Included within the scope of the present invention are functional equivalents of the polypeptides and nucleic acid molecules defined herein. The term “functional equivalent” is intended to include fragments, mutants, hybrids, variants, analogs, or chemical derivatives of a nucleic acid molecule or protein as defined herein, which shares at least one structural characteristic of the native nucleic acid molecule or functional characteristic of the protein.
A functional fragment of a nucleic acid molecule or polypeptide as defined herein may include any portion of an amino acid or nucleic acid sequence which shares at least one functional or structural characteristic that is substantially similar to the subject polypeptide or nucleic acid molecule. A structural or functional characteristic may include binding characteristics, the ability to regulate a downstream signalling pathway, to mediate one or more phenotypic effects, of the native nucleic or polypeptide.
As used herein, the term “hybridizes under stringent conditions” describes conditions for hybridization and washing. Stringent conditions are known to those skilled in the art and can be found in available references (e.g., Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 1989, 6.3.1-6.3.6). Aqueous and non-aqueous methods are described in that reference and either can be used.
Sequence identity (e.g., percent homology) can be determined using any homology comparison software, including for example, the BlastN software of the National Center of Biotechnology Information (NCBI) such as by using default parameters.
A variant of a polypeptide or protein defined herein may be one which is altered by one or more amino acids. The variant may have “conservative” changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have “non-conservative” changes, e.g., replacement of a glycine with a tryptophan. Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted may be found using computer programs well known in the art, for example, DNASTAR© software. A functional variant of a polypeptide defined herein will preferably retain at least one structural or functional characteristic of the non-variant polypeptide.
By “vascular tissue” is meant the conductive and supportive tissue in a plant.
By “specifically” or “specific expression or activity” is meant that the nucleic acid molecule is preferentially expressed, or the protein is preferentially active, in one cell type, tissue, or developmental than another.
Herein, the “growth” of a plant refers to the size of a plant, preferably the secondary growth, and preferably the amount of vascular and/or interfasicular tissue, more preferably the amount of xylem cells, also referred to as the woody tissue or biomass of a plant.
“Radial diameter” is a measure of the circumference of a plant, and indicative of growth and division of vascular tissue.
“Biomass” refers to the amount of tissue produced by a plant, for example in one growing season. The “leaf tissue” is the quantity of leaves, expressed in weight.
“Vigour” refers to the amount, by weight, of tissue produced by a plant in a given time.
“Growth rate” is a measure the amount of growth, for example weight or radial growth, in a specified time period.
“Seed yield” is the amount of seeds, for example by weight, harvested from a plant, for example in a given growing season.
The “structure” of a plant refers to the organisation of tissue in a plant, preferably the vascular tissue, most preferably the polarity of the phloem and xylem cells.
By “identical conditions” is meant conditions which are the substantially the same in terms of temperature, light, and availability of nutrients and water. By substantially is meant that the conditions may vary slightly, but not to an extent to which is known to affect the growth of a plant. The term “identical conditions” also encompasses comparing plants of the same species, of the same pre-selected developmental stage.
PXY
Herein, PXY refers to a receptor-like kinase which in nature binds to CLE41 or CLE42. Preferably, the term PXY refers to a receptor like kinase which comprises an extracellular domain comprising leucine rich repeats (LRR). Upon binding by CLE41 and/or CLE42 it mediates the activation of a signalling pathway which results in division of undifferentiated cambial cells. Herein, preferably the term PXY refers to a member of the XI family of Arabidopsis thaliana RLK proteins. PXY is also known in the art as TDR.
Herein, a PXY polypeptide includes i) a polypeptide comprising a conserved region in the kinase domain wherein the conserved region comprises the consensus sequence of FIG. 13, and is capable of binding CLE41 and/or CLE42 and mediating the activation of a signalling pathway which results in division of undifferentiated cambial cells; ii) a polypeptide having an amino acid sequence as shown in FIG. 5A, 14, 15. Alternatively, a PXY polypeptide as defined herein may be a functional orthologue of such a polypeptide, derived from another plant, such as a woody plant. Such orthologues will preferably be capable of binding to CLE41 or CLE42 and mediating the activation of a signalling pathway which results in division of undifferentiated cambial cells. Such orthologues will preferably comprise the PXY polypeptide consensus sequence of FIG. 13.
References herein to PXY include functional equivalents of the polypeptide. Equivalents include fragments and variants (including orthologues) of a PXY polypeptide as described herein. A functional equivalent of PXY for use in the present invention will have some or all of the desired biological activity of the native polypeptide, preferably the ability to bind to CLE41 and/or CLE42 and regulate growth and/or differentiation of the vascular tissue. Functional equivalents may exhibit altered binding characteristics to CLE41 and/or CLE42 compared to a native PXY polypeptide. Preferred functional equivalents may show reduced non-desirable biological activity compared to the native protein. A functional equivalent will preferably comprise at least 70% sequence identity to a PXY polypeptide of FIG. 5A, 14, or 15, more preferably at least 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a PXY polypeptide of FIG. 5A, 14, or 15. A functional equivalent will preferably comprise a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the PXY consensus sequence of FIG. 13.
A functional fragment of a PXY polypeptide is a portion of a polypeptide sequence or variant thereof as defined herein. A functional fragment preferably retains some or all of the biological activity of the full length PXY polypeptide. Preferably, a functional fragment of PXY retains the ability to bind CLE41 or CLE42 and regulate the growth and/or differentiation of the vascular tissue of a plant. Preferably, a fragment will comprise at least a portion of the kinase domain, preferably a biologically active portion thereof, up to the full length kinase domain. Most preferably, a fragment will further comprise at least a portion of the extracellular domain, and will preferably comprise at least a portion of the LLR region. A fragment may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length PXY polypeptide.
A nucleic acid encoding PXY may comprise a sequence which encodes a polypeptide of FIG. 5A. A nucleic acid sequence encoding PXY may be as shown in FIG. 14 or FIG. 15. Nucleic acid sequences encoding PXY are available from Genbank, under references PXY=At5g61480 (TAIR), PXL1=At1g08590 (TAIR), and PXL2=At4g28650 (TAIR); and Genbank Accession No. KP682331 version 1 (PttPXY).
Also included are functional equivalents of the nucleic acid molecules defined herein, which encode a polypeptide or a functional equivalent thereof as defined herein. A functional equivalent may be a sequence variant and/or a functional fragment of a PXY nucleic acid sequence as defined herein. A variant will preferably encode a polypeptide which has the ability to bind CLE41 or CLE42 and regulate the growth and/or differentiation of the vascular tissue of a plant, and preferably shares at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity with a nucleic acid sequence of FIG. 14 or 15, or encodes a polypeptide having at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity with a polypeptide of FIG. 5A or 14 or FIG. 15, or encodes a polypeptide comprising a consensus sequence of FIG. 13. Alternatively, a variant may be defined as a sequence which hybridises under stringent conditions to a complement of the nucleic acid sequence of FIG. 14 or 15. A fragment may encode a functional fragment of a PXY polypeptide as defined above. A fragment of a nucleic acid molecule may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length PXY nucleic acid sequence of FIG. 14 or 15.
CLE
Herein, CLE refers to a ligand which is able to activate a kinase receptor, and result in phosphorylation of itself or its target. Preferably, the term CLE refers to a signalling protein, preferably of less than 15 kDa in mass, and preferably comprising a hydrophobic region at the amino terminus. Upon binding to PXY it mediates the activation of a signalling pathway which results in division of undifferentiated cambial cells. The term CLE includes CLE41, CLE42 and CLE44, and the aspects of the invention may relate to increased expression and/or activity of CLE41, CLE42 or CLE44. CLE41 is also known in the art as TDIF.
