Methods of Increasing Plant Growth

This invention relates to methods of increasing the growth of plants .

Long-term energy security is both a challenge and an opportunity. From the wide range of options currently available, renewable biomass resources offer a very real alternative to fossil fuels. Biomass production provides a carbon-neutral, renewable means of supplying bioenergy and biomaterial resources. Biomass production in short -rotation biomass plantations in Sweden currently averages 8 dry Mg ha ^"1 year^and in the United States 10 to 22 dry Mg ha ^"1 year ^"1 (for short-rotation woody crops) (Ragauskas et al . 2006. Science. Vol. 113, 484-489) .

Plants, like animals, require a supply of new cells for growth and development. Biomass accumulation in woody plant species begins with cell divisions in the primary and secondary meristems. Meristems are the plant stem cell niches. The primary, apical meristems provide cells for root and shoot tip growth. A circumferential secondary meristem, called the vascular cambium (VC) , is located towards the exterior of trees and other woody species. Wood is derived from cell proliferation in the VC. Surprisingly, given its importance to commerce and the environment, although there has been a long tradition of anatomical and physiological analysis of the VC, almost nothing is known of the molecular bases of its establishment and function. Even the cellular components that comprise the VC stem cell niche have remained unidentified.

The Arabidopsis SHORT-ROOT (AtSHR) gene has been shown to play critical roles in the establishment and maintenance of the root apical meristem. In the Arabidopsis root, the SHORT-ROOT (AtSHR) protein, along with SCARECROW (AtSCR) and the PLETHORA (AtPLTl and AtPLT2) proteins, is essential for the specification and maintenance

of the QC and the abutting stem cell populations (Levesque et al . PLoS Biol. 2006 4(5) :el43) . Loss-of - function shr mutants exhibit a progressive disorganization of the QC, a loss of stem cell activity and the cessation of root tip growth (Levesque et al . , PLoS Biol. 2006 May; 4(5) : el43. ) . AtSHR is transcribed in the inner layers of the stele and the protein moves to the nucleus of cells in the adjacent AtSCi?-expressing, QC, endodermis and endodermal/cortex initials and daughter cells (Nakajima et al . , Nature. 2001 Sep 20; 413 (6853) :307-ll) .

In combination with the role in QC and stem cell fate determination, AtSHR is a key regulator of asymmetric division in the Arabidopsis root apex. Together with AtSCR, AtSHR is essential for the asymmetric periclinal division of the cortex/endodermal stem cell daughter (Helariutta et al . , Cell. 2000 May 26; 101 (5) : 555-67) .

Cortical/endodermal cell fate separation of the daughter cells of this division relies on the degradation of AtSHR and AtSCR in the outer cell and the maintenance of AtSHR in the inner, endodermal cell (Helariutta et al . , Cell. 2000 May 26; 101 (5) : 555-67) . Finally, as the root ages, a second cortical layer is produced through the asymmetric division of the endodermis. AtSHR is critical for this division and in this instance it operates through an AtSCR- independent mechanism (Paquette and Benfey Plant Physiol. 2005 Jun; 138 (2) :636-40) .

A complete loss of AtSHR function is clearly detrimental to growth in Arabidopsis, as both the roots and shoots of shr mutants are severely dwarfed compared to the WT (Benfey et al . , Development. 1993 Sep; 119(1) :57-70) .

It has also been reported that ectopic expression of AtSHR in transgenic Arabidopsis plants, driven by either the CaMV 35S, AtSCR or WEREWOLF (AtWER) promoters, can result in a modification of radial patterning in Arabidopsis root tips (Benfey et al . , Cell,

Vol. 101, 2000; Nakaj ima et al . , 2001 Nature; Sena et al . , 2004, Development) . This radial patterning defect occurs through a multiplication of cell layers originating m the root meristem (Benfey et al . , Cell, Vol. 101, 555-567, 2000). Sena et al . (2004) suggested that the supernumerary layers in AtWERpromoter. AtSHR transgenic plants originated from the ectopic expression of AtSHR occurring in the epidermal/lateral root cap initials (stem cells) rather than in the progeny of these cells.

Although AtSHR is essential for root development, the mechanisms involved in cell-to-cell movement of the protein and its precise cell autonomous and non-cell -autonomous modes of action have yet to be determined.