Herein, a CLE41 or CLE42 polypeptide includes i) a polypeptide comprising a conserved region in the kinase domain having the sequence comprising the consensus sequence of FIG. 16, and being capable of binding PXY to mediate the activation of a signalling pathway which results in division of undifferentiated cambial cells; ii) a polypeptide having an amino acid sequence as shown in FIG. 5B, 17 or 18, or 19. Alternatively, a CLE41 or CLE 42 polypeptide as defined herein may be a functional orthologue of such a polypeptide, derived from another plant, such as a woody plant. Such orthologues will preferably be capable of binding to PXY and mediating the activation of a signalling pathway which results in division of undifferentiated cambial cells. Such orthologues will preferably comprise the CLE polypeptide consensus sequence of FIG. 16.
References herein to CLE41, CLE42 and CLE44 include functional equivalents of the polypeptides. Equivalents include fragments and variants (including orthologues) of a CLE41 or CLE42 polypeptide as described herein. A functional equivalent of CLE for use in the present invention will have some or all of the desired biological activity of the native polypeptide, preferably the ability to bind to PXY and regulate growth and/or differentiation of the vascular tissue. Functional equivalents may exhibit altered binding characteristics to PXY compared to a native CLE polypeptide. Preferred functional equivalents may show reduced non-desirable biological activity compared to the native protein. A functional equivalent will preferably comprise at least 70% sequence identity to a CLE41 polypeptide of FIG. 5B, FIG. 17 or FIG. 18, more preferably at least 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a CLE41 polypeptide of FIG. 5B, FIG. 17 or FIG. 18. A functional equivalent will preferably comprise a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the CLE41 consensus sequence of FIG. 16. A functional equivalent will preferably comprise at least 70% sequence identity to a CLE42 polypeptide of FIG. 16, FIG. 17 or FIG. 19, more preferably at least 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with a CLE42 polypeptide of 16, FIG. 17 or FIG. 19. A functional equivalent will preferably comprise a sequence having at least 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the CLE42 consensus sequence of FIG. 1.6.
A functional fragment of a CLE polypeptide is a portion of a CLE41 or CLE42 polypeptide sequence or a variant thereof as defined herein. A functional fragment preferably which retains some or all of the biological activity of the full length CLE polypeptide. Preferably, a functional fragment of CLE retains the ability to bind PXY and regulate the growth and/or differentiation of the vascular tissue of a plant. A fragment may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length CLE41 or CLE42 polypeptide. Preferably, a fragment may be at least 7 amino acids in length, preferably at least 8, 9, or 10, 15, 20, 25, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids in length, up to the full length CLE41 or CLE42 polypeptide. Most preferably, a fragment will comprise the conserved region consisting of amino acids 124 to 137 of the consensus sequence of FIG. 16.
A nucleic acid encoding CLE41 may comprise a sequence which encodes a polypeptide of FIG. 5B, FIG. 17 or FIG. 18. A nucleic acid sequence encoding CLE41 may be as shown in FIG. 17 or 18. The nucleic acid and amino acid sequence of PttCLE41 are available under Genbank Accession No. KP682332, version 1.
A nucleic acid encoding CLE42 may comprise a sequence which encodes a polypeptide of Figure SB or FIG. 19. A nucleic acid sequence encoding CLE42 may be as shown in FIG. 20.
Also included are functional equivalents of the nucleic acid sequences defined herein, which encode a polypeptide as defined herein or an orthologues and functional equivalents of the above mentioned polypeptides, as defined herein. A functional equivalent may be a sequence variant and/or a functional fragment of a CLE41 or CLE42 nucleic acid sequence as defined herein. A variant will preferably encode a CLE41 or CLE42 polypeptide or functional equivalent thereof which has the ability to bind PXY and regulate the growth and/or differentiation of the vascular tissue of a plant, and preferably shares at least 70%, 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, sequence identity with a nucleic acid sequence of FIG. 17 or 18, or 20. Alternatively, a variant may be defined as a sequence which hybridises under stringent conditions to a complement of the nucleic acid sequence of FIG. 17 or 18, or 20. A fragment may encode a functional fragment of a CLE41 or CLE42 polypeptide as defined above. A fragment of a nucleic acid molecule may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length CLE41 or CLE42 nucleic acid sequence. A fragment of a nucleic acid molecules encoding CLE41 or CLE42 will preferably comprise at least 21 nucleotides in length, more preferably at least 24, 27, 30 or 33 nucleotides, up to the total number of nucleotide residues in a full length sequence of FIG. 17 or 18, or 20.
Functional Equivalents
A variant of a nucleic acid molecule as defined herein may include a sequence which hybridises under stringent conditions to a complement of the reference sequence, or a sequence which has a specified degree of sequence identity with the reference sequence. Two nucleic acid or amino acid sequences are orthologs of each other if they share a common ancestral sequence and diverged when a species carrying that ancestral sequence split into two species, sub-species, or cultivars. Orthologous sequences are also homologous sequences. Orthologous sequences hybridize to one another under high-stringency conditions.
A preferred example of high stringent hybridization conditions are hybridization in 6× sodium chloride/sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 50° C. Another example of stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 55° C. A further example of stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 60° C. Preferably, stringent hybridization conditions are hybridization in 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% (w/v) SDS at 65° C. Particularly preferred stringency conditions (and the conditions that should be used if the practitioner is uncertain about what conditions should be applied to determine if a molecule is within a hybridization limitation of the invention) are 0.5 molar sodium phosphate, 7% (w/v) SDS at 65° C., followed by one or more washes at 0.2×SSC, 1% (w/v) SDS at 65° C.
Sequence identity may be determined as defined herein, across a pre-defined window of comparison. A comparison window may be 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% of the full length reference sequence.
Manipulating Plant Growth/Structure
By “manipulate” is meant altering the native growth pattern of a plant, such that a plant manipulated according to an aspect of the present invention will exhibit an altered growth pattern and/or structure compared to a wild type (non-manipulated) plant of the same species, maintained under identical conditions. The manipulation is preferably effected by a method of the first aspect as defined herein.
The manipulation preferably includes increasing the expression and/or activity of PXY and/or CLE in a plant.
Increased expression will generally result in an increased amount of the protein in a cell or tissue compared to the amount in a corresponding cell or tissue in a wild type plant of the same species maintained under identical conditions. Increased expression may be determined either by measuring the relative amounts of the gene product in a cell or tissue extracted from the modified plant and comparing it to the level in a corresponding cell or tissue from a wild type plant using techniques available in the art such as activity assays, Western blots using antibodies capable of specifically binding the polypeptide, Enzyme-Linked ImmunoSorbent Assay (ELISA), radio-immuno-assays (RIA), immunohistochemistry, immunocytochemistry, immunofluorescence, Northern blot analysis, reverse transcription polymerase chain reaction (RT-PCR) analysis (including quantitative, semi-quantitative or real-time RT-PCR) and RNA-in situ hybridization. Increased expression may also be determined by measuring the phenotypic effects of the protein, and determining whether there is an increase in a phenotypic effect compared to the corresponding phenotype in a wild type plant maintained under identical conditions.
Increased protein activity may be determined herein by measuring the phenotypic effects on the modified plant and comparing the phenotypic effects to the same phenotypes of a wild type plant maintained under identical conditions. A statistically significant improvement in a phenotypic effect in the modified plant compared to the wild type plant is indicative of an increase in the expression and/or activity of the protein.
It is envisaged that where a plant naturally expresses PXY or CLE, their modulation may be achieved by altering the expression pattern of the native gene(s) and/or production of the polypeptide. This may be achieved by any suitable method, including altering transcription of the gene, and/or translation of the mRNA into polypeptide, and post-translational modification of the polypeptide.
Tissue specific expression or activity means that the nucleic acid molecule is preferentially expressed in a particular tissue compared to another tissue of the same plant, or the protein is preferentially active in one tissue compared to another. Specific expression may be achieved using a specific regulatory element to control expression of the nucleic acid molecule.
Altering the expression pattern of a native gene may be achieved by placing it under control of a heterologous regulatory sequence, which is capable of directing the desired expression pattern of the native gene as defined herein. Suitable regulatory sequences are described herein.
Alternatively, regulation of expression of the native gene may be altered through changing the pattern of transcription factors which mediate expression of the gene. This may require the use of modified transcription factors, whose binding pattern is altered to obtain a desired expression pattern of the gene.
Alternatively, the copy number of the native gene may be increased or decreased, in order to change the amount of expression of the gene, for example by introducing into a plant cell an expression cassette comprising the gene. The gene may be under control of a suitable regulatory element to achieve the desired tissue specific expression, as described herein.
Suitable methods for carrying out these embodiments of the invention are known to persons skilled in the art, and may employ the use of an expression construct according to the invention.
In addition, modulating the activity mediated by CLE and/or PXY may be achieved by altering their binding pattern, in order to up regulate the downstream signalling pathway. The binding pattern may be altered in any suitable way, for example by altering the structure, binding affinity, temporal binding pattern, selectivity and amount available for binding on the cell surface of CLE and/or a PXY. A binding pattern may be altered by making appropriate variations to the ligand polypeptide, for example to change its binding site to the receptor, using known methods for mutagenesis. Alternatively, non-protein analogues may be used. Methods for manipulating a polypeptide used in the present invention are known in the art, and include for example altering the nucleic acid sequence encoding the polypeptide. Methods for mutagenesis are well known. Preferably, where variants are produced using mutagenesis of the nucleic acid coding sequence, this is done in a manner which does not affect the reading frame of the sequence and which does not affect the polypeptide in a manner which affects the desired biological activity.
In selecting suitable variants for use in the present invention, routine assays may be used to screen for those which have the desired properties. This may be done by visual observation of plants and plant material, or measuring the biomass of the plant or plant material.
The manipulation may be stable or transient.
Over expression of PXY and/or CLE in the vascular tissue of a plant may be used to increase the number of cells in the vascular tissue of a plant, but without increasing the actual biomass of the plant (i.e. the number of cells may be increased, but the size of these cells is smaller). This has the effect of increasing the density of the vascular tissue, and therefore producing a harder wood. Thus, the invention includes methods for the production of a wood product having a particular density, comprising the steps of the first aspect as defined herein. In addition, it is envisaged that by manipulating plant cells to differentiate their vascular tissue, and therefore grow, environmental growth signals may be bypassed and the present invention may be used to extend the growth season of plants, beyond that which would be possible in a native plant.
Regulatory Elements
A regulatory element controls expression of a gene to which is operably linked, for example the spatial and/or temporal expression.
Regulatory elements include, without limitation, promoters, 5′ and 3′ UTR's, enhancers, transcription factor or protein binding sequences, start sites and termination sequences, ribozyme binding sites, recombination sites, polyadenylation sequences, and sense or antisense sequences. As used herein, the term “promoter” refers to a region of DNA which lies upstream of the transcriptional initiation site of a gene to which RNA polymerase binds to initiate transcription of RNA. A regulatory element may be DNA, RNA or protein. Preferably, a regulatory element is a nucleic acid sequence which is capable of directing tissue specific expression of a coding sequence to which it is operably linked.
A regulatory element is therefore preferably tissue specific, vascular tissue specific. It may be specific for directing expression in the cambium, xylem and/or phloem tissue of a plant. Preferred regulatory elements are cambium or phloem specific.
A tissue specific regulatory element need not direct expression exclusively in the relevant tissue, but may direct expression in non-vascular tissue (or non-cambium or non-phloem) tissue, but may direct limited or absent expression or activity in the non-vascular (e.g. non-cambium or non-phloem) tissue.
A regulatory element may preferably be plant derived, in order to provide the desired tissue specificity. Preferably, a regulatory element may be derived from the same species of plant as the plant being modulated. However, it is envisaged that non-plant regulatory sequences may be suitable for use in the invention where they are capable of providing tissue specific expression, for example when used in conjunction with another tissue specific regulatory element. Such promoters include viral, fungal, bacterial, animal and plant-derived promoters capable of functioning in plant cells.
A regulatory element may be inducible and may direct expression in response to environmental or developmental cues, such as temperature, chemicals, drought, and others. It may be developmental stage specific. A regulatory element may be constitutive, whereby it directs expression under most environmental or developmental conditions. In a preferred aspect, the promoter is an inducible promoter or a developmentally regulated promoter.
Phloem specific promoters include SUC2, APL, KAN1, KAN2, At4g33660, At3g61380, and At1g79380, and PP2. Preferably, the promoter is a sequence present in the upstream region of the PP2 gene of Populus trichocarpa, preferably within 1999 bp upstream of the start codon of the PP2 gene. The promoter may be obtained using primers: (pPtPP2-FatccctaggcctgcaggTAAGCTATGTACGTTTTGG (SEQ. ID. 20) and pPttANT-R atcactagtGACAAGCTGAGAGACTG (SEQ. ID. 21)).
Xylem specific promoters include REV, IRX1 COBL4, KOR, At2g38080, and Atlg2744.
Cambium tissue specific promoters include ANT. Preferably, the promoter is a sequence present in the upstream region of the ANT gene of hybrid aspen from 1156 bp upstream of the start codon to 906 bp upstream of the start codon. The promoter may be obtained using primers primers: pPttANT-F:atcgggcccCCGAAGTTGCTCACTTC (SEQ. ID. 22) and pPttANT-R:atcactagtGACAAGCTGAGAGACTG (SEQ. ID. 23).
Also included are functional equivalents (fragments and variants) of a regulatory element as defined herein, wherein such equivalents are capable of directing vascular tissue specific expression of a coding region to which they are operably linked, preferably cambium or phloem specific expression. A preferred functional equivalent may show reduced non-desirable activity compared to the native regulatory element. A functional variant will preferably comprise at least 70% sequence identity to the PttANT or PttPP2 promoter sequence defined herein, more preferably at least 75%, 80%, 82%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the PttANT or PttPP2 promoter sequence defined herein.
A functional fragment is a portion of a regulatory element or variant thereof as defined herein, preferably which retains some or all of the biological activity of the full length regulatory element. Preferably, a functional fragment of a regulatory element as defined herein is capable of directing vascular tissue specific expression of a coding region to which they are operably linked, preferably cambium or phloem specific expression. A fragment may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length regulatory element. A fragment may be 40%, 50%, 60%, 70%, 80%, 90%, or 95% of the length of the full length PttANT or PttPP2 promoter sequence defined herein. Preferably, a fragment may be at least 30 amino acids in length, preferably at least 50, 70, 90, 100, 200, 300, 400 or 500 base pairs in length.
Growth and Structure