The present inventors have discovered that, whereas a complete loss- of -function of AtSHR leads to the degeneration of the root apical meristem and a dwarfing or Arabidopsis shoots, a partial suppression of the steady- state mRNA levels of AtSHR or the Poplar SHORT-ROOT1 (PtSHRl) resulted in a dramatic and sustained increase in the activities of the shoot primary (tip growth) and secondary (girth growth) meristems. Manipulation of SHORT-ROOT expression may therefore be useful in accelerating growth and increasing biomass production in transgenic plants

An aspect of the invention provides a method of increasing the growth and/or biomass of a plant comprising; altering the expression of a SHORT-ROOT (SHR) polypeptide within cells of said plant.

Expression of the SHORT-ROOT (SHR) polypeptide may be altered relative to control plants, for example wild-type plants.

Alteration of SHORT-ROOT (SHR) expression may increase the rate of post -germination development of plants. For example, alteration of

SHORT-ROOT (SHR) polypeptide expression within cells of the plant may increase the rate of primary (height) and secondary (girth) growth and/or the rate of accumulation of biomass in plant.

Increased growth or biomass may occur in the above-ground portion of a plant e.g. in the stems and other aerial structures of the plant, relative to control plants. The above-ground portion of a plant may therefore have increased primary (height) and secondary (girth) growth and/or increased biomass relative to control plants. In a woody plant, the wood density may be increased.

Alteration of SHORT-ROOT (SHR) polypeptide expression within cells of the plant may also increase the rate of germination of the plant.

Growth may be increased by an overall increase in cell number, for example, the pith, vasculature and cortex may be proportionately greater in plants treated as described herein than in control plants .

SHORT-ROOT (SHR) polypeptides are members of the GRAS superfamily of transcription factors. Transcription factors of the GRAS superfamily share a variable amino- terminus and a highly conserved carboxyl- terminus that contains a variety of recognisable motifs (Bolle C, Planta. 2004 Mar,- 218 (5) : 683 - 92) . SHR polypeptides, such as AtSHR, PtSHRl, PtSHR2A and PtSHR2B share a number of conserved sequences between them that are not shared by other GRAS functional classes (see Figure 3 for conserved sequences and Bolle C, Planta. 2004 Mar; 218 (5) : 683-92) for GRAS domains).

A SHORT-ROOT (SHR) polypeptide may fall within the SHR clade, as shown in Figure 1 for AtSHR, PtSHRl, PtSHR2A and PtSHR2B, in a cladogram of other GRAS protein sequences, in particular sequences of Scarecrow-like (SCL) proteins such as PtSCL35b, PtSCL53b, PtSCL62, PtSCL69b, PtSCL92b, PtSCL97b, AtSCL29 and AtSCL32. A

cladogram may be produced using conventional techniques. For example, a cladogram may be calculated using ClustalW to align the protein sequences, Phylip format for tree output, with 1000 bootstrap replicates and TreeViewX (version 0.5.0) for visualisation.

A suitable SHORT-ROOT (SHR) polypeptide may have the amino acid sequence of any one of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14 or 16 or may be a fragment or variant of this sequence which retains SHR activity.

In some preferred embodiments, a SHORT-ROOT (SHR) polypeptide may have the amino acid sequence of SEQ ID NO: 2

(PU04350_eugene3.01860017) , SEQ ID NO:4 (eugene3.00070144 ) or SEQ ID NO: 6 (eugene3.00640143 ) or may be a fragment or variant of this sequence which retains SHR activity.

A SHORT-ROOT (SHR) polypeptide which is a variant a reference SHR sequence set out herein, such as SEQ ID NO: 2, may comprise an amino acid sequence which shares greater than 30% sequence identity with the reference SHR amino acid sequence, preferably greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 70%, greater than 80%, greater than 90% or greater than 95%.

Particular amino acid sequence variants may differ from a known SHR polypeptide sequence as described herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20- 30, 30-50, or more than 50 amino acids.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA) . GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the

number of gaps. Generally, default parameters are used, with a gap creation penalty = 12 and gap extension penalty = 4.

Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al . (1990) J. MoI. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. MoI Biol. 147: 195-197), or the TBLASTN program, of Altschul et al . (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequence described herein, or may be over a contiguous sequence (i.e. a 'window') of at least 50, 75, 100, 150 or more amino acids or nucleotide triplets, compared with the relevant amino acid sequence or nucleotide sequence.

Certain domains of a SHORT-ROOT (SHR) polypeptide may show an increased level of identity with domains of a SHR reference sequence, such as SEQ ID NO: 2, 4 or 6 , relative to the SHORT-ROOT (SHR) polypeptide sequence as a whole. For example, a SHORT-ROOT (SHR) polypeptide may comprise one or more domains or motifs having an amino acid sequence which has at least 80%, at least 90%, at least 95%, or at least 98% sequence identity or similarity, with an amino acid sequence selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 21. In some preferred embodiments, a SHORT-ROOT (SHR) polypeptide may comprise one or more domains or motifs having an amino acid sequence which his selected from the group consisting of SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20 and SEQ ID NO:21.