Preferably, altered growth is improved or increased growth, preferably vascular growth. Growth of a manipulated plant may be compared to growth of a wild type plant which has been maintained under identical conditions. Indicators of growth used are the radial diameter, vigour, growth rate, the amount of leaf tissue, the amount of biomass, and seed yield.
Radial diameter may be measured at breast height, for saplings and mature trees. The radial diameter may be used as an indication of biomass volume. It may be expressed as a unit of length. Vigour may be calculated by the increase in growth parameters, such as leaf area, fibre length, rosette diameter, plant fresh weight, and the like, per specified time period. An increase in vigour may be used to determine the plant yield, or may impact the plant yield (amount of tissue produced per plant per growing season). Growth rate can be measured using digital analysis of growing plants for example. Images of plants may be captured at regular intervals and the rosette area calculated by digital analysis. Rosette area growth is calculated using the difference between in area between the days of sampling divided by the difference in days between sampling. Alternatively, biomass produced, leaf size, root length etc. can be used as indicators of growth rate. Seed yield can be obtained by collecting the total seeds from a number of plants (e.g. 846), weighing them and dividing the total weight by the number of plants. Leaf tissue is preferably harvested and measured during summer, prior to leaf fall.
An altered structure may be a result of altered growth, or may be exhibited as the order of the vascular tissue. Wild type structure may be recognised by the ordered layout of the cells in defined rows, in contrast to an unordered structure where vascular tissue cells are present randomly without any recognisable pattern. A plant modified by the present invention will preferably show an ordered vascular tissue structure.
The vascular tissue comprises xylem, phloem and cambium cells. The phloem comprises living cells, responsible for transport within the plant. Phloem tissue may comprise conductive cells, parenchyma cells and supportive cells. The cambium lies between the phloem and xylem, and is a source of phloem and xylem cells. Xylem cells are also responsible for transport. Xylem cells are typically dead, and transport water within a plant.
The altered growth may be achieved by increasing the levels of PXY and/orCLE in a tissue specific manner in the vascular tissue of a plant. A manipulated plant may have increased levels/activity of PXY and/or CLE (or functional equivalents thereof) in the vascular tissue and at a pre-selected developmental stage, compared to the level/activity in the same tissue of a wild type plant of the same species, at the same developmental stage and grown in identical conditions.
Preferably, the levels of PXY are increased by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% compared to the level of PXY in a wild type plant maintained under identical conditions. Preferred levels of CLE are increased by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% compared to the level of CLE in a wild type plant maintained under identical conditions.
The increase in level of PXY may directly increase the activity of PXY in the tissue, or the activity may be increased independently of the amount of PXY present, for example through modulation of interaction between PXY and its ligand, CLE. Preferably, the activity of PXY is increased by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% compared to the activity of PXY in a wild type plant maintained under identical conditions. Preferred activity of CLE is increased by at least about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% compared to the activity of CLE in a wild type plant maintained under identical conditions.
Increased growth may be defined as at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80% or greater increase in radial diameter, vigour, growth rate, the amount of leaf tissue, the amount of biomass, and/or seed yield as compared to a wild type plant maintained under identical conditions.
Alterations in growth and/or structure may be assessed at periodic intervals during the lifetime of a plant, and particularly in early development. For example, expression levels of PXY and/or CLE may be sampled at 3 or 6 monthly intervals, or annually.
The source of biomass in plants is their woody tissue, derived from the vascular meristems of the plant such as the cambium and procambium, which divide to form the phloem and xylem cells of the vascular tissue within the plant stems and roots. The cambium and procambium (collectively known as the vascular meristems) are growth zones which enable the plant to grow laterally, thus generating the majority of biomass. It has been shown that increasing levels of PXY and/or CLE in the vascular tissue of a plant enhances lateral growth, thereby leading to an increase in the plant biomass. This may provide an additional source of biomass for various industries dependent upon plant derived products, such as the biofuel or paper industries.
Expression Cassette
A regulatory element for directing tissue specific expression may be provided in an expression cassette, as described herein. In addition to a regulatory element for directing expression of PXY or CLE in the vascular tissue as described herein, an expression cassette may comprise terminator fragments, polyadenylation sequences, enhancer sequences, introns, and other sequences. These elements must be compatible with the remainder of the expression cassette. These elements may not be necessary for the expression or function of the gene but may serve to improve expression or functioning of the gene by affecting transcription, stability of the mRNA, or the like. Therefore, such elements may be included in the expression construct to obtain the optimal expression and function of PXY and CLE in the plant.
An expression cassette may further comprise additional region(s) that allows protein targeting, stabilization, and/or purification. The open reading frame may be orientated in either a sense or anti-sense direction. An expression cassette may be provided as part of a vector or expression construct.
An expression cassette may further comprise a second regulatory element for directing tissue specific expression of a second gene, e.g. PXY or CLE. The second regulatory sequence may be operably linked to the second gene, as described herein.
Where two or more coding sequences are operably linked to the same regulatory element, the coding sequences may be inter-linked via an internal ribosome entry site (IRES) sequence which facilitates translation of polynucleotide sequences positioned downstream of the IRES sequence.
Vectors
Herein, a vector is the vehicle used to transport the expression cassette into the cell, to produce a transformed or transgenic cell. Therefore a vector may comprise genetic material in addition to the expression cassette, for example one or more nucleic acid sequences that permit it to replicate in one or more host cells, such as origin(s) of replication, selectable marker genes and other genetic elements known in the art (e.g., sequences for integrating the genetic material into the genome of the host cell, and so on). The vector may be an expression vector.
Vectors include viral derived vectors, bacterial derived vectors, plant derived vectors and insect derived vectors. A vector will preferably be capable of propagating in both a bacterial host cell, such as E. coli, and be compatible with propagation in a plant cell. A vector may be a phagemid, plasmid, a phage, a virus, or an artificial chromosome.
A typical vector may comprise one or more of a promoter, selection marker, signal sequence, regulatory element (e.g. polyadenylation sequences, untranslated regions, 3′ untranslated regions, termination sites and enhancers). Such companion sequences may be of plasmid or viral origin, and may provide the necessary characteristics to enable the vector to be generated in bacteria and introduced to a plant cell. A bacterial/plant vector may preferably comprise a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T-DNA sequences for Agrobacterium-mediated transfer to plant chromosomes.
A cloning vector is designed so that a coding sequence (e.g. PXY or CLE) is inserted at a particular site and will be transcribed and translated. The basic bacterial/plant vector construct may preferably comprise a broad host range prokaryote replication origin; a prokaryote selectable marker; and, for Agrobacterium transformations, T-DNA sequences for Agrobacterium-mediated transfer to plant chromosomes. A vector may also comprise suitable sequences for permitting integration of the expression cassette into the plant genome. These might include transposon sequences, Cre/lox sequences and host genome fragments for homologous recombination, as well as Ti sequences which permit random insertion of an expression cassette into a plant genome.
A vector of the present invention may comprise a transcriptional termination region at the opposite end of the gene from the transcription initiation regulatory region. The transcriptional termination region may normally be associated with the transcriptional initiation region or derived from a different gene. The transcriptional termination region to be used may be selected, particularly for stability of the mRNA, to enhance expression. Examples of termination regions include the NOS terminator from Agrobacterium Ti plasmid and the rice α-amylase terminator.