In some embodiments, expression of the SHORT-ROOT (SHR) polypeptide may be reduced within cells of said plant. Reduction in this context

excludes complete abolition of expression. For example, expression of the SHORT-ROOT (SHR) polypeptide may be reduced within cells of said plant by up to 90%, up to 80%, up to 70%, up to 60%, up to 50% up to 40% or up to 30%. In other words, expression of the SHORT-ROOT (SHR) polypeptide in the plant may be 0.1 fold, 0.2 fold, 0.3 fold, 0.4 fold, 0.5 fold, 0.6 fold or 0.7 fold of the expression within cells of control plants.

Reduction of SHORT-ROOT (SHR) polypeptide expression as described herein may lead to an increase in plant growth or biomass. In some embodiments, the growth or biomass of the above-ground portion of the plant may be increased.

Expression of the SHORT-ROOT (SHR) polypeptide may be reduced within cells of said plant by any convenient method.

In some embodiments, expression of the SHORT-ROOT (SHR) polypeptide may be reduced by expressing a heterologous nucleic acid which encodes or transcribes a suppressor nucleic acid, for example a suppressor RNA molecule, within cells of said plant.

Nucleic acids as described herein may be wholly or partially synthetic. In particular, they may be recombinant in that nucleic acid sequences which are not found together in nature (do not run contiguously) have been ligated or otherwise combined artificially. Alternatively, they may have been synthesised directly e.g. using an automated synthesiser.

The nucleic acid may of course be double- or single-stranded, cDNA or genomic DNA, or RNA. The nucleic acid may be wholly or partially synthetic, depending on design. Naturally, the skilled person will understand that where the nucleic acid includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T.

"Heterologous" indicates that the gene/sequence of nucleotides in question or a sequence regulating the gene/sequence in question, has been introduced into said cells of the plant or an ancestor thereof, using genetic engineering or recombinant means, i.e. by human intervention. Nucleotide sequences which are heterologous to a plant cell may be non-naturally occurring in cells of that type, variety or species (i.e. exogenous or foreign) or may be sequences which are non-naturally occurring in that sub-cellular or genomic environment of the cells or may be sequences which are non-naturally regulated in the cells i.e. operably linked to a non-natural regulatory element .

The suppression of the expression of target polypeptides in plant cells is well-known in the art. A suitable suppressor nucleic acid may be a copy of all or part of the target SHR gene inserted in antisense or sense orientation or both relative to the SHR gene, to achieve reduction in expression of the SHR gene. See, for example, van der Krol et al . , (1990) The Plant Cell 2, 291-299; Napoli et al., (1990) The Plant Cell 2, 279-289; Zhang et al . , (1992) The Plant Cell 4, 1575-1588, and US-A-5 , 231 , 020. Further refinements of this approach may be found in WO95/34668 (Biosource) ; Angell & Baulcombe (1997) The EMBO Journal 16, 12:3675-3684; and Voinnet & Baulcombe (1997) Nature 389: pg 553.

In some embodiments, the suppressor nucleic acid may be a sense suppressor of expression of the SHORT-ROOT (SHR) polypeptide.

A suitable sense suppressor nucleic acid may be a double stranded RNA (Fire A. et al Nature, VoI 391, (1998)). dsRNA mediated silencing is gene specific and is often termed RNA interference (RNAi) . RNAi is a two step process. First, dsRNA is cleaved within the cell to yield short interfering RNAs (siRNAs) of about 21-23nt length with 5' terminal phosphate and 3' short overhangs (~2nt) . The siRNAs target the corresponding mRNA sequence specifically for

destruction (Zamore P. D. Nature Structural Biology, 8, 9, 746-750, (2001)

siRNAs (sometimes called microRNAs) down-regulate gene expression by binding to complementary RNAs and either triggering mRNA elimination (RNAi) or arresting mRNA translation into protein. siRNA may be derived by processing of long double stranded RNAs and when found in nature are typically of exogenous origin. Micro- interfering RNAs (miRNA) are endogenously encoded small non-coding RNAs, derived by processing of short hairpins. Both siRNA and miRNA can inhibit the translation of mRNAs bearing partially complementary target sequences without RNA cleavage and degrade mRNAs bearing fully complementary sequences.

Accordingly, the present invention provides the use of RNAi sequences based on the SHORT-ROOT (SHR) nucleic acid sequence for suppression of the expression of the SHORT-ROOT (SHR) polypeptide. For example, an RNAi sequence may correspond to a fragment of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or a variant thereof.

siRNA molecules are typically double stranded and, in order to optimise the effectiveness of RNA mediated down- regulation of the function of a target gene, it is preferred that the length and sequence of the siRNA molecule is chosen to ensure correct recognition of the siRNA by the RISC complex that mediates the recognition by the siRNA of the mRNA target and so that the siRNA is short enough to reduce a host response.

miRNA ligands are typically single stranded and have regions that are partially complementary enabling the ligands to form a hairpin. miRNAs are RNA sequences which are transcribed from DNA, but are not translated into protein. A DNA sequence that codes for a miRNA is longer than the miRNA. This DNA sequence includes the miRNA sequence and an approximate reverse complement. When this DNA sequence is transcribed into a single-stranded RNA molecule, the

miRNA sequence and its reverse-complement base pair to form a partially double stranded RNA segment. The design of microRNA sequences is discussed on John et al, PLoS Biology, 11(2), 1862- 1879, 2004.

Typically, the RNA molecules intended to mimic the effects of siRNA or miRNA have between 10 and 40 ribonucleotides (or synthetic analogues thereof), more preferably between 17 and 30 ribonucleotides, more preferably between 19 and 25 ribonucleotides and most preferably between 21 and 23 ribonucleotides. In some embodiments of the invention employing double -stranded siRNA, the molecule may have symmetric 3' overhangs, e.g. of one or two (ribo) nucleotides, typically a UU of dTdT 3' overhang. Based on the disclosure provided herein, the skilled person can readily design suitable siRNA and miRNA sequences, for example using resources such as Ambion's siRNA finder, see http : //www. ambion. com/techlib/misc/siRNA_finder . html . siRNA and miRNA sequences can be synthetically produced and added exogenously to cause gene downregulation or produced using expression systems (e.g. vectors) . In a preferred embodiment, the siRNA is synthesized synthetically.

Longer double stranded RNAs may be processed in the cell to produce siRNAs (see for example Myers (2003) Nature Biotechnology 21:324- 328) . The longer dsRNA molecule may have symmetric 3' or 5 ' overhangs, e.g. of one or two (ribo) nucleotides, or may have blunt ends. The longer dsRNA molecules may be 25 nucleotides or longer. Preferably, the longer dsRNA molecules are between 25 and 30 nucleotides long. More preferably, the longer dsRNA molecules are between 25 and 27 nucleotides long. Most preferably, the longer dsRNA molecules are 27 nucleotides in length. dsRNAs 30 nucleotides or more in length may be expressed using the vector pDECAP (Shinagawa et al . , Genes and Dev. , 17, 1340-5, 2003).

Another alternative is the expression of a short hairpin RNA molecule (shRNA) in the cell. shRNAs are more stable than synthetic siRNAs. A shRNA consists of short inverted repeats separated by a small loop sequence. One inverted repeat is complementary to the gene target. In the cell the shRNA is processed by DICER into a siRNA which degrades the target gene mRNA and suppresses expression. In a preferred embodiment the shRNA is produced endogenously (within a cell) by transcription from a vector. shRNAs may be produced within a cell by transfecting the cell with a vector encoding the shRNA sequence under control of a RNA polymerase III promoter such as the human Hl or 7SK promoter or a RNA polymerase II promoter. Alternatively, the shRNA may be synthesised exogenously (in vitro) by transcription from a vector. The shRNA may then be introduced directly into the cell. Preferably, the shRNA molecule comprises a partial sequence of SHR. For example, the shRNA sequence is between 40 and 100 bases in length, more preferably between 40 and 70 bases in length. The stem of the hairpin is preferably between 19 and 30 base pairs in length. The stem may contain G-U pairings to stabilise the hairpin structure.

siRNA molecules, longer dsRNA molecules or miRNA molecules may be made recombinantly by transcription of a nucleic acid sequence, preferably contained within a vector. Preferably, the siRNA molecule, longer dsRNA molecule or miRNA molecule comprises a partial sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or a variant thereof .

In other embodiments, the suppressor nucleic acid may be an anti- sense suppressor of expression of the SHORT-ROOT (SHR) polypeptide. In using anti-sense sequences to down-regulate gene expression, a nucleotide sequence is placed under the control of a promoter in a "reverse orientation" such that transcription yields RNA which is complementary to normal mRNA transcribed from the "sense" strand of the target gene. See, for example, Rothstein et al , 1987; Smith et

al, (1988) Nature 334, 724-726; Zhang et al, (1992) The Plant Cell 4, 1575-1588, English et al . , (1996) The Plant Cell 8, 179-188. Antisense technology is also reviewed in Bourque, (1995), Plant Science 105, 125-149, and Flavell (1994) PNAS USA 91, 3490-3496.