Selectable markers encode easily assayable marker proteins are well known in the art. In general, a selectable marker is a gene which is not present or expressed by the recipient organism or tissue and which encodes a protein whose expression is manifested by some easily detectable property, e.g. phenotypic change or enzymatic activity.
Suitable selectable marker may be used to facilitate identification and selection of transformed cells. These will confer a selective phenotype on the plant or plant cell to enable selection of those cells which comprise the expression cassette. Preferred genes include the chloramphenicol acetyl transferase (cat) gene from Tn9 of E. coli, the beta-gluronidase (gus) gene of the uidA locus of E. coli, the green fluorescence protein (GFP) gene from Aequoria victoria, and the luciferase (luc) gene from the firefly Photinus pvralis. If desired, selectable genetic markers may be included in the vector, such as those that confer selectable phenotypes such as resistance to antibodies or herbicides (e.g. kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate). A selectable marker may be provided on the same expression cassette or vector as the tissue specific regulatory element, or may be provided on a separate expression cassette and co-transformed with the expression cassette of the invention. A selectable marker and/or reporter gene may be flanked with appropriate regulatory sequences to enable their expression in a plant cell.
An expression cassette may be utilized to stably or transiently transform plant cells. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
For further details see, for example, Molecular Cloning: Laboratory Manual: 2^ndedition, Sambrook et al. 1989, Cold Spring Habor Laboratory Press or Current Protocols in Molecular Biology, Second Edition, Ausubel et al. Eds., John Wiley & Sons, 1992.
Methods of Transformation
Methods to transform woody species of plant are well known in the art. For example the transformation of poplar is disclosed in U.S. Pat. No. 4,795,855 and WO91 18094. The transformation of eucalyptus is disclosed in EP1050209 and WO9725434.
An expression cassette or vector of the present invention may be used to stably or transiently transform a plant cell. In stable transformation, the exogenous polynucleotide is integrated into the plant genome and as such it represents a stable and inherited trait. In transient transformation, the exogenous polynucleotide is expressed by the cell transformed but it is not integrated into the genome and as such it represents a transient trait.
Stable integration may include i) Agrobacterium-mediated gene transfer (ii) Direct DNA uptake. The latter may include including methods for direct uptake of DNA into protoplasts, DNA uptake induced by brief electric shock of plant cells: DNA injection into plant cells or tissues by particle bombardment by the use of micropipette systems; glass fibres or silicon carbide whisker transformation of cell cultures, embryos or callus tissue, or by the direct incubation of DNA with germinating pollen.
The Agrobacterium system includes the use of plasmid vectors that contain defined DNA segments that integrate into the plant genomic DNA. Methods of inoculation of the plant tissue vary depending upon the plant species and the Agrobacterium delivery system. A widely used approach is the leaf disc procedure which can be performed with any tissue explant that provides a good source for initiation of whole plant differentiation. A supplementary approach employs the Agrobacterium delivery system in combination with vacuum infiltration. The Agrobacterium system is especially viable in the creation of transgenic dicotyledonous plants.
The regenerated transformed plants can then be cross-bred and resultant progeny selected for superior growth, biomass, yield and/or vigor traits, using conventional plant breeding techniques.
Routine assays may be used to screen for those which have the desired properties. This may be done by visual observation of plants and plant material, or measuring the biomass of the plant or plant material.
Plant
A transgenic plant will include a plant that is grown from a recombinant plant cell, and all ancestors and progeny of that plant that contain the recombinant nucleic acid. This includes offspring produced sexually or asexually. It is understood that the term transgenic plant encompasses the entire plant or tree and parts of the plant or tree, for instance grains, seeds, suspension cultures, flowers, leaves, roots, fruit, pollen, callus tissue, gametophytes, sporophytes, stems, embryos, microspores etc.
Preferred plants for use in the present invention are those which are targets for biomass, and/or are readily grown, exhibit high growth rates, are easily harvested, and can be readily converted to a biofuel. Preferred plants include grasses, trees, crops, and shrubs.
According to some embodiments of the invention, the plant used by the method of the invention is a crop plant such as rice, maize, wheat, barley, peanut, potato, sesame, olive tree, palm oil, banana, soybean, sunflower, canola, sugarcane, alfalfa, millet, leguminosae (bean, pea), flax, lupinus, rapeseed, tobacco, popular and cotton.
Suitable plants for use in the present invention are those which in their native form produce a high yield of feedstock, for paper or fuel production. Examples of suitable plant types include perennial fast growing herbaceous and woody plants, for example trees, shrubs (such as tobacco) and grasses. Trees for use in the invention include birch, spruce, pine, poplar, hybrid poplar, willow, silver maple, black locust, sycamore, sweetgum and eucalyptus. Perennial grasses include switchgrass, reed canary grass, prairie cordgrass, tropical grasses, Brachypodiumdistachyon, and Miscanthes. Crops include cereals and pulses, wheat, soybean, alphalpha, corn, rice, maize, and sugar beet, potatoes, tapioca, sorghum, millet, cassava, barley, pea, and other root, tuber or seed crops. Important seed crops are oil-seed rape, sugar beet, maize, sunflower, soybean, and sorghum. According to a further embodiment of the invention said plant is a woody plant selected from: poplar; eucalyptus; Douglas fir; pine; walnut; ash; birch; oak; teak; spruce. Horticultural plants to which the present invention may be applied may include lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums. The present invention may be applied in tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum. Grain plants that provide seeds of interest include oil-seed plants and leguminous plants. Seeds of interest include grain seeds, such as corn, wheat, barley, rice, sorghum, lye, etc. Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants include beans and peas. Beans include guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.
In a further embodiment of the invention said plant is selected from: corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cerale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuas), wheat (Tritium aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Lopmoea batatus), cassava (Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera), pineapple (Anana comosus), citrus tree (Citrus spp.) cocoa (Theobroma cacao), tea (Camellia senensis), banana (Musa spp.), avocado (Persea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifer indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia intergrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris), oats, barley, vegetables and ornamentals.
Plant Derived Product
The present invention has uses in methods which require increased biomass in plants, for example where plant biomass is used in the manufacture of products such as biofuels and paper. The invention is not limited to methods of making these particular products, and it is envisaged that the invention will be applicable to the manufacture of a variety of plant based products. In addition, the invention is also useful in altering the characteristics of plant material, such that the plant material may be adapted for particular purposes (such as disclosed in WO2010029357, which is incorporated in its entirety by reference).
A plant-derived product may include seed, biomass, fibres, forage, biocomposites, biopolymers, wood, biofuel, board or paper.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other components. Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features described in conjunction with a particular embodiment of the invention are to be understood to be applicable to any other embodiment described herein unless incompatible therewith. All of the features disclosed in this specification (including any accompanying claims and drawings) may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims and drawings).
The invention is further described in the following examples with reference to the Figures, in order to illustrate the invention in a non-limiting manner:

EXAMPLES

Binary Vectors for Plant Transformation
For poplar 35S overexpression vectors, primers were designed against Populus trichocarpa (Tuskan et al (2006). Science 313, 1596-1604)

	CLE41
	(Potri.012G019400):
	PttCLE41-F:
	(SEQ. ID. 24)
	CACCTAGCTAGCCTTGGTGCTGGT

	PttCLE41-R:
	(SEQ. ID. 25)
	ACCCCTTAATTCCCCCATTA
	and

	PXY (Potri.003G107600):
	PttPXYF:
	(SEQ. ID. 26)
	CACCATGAAACTCCCTTTTCTTTT

	PttPXY-R
	(SEQ. ID. 27)
	ACATTCGACTGCAGGCTTTT

and used to amplify sequences from DNA extracted from hybrid aspen (Populus tremula×tremuloides clone T89). These were subsequently cloned into pK2GW7 (Karimi, et al (2002). Trends in Plant Science 7, 193-195.2) via pENTR-D-TOPO. For the rolD::PttPXY 35S::CLE41 construct, PttCLE41 was subcloned into pDONRP4-P3 which was combined with pENTR-D-TOPO-PttPXY and pK7m34GW2-8m21GW3 (Karimi et al (2007) Plant Physiology 145, 1183-1191) using an LR clonase reaction. Cloned PttPXY and PttCLE41 were sequenced either in entry clones or expression clones. Sequences were annotated by aligning with P. trichocarpa sequences. Annotated sequences for PttPXY (accession number, KP682331) and PttCLE41 (accession number, KP682332) were submitted to NCBI. During the cloning a mutation was accidently introduced that had removed the stop codon at the end of the PttPXY gene and resulted in a 44 amino acid extension encoded by the vector being added to the C terminus.

For tissue specific expression, PttPXY and PttCLE41 pENTR-D/TOPO entry clones were used in an LR clonase reaction in combination with custom Gateway destination vectors, pVX31 (ApaI-pPttANT1-SpeI-R1R2 Gateway Cassette-t35S-SbfI) and pVX33 (SbfI-pPtPP2-SpeI-R1R2 Gateway Cassette-t35S-SbfI), which were constructed in a pCambia2300 backbone using restriction based cloning. The promoter sequences were chosen on the basis of poplar expression data. PttANT (Potri.002G114800) regulatory sequences were used for cambium specific expression and a PtPP2 (Potri.015G120200) promoter was used for phloem specific expression. For the PtPP2 promoter from Populus trichocarpa primers (pPtPP2-F atccctaggcctgcaggTAAGCTATGTACGTTTTGG (SEQ. ID. 20), pPttANT-R atcactagtGACAAGCTGAGAGACTG (SEQ. ID. 21)) were used to amplify a fragment of 1999 bp upstream of the start codon. For the PttANT1 promoter, primers (pPttANT-F atcgggcccCCGAAGTTGCTCACTTC (SEQ. ID. 22), pPttANT-R atcactagtGACAAGCTGAGAGACTG (SEQ. ID. 23) were used to amplify a sequence 1156 bp upstream of start codon to 904 bp downstream of the start codon that drove expression in vascular tissue. To create the double tissue specific expression construct, SbfI-pPtPP2-PttCLE41-t35S-SbfI cassette was excised and cloned into SbfI site of ApaI-pPttANT1-PttPXY-t35S-SbfI. Transcriptional reporter lines for pPttANT and pPtPP2 were generated by cloning a fragment encoding eGFP-GUS in in pVX31 and pVX33 using an LR clonase reaction resulting in pPttANT::eGFP-GUS and pPtPP2::eGFP-GUS constructs. Hand sections were stained using a variation on the method described in Rodrigues-Pousada ((1993) The Plant Cell Online 5, 897-911) and were viewed following clearing by overnight incubation at 4° C. in chloral hydrate solution (Berleth, et al (1993) Development 118, 575-587).
Plant Transformation
Arabidopsis transformation was carried out using the method of Clough and Bent (Clough, S. J., and Bent, A. F. (1998). Plant Journal 16, 735-743). For transformation of hybrid aspen (clone T89), a method based on that of Nilsson et al ((1992) Transgenic Research 1, 209-220) was used. Briefly, Agrobacterium strain GV3101 harbouring a binary vector was grown to an OD600 of 0.6, collected by centrifugation in a 50 ml tube and resuspended in MS medium, pH5.8 supplemented with acetosyringone to a final concentration of 25 μM at room temperature. Leaf and petiole sections were cut from hybrid aspen grown under sterile conditions and incubated in the resuspended Agrobacterium for 1 hour and placed on MS agar supplemented with 2% sucrose, BAP (0.2 mg/L), IBA 0.1 mg/L, and TDZ (0.1 mg/L) prior to incubation in dark for 48 hours. Subsequently, plant pieces were rinsed in MS and placed in the light on IVIS agar supplemented with 250 μg/ml cefatoxime and 100 μg/mi kanamycin. Following shoot initiation calli were transferred to woody plant medium (WPM) supplemented with sucrose (2%), BAP (0.2 mg/L), IBA (0.1 mg/L), kanamycin (100 μg/ml) for shoot elongation and subsequently to WPM for rooting. In order to synchronise plant growth for subsequent analysis the top 2 cm of each plantlet to be used was removed and re-rooted on the same day. All plants used for growth analysis were grown side by side in the same incubator and transferred to soil on the same day once roots were established. For long term growth, plants were transferred to a greenhouse in April and maintained for up to 12 months.
Determination of Plant Growth Characteristics
Vascular organization was determined using plant material fixed in FAA, dehydrated through an ethanol series before infiltration and embedding with JB4 embedding media. 5 μM sections were stained with 0.05% aqueous toluidine blue, mounted in Cytoseal and visualised on a Leica 5500 microscope. Vascular tissue was considered to be ordered if xylem could be incorporated in an elliptical shape that excluded the phloem. Xylem cell counts and determination of cell wall area was performed on cross sections from the entire cross section of tissue culture plants 3 weeks post rooting or from the 50th internode of greenhouse grown plants in which case only a 40° segment of the stem was used (FIG. 56A). Cell counting was carried out using Cellprofiler (Carpenter et al (2006) Genome Biol 7, R100) as outlined in Figure S6. For greenhouse grown plants, 10 cm segments were sampled from base of the plant, from 50th internode and from the top of the plants, 12 months following transfer to soil. Material was dried at 50° C. for 4 weeks before weighing.