An anti-sense suppressor nucleic acid may comprise an anti-sense sequence of at least 10 nucleotides from a nucleotide sequence is a fragment of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15 or a variant thereof .

It may be preferable that there is complete sequence identity in the sequence used for down-regulation of expression of a target sequence, and the target sequence, although total complementarity or similarity of sequence is not essential. One or more nucleotides may differ in the sequence used from the target gene. Thus, a sequence employed in a down- regulation of gene expression in accordance with the present invention may be a wild-type sequence (e.g. gene) selected from those available, or a variant of such a sequence .

The sequence need not include an open reading frame or specify an RNA that would be translatable. It may be preferred for there to be sufficient homology for the respective anti-sense and sense RNA molecules to hybridise. There may be down regulation of gene expression even where there is about 5%, 10%, 15% or 20% or more mismatch between the sequence used and the target gene. Effectively, the homology should be sufficient for the down-regulation of gene expression to take place.

In other embodiments, expression of the SHORT-ROOT (SHR) polypeptide may be reduced in a plant by selective plant breeding methods which employ the SHORT-ROOT (SHR) amino acid or nucleic acid sequence as a molecular marker in order to produce a plant having increased above- ground growth and/or biomass.

A method of producing a plant having increased growth and/or biomass may comprise: providing a population of plants, determining the amount of expression of an SHR polypeptide in one or more plants in the population, and identifying one or more plants in the population with reduced expression of the SHR polypeptide relative to other members of said population.

The identified plants may be further propagated or crossed, for example, with other plants having reduced SHR expression or self- crossed to produce inbred lines. The expression of an SHR polypeptide in populations of progeny plants may be determined and one or more progeny plants with reduced expression of the SHR polypeptide identified.

The expression of an SHR polypeptide in a plant may be determined by any convenient method. In some embodiments, the amount of expression of the SHR polypeptide may be determined at the protein level. A method of producing a plant having increased growth and/or biomass may comprise: providing a population of plants, determining the amount of SHR polypeptide in one or more plants of said population, and identifying one or more plants in the population with reduced amount of an SHR polypeptide relative to other members of said population.

The amount of SHR polypeptide may be determined in one or more cells of the plant, preferably cells from an above-ground portion or tissue of the plant, such as the vasculature and primary and secondary meristems in the shoot, in particular, the cambial zone

The amount of SHR polypeptide may be determined using any suitable technique. Conveniently, immunological techniques, such as Western blotting may be employed, using antibodies which bind to the SHR polypeptide and show little or no binding to other antigens in the plant. For example, the amount of an SHR polypeptide in a plant cell may be determined by contacting a sample comprising the plant cell with an antibody or other specific binding member directed against the SHR polypeptide, and determining binding of the SHR polypeptide to the sample. The amount of binding of the specific binding member is indicative of the amount of SHR polypeptide which is expressed in the cell.

In other embodiments, the expression of the SHR polypeptide may be determined at the nucleic acid level. For example, the amount of nucleic acid encoding an SHR polypeptide may be determined. A method of producing a plant having increased above-ground growth and/or biomass may comprise: providing a population of plants, determining the level or amount of nucleic acid, for example mRNA, encoding the SHR polypeptide in a cell of one or more plants of said population, and, identifying one or more plants in the population with reduced amount of an SHR encoding nucleic acid relative to other members of said population.

The level or amount of encoding nucleic acid in a plant cell may be determined for example by detecting the amount of transcribed encoding nucleic acid in the cell. This may be performed using standard techniques such as Northern blotting or RT-PCR.

A suitable cell may be from an above-ground portion or tissue of the plant, such as the vasculature and primary and secondary meristems in the shoot, in particular, the cambial zone.

Alternatively, the presence of sequence variations which affect the expression or activity of an SHR polypeptide may be determined.

Another method of producing a plant having increased growth and/or biomass may comprise: providing a population of plants, determining the presence of one or more sequence variations, for example, polymorphisms, mutations or regions of hypermethylation, in a nucleic acid encoding an SNR polypeptide in a cell in one or more plants of said population, wherein said one or more sequence variations which reduce but not abolish the expression or activity of the encoded SHR polypeptide, and identifying one or more plants in the population with one or more sequence variations which reduce the expression or activity of an SHR polypeptide relative to other members of said population.

SHR