Results and Discussion

The PXY-CLE signalling pathway is conserved in trees and acts to regulate secondary growth:
Wood is composed of xylem cells that arise from divisions of stem cells that reside within the vascular meristem, known as the cambium or procambium. One mechanism that promotes cell division in vascular meristems of Arabidopsis involves phloem-specific expression of CLE41 that encodes a peptide ligand known as TDIF. TDIF is perceived by a receptor kinase, PXY (also known as TDR) that is expressed in the adjacent stem cells of the procambium (Etchells, and Turner (2010) Development 137, 767-774; Fisher and Turner (2007) Current Biology 17, 1061-1066; Hirakawa (2008) Proceedings of the National Academy of Sciences, USA 105, 15208-15213; Kondo et al (2006) Science 313, 845-848). PXY controls both the orientation (Etchells and Turner (2010) Development 137, 767-774) and rate of cell division in procambial stem cells (Etchells et al, (2013) Development 140, 2224-2234; Hirakawa et al (2010) Plant Cell 22, 2618-2629) and inhibits their differentiation into xylem (Hirakawa et al (2008) Proceedings of the National Academy of Sciences, USA 105, 1520845213; Kondo et al (2014) Nat Commun 5). Consequently, while ectopically overexpressing CLE41 in Arabidopsis increases the number of cells in vascular bundles, these increases are accompanied by repression of xylem differentiation and loss of vascular organization (Etchells and Turner (2010) supra; Hirakawa, et al (2008) supra; Whitford et al (2008) Proceedings of the National Academy of Sciences USA 105, 18625-18630). Furthermore, output from the pathway is regulated by a negative feedback loop in which CLE41 expression results in down regulation of PAT (Etchells and Turner (2010) supra). To determine whether PXY-CLE41 signalling is conserved in poplar, putative orthologous of PXY and CLE41 genes were cloned from the hybrid aspen (Populus tremula×P. tremuloides) referred to hereafter as PttPXY and PttCLE41 respectively (FIG. 5). When overexpressed in Arabidopsis, 35S::PttCLE41 lines demonstrated a loss of vascular organisation, increased numbers of cells per vascular bundle and decreased plant height (FIGS. 6A,B, 7A,B). The 35S::PttPXY construct complemented the Arabidopsis pxy mutant phenotype (FIGS. 6C-D, 7C-E) and this complemented line also restored the ability of the plants to respond to overexpression of the AtCLE41 ligand (FIG. 7C,D). As such, both PttCLE41 and PttPXY clones act as functional orthologues of their respective Arabidopsis genes. Furthermore expression of PttPXY in Arabidopsis plants already engineered for tissue-specific AtCLE41 over-expression resulted in increased plant biomass (FIG. 7E).
Ectopic expression of PttCLE41 or PttPXY leads to abnormal vascular tissue development in trees.
The consequence of constitutively over-expressing these genes in trees was investigated by making use of the 35S promoter that is known to give widespread expression in hybrid aspen (Nilsson et al (1996) Plant Mol. Biol. 31, 887-895). The 35S::PttPXY and 35S::PttCLE41 constructs (see above) were individually over-expressed or over-expressed both genes together in a single binary plasmid containing that contained 35S::PttCLE41 and rolD::PttPXY cassettes. To varying degrees, all independent lines (n=15) of 35S::PttCLE41 hybrid aspen had intercalated xylem and phloem (FIG. 35S::PttPXY lines (n 10) also demonstrated disrupted organisation in parts of the xylem, but to a much lesser extent than seen in 35S::PttCLE41 (FIG. 1A). 7 out of 15 35S::PttCLE41-rolD::PttPXY lines appeared normal while the remaining 8 exhibited varying degrees of tissue disruption (FIG. 1A). None of these lines led to significant increases in tree growth, in fact 35S::PttCLE41 lines were significantly shorter than wild type, exhibiting various growth abnormalities (FIG. 1B and FIG. 8).
Tissue specific expression of PttPXY and PttCLE41 increases vascular cell division and retain normal vascular tissue organization.
It was hypothesized that the tissue-specific expression of both PttPXY and PttCLE41 might be important both for tissue organization and to maximize cambial cell division. Transcriptomic data shows that in poplar, PXY is expressed predominantly in the cambium and at a low level in the xylem (Schrader et al (2004) Plant Cell 16, 2278-2292). Poplar microarray data identified the ANTEGUMENTA (ANT) gene as highly expressed only within the division zone (Schrader et al (2004) supra). Using an early draft of the Populus trichocarpa genome (Tuskan et al (2006) supra) as a guide, we identified and cloned a putative promoter from hybrid aspen (PttANT), although better annotation of the genome subsequently suggested the PttANT promoter fragment contained sequences both upstream and downstream of the putative transcriptional start site. Analysis of leaves from PttANT::GUS plants showed clear vascular specific GUS expression, while in the stems, GUS activity was restricted to the dividing cambial zone (FIG. 2B) consistent with our initial interpretation of the expression data. We also identified and cloned regulatory sequences from a phloem specific lectin gene, PHLOEM PROTEIN2 (PP2), from Populus trichocorpa (PtPP2). GUS analysis verified this promoter as vascular tissue specific in the leaves and giving excellent phloem-specific expression in stems (FIG. 2C). These promoters were used to generate 3 constructs designed to give tissue specific increases in expression: PttANt:PttPXY, PtPP2::PttCLE41 and PtPP2::PttCLE41-PttANT::PttPXY. In contrast to 35S::PttCLE41 (FIG. 1A), PtPP2::PttCLE41 lines demonstrated highly organized vasculature in all 14 lines examined (FIG. 1C). 7 out of 15 PttANT::PttPXY lines demonstrated minor disruptions in xylem morphology (FIG. 1C; arrow) similar to those observed in 35S::PttPXY trees (FIG. 1A), however all 12 independent PtPP2::PttCLE41-PttANT::PttPXY double overexpression lines analysed exhibited highly organized vascular tissue comparable to that of wild-type controls (FIG. 1C). Strikingly, PttPP2::PttCLE41, PttANT::PttPXY and PtPP2::PttCLE41-PttANT:PttPXY double overexpression lines clearly demonstrated increases in the number of vascular cells as early as 3 weeks post rooting in tissue culture (FIG. 1C and FIG. 55A).
Tissue specific expression of PttPXY and PttCLE41 results in trees that grow faster.
The growth of these transgenic hybrid aspen trees was further monitored following transfer to soil and maintenance in the greenhouse. Over a 6 month period PtPP2::PttCLE41, PttANT::PttPXY and PtPP2::PttCLE41-PttANT::PttPXY plants grew normally (FIG. 1C) and were consistently larger than the control plants with both greater stem diameter and plant height (FIG. 3A,B). PtPP2::PttCLE41-PttANT::PttPXY lines gave the largest increase in radial growth and after 6 months in the greenhouse exhibited a 35% increase in stem diameter compared to untransformed controls and an increase of 10% compared to PtPP2::PttCLE41, the next best performing genotype (FIG. 3A). The PtPP2::PttCLE41-PttANT::PttPXY lines also demonstrated a 56% increase in height over their wild-type counterparts and a 12% increase in height over the next-best performing transgenic line (PttANT::PttPXY) (FIG. 3B). This increase was due to a generally faster growth rate with PtPP2::PttCLE41-PttANT::PttPXY plants having on average 90 internodes compared to the a mean of 60 for control plants (FIG. 3C), as well as an increase in internode length (FIG. 3D). While the plants appeared morphologically normal (FIG. 1C), the PtPP2::PttCLE41-PttANT::PttPXY lines also exhibited increases in leaf area (FIG. 3E) with the average leaf area increased by almost 2 fold. These increases in growth reflect PXY/CLE signalling acting on other aspects of plant development or be a consequence of increases in sink strength. They contribute to a general increase in biomass that is likely to further improve the effectiveness of any biotechnological application of these discoveries.
Tissue specific expression of PttPXY and PttCLE41 results in large increases in wood and biomass formation.
To better understand the cause of the increases in stem diameter in PtPP2::PttCLE41-PttANT::PttPXY lines, at 33 weeks half of the trees from each line were harvested and sectioned for stem material in order to perform cell counts for each line as described in FIG. 10. In order to examine material from a similar developmental stage and to account for the differing sizes of the trees examined, the analysis was carried out on material from the 50th internode. A dramatic increase in xylem cell numbers was observed, that correlated with the increase in stem diameter with PtPP2::PttCLE41-PttANT::PttPXY lines having the largest number of xylem cells, 189% that of control plants (FIG. 3F). Within individual lines there was also a correlation between cell numbers and PttCLE41 expression and to a lesser extent with PttPXY expression (FIG. 11). To determine whether it was possible to increase wood formation without altering xylem morphology, Cellprofiler was adapted (Carpenter et al. (2006) supra) to measure a number of morphological characteristics of the xylem (FIG. 10). The analysis revealed no significant differences in average cell size, average cell lumen size, average cell wall area and vessel numbers as a proportion of total xylem cells in PtPP2::PttCLE41-PttANT::PttPXY compared to controls lines (Table 1) indicating that the increased wood production did not alter wood morphology.

TABLE 1

Analysis of transverse section of xylem from the 50th
internode of control and PtPP2::CLE41-PttANT::PXY
plants. Mean of 5 independent lines are shown with
the standard error. Area measurements are in arbitrary
units. Statistical analysis was carried out using
a T-test, no significant differences were found.

		PtPP2::CLE41-
	Control	PttANT::PXY

Average cell size	607 ± 13	577 ± 19
Average lumen size	312 ± 13	346 ± 9
Average cell wall area	265 ± 24	260 ± 16
Vessels per 1000 cells	50 ± 2	49 ± 6

To determine whether the improved growth characteristics led to increased woody biomass, the remaining trees were allowed to grow for a further 6 month period after which we determined dry weight (FIG. 3G) and wet weight (FIG. 9) at various points along the stem. Consistent with previous observations, measurement at the base, 50th internode (middle) and at the top of the stem demonstrated that PtPP2::PttCLE41-PttANT::PttPXY lines exhibited significant increases in dry weight in comparison to other lines used in this study. In particular, at the middle and base of trees, the dry weight of PtPP2::PttCLE41-PttANT::PttPXY stem segments were on average more than twice the weight of the control plants.
In order to ensure that the differences observed were reproducible, material from six independent PtPP2::PttCLE41-PttANT::PttPXY lines was clonally propagated. The growth of these plants was monitored weekly, starting shortly after transfer to soil. The diameter of several clones was significantly bigger than wild type at all stages monitored (FIG. 4). There was also variation between clones such that plants from line 2 were both significantly taller and exhibited a significantly larger diameter than plants from line 3 at all 5 time points examined (FIG. 4).

CONCLUSIONS

Trees represent a huge natural resource used for the production of paper, fuel and materials, and are an increasingly important carbon sink (Stephenson et al (2014) Nature 507, 90-93) that can help to ameliorate anthropogenic increases in atmospheric CO2. Recently, trees have also been the focus of intense interest as a renewable source of plant biomass that may be converted into bioethanol (Somerville et al (2006) Science 312, 1277) and other chemicals for the rapidly expanding field of industrial biotechnology (Raunikar et al (2010). Forest Policy and Economics 12, 48-56). The majority of biomass in trees is derived from radial growth that is characterised by growth rings in the wood. The size of each growth ring is intimately linked to the environmental conditions during the growing season that year. The data provided herein data suggests that the PXY-CLE pathway functions in trees to regulate secondary growth and is likely to be central to the way in which trees evolved secondary growth Together, the analysis demonstrates that by engineering the PXY-CLE pathway we were able to dramatically increase secondary growth in plants shortly after they were first rooted (FIG. 3 and FIG. 9), the earliest point they could be analysed, and the increase in xylem was maintained in plants grown for up to a year (FIG. 4 and FIG. 9). These results indicate that this pattern of growth continues during the lifetime of the tree, thereby providing a means of dramatically increasing tree productivity that would help to meet the increasing demand for renewable resources. While tree productivity may benefit from anthropogenic increases in atmospheric CO2, climate models and recent changes in weather pattern strongly suggest that we are entering a period in which large parts of the globe experience more frequent exposure to extreme and changeable weather (Palmer, et al (2014) Science 344, 803-804) that is likely to have detrimental effects on growth. It will be important to establish whether manipulating PXY-CLE signalling will enable us override the environmental cues that normally regulate plant growth and so enable us to generate trees that are able to maintain high productivity even when exposed to more extreme environmental conditions.

Claims

1. A method for manipulating the growth and/or structure of a plant comprising modifying the plant to specifically increase the expression and/or activity of CLE in the vascular tissue of the plant compared to the expression and/or activity of CLE in the vascular tissue of a wild type plant of the same species maintained under identical conditions, the process comprising:

introducing into the plant a regulatory element which specifically directs expression of CLE in the vascular tissue of the plant,

wherein expression and/or activity of CLE modified in the vascular tissue remains substantially unaltered in non-vascular tissue of the modified plant,

wherein the regulatory element comprises an PP2 promoter sequence produced by using the primer sequence of SEQ. ID. NO. 20, and

wherein the CLE comprises the polypeptide sequence of SEQ. ID. NO. 39.

2. The method according to claim 1, wherein CLE is CLE41 or CLE42 or CLE44.

3. The method according to claim 1, further comprising modifying the plant to specifically increase the expression and/or activity of one or more of CLE41, CLE42 or CLE44 in the vascular tissue of the plant compared to the expression and/or activity of the respective CLE in the vascular tissue of a wild type plant of the same species maintained under identical conditions, wherein expression and/or activity of the one or more of CLE41, CLE42 or CLE44 modified in the vascular tissue remains substantially unaltered in non-vascular tissue of the modified plant.

4. The method according to claim 1, wherein the method comprises introducing into the plant a regulatory element which specifically directs expression of one or more of CLE41, CLE42 or CLE44 in the phloem of the plant.

5. The method according to claim 1, wherein the regulatory element is derived from hybrid Aspen.

6. The method according to claim 1, wherein the regulatory element is provided in an expression cassette, operably linked to a nucleic acid molecule encoding CLE.

7. The method according to claim 1, wherein the regulatory element is provided in an expression cassette which comprises sequences for insertion of the regulatory element into the plant genome, operably linked to the native CLE gene.

8. The method according to claim 7, wherein the expression cassette is provided in a vector.

9. The method according to claim 8, wherein the vector is capable of propagating in both a bacterial host cell and a plant cell.

10. The method according to claim 8 further comprising the step of:

introducing the expression cassette or vector into a plant cell.

11. The method according to claim 1 further comprising:

measuring expression of CLE by measuring the relative amounts of the gene product(s) in a cell or tissue extracted from the modified plant and comparing it to the level of said gene products(s) in a corresponding cell or tissue from a wild type plant of the same species maintained under identical conditions.

12. The method according to claim 11, wherein the levels of CLE are increased by at least about 20% compared to the level of CLE in a wild type plant maintained under identical conditions.

13. The method according to claim 11, wherein the activity of CLE is increased by at least about 5% compared to the activity of CLE in a wild type plant maintained under identical conditions.

14. The method according to claim 11, further comprising:

measuring increased growth by measuring radial diameter, vigour, growth rate, the amount of leaf tissue, the amount of biomass, and/or seed yield in the modified plant comparing it to the corresponding phenotype in a corresponding cell or tissue from a wild type plant.

15. The method according to claim 14, wherein increased growth is at least about 20% increase in radial diameter, vigour, growth rate, the amount of leaf tissue, the amount of biomass, and/or seed yield as compared to a wild type plant maintained under identical conditions.

16. The method according to claim 1, wherein CLE is CLE41 or CLE42 and wherein the nucleic acid encoding CLE41 comprises SEQ. ID. 2 or 5 and wherein the nucleic acid encoding CLE42 is SEQ. ID. 29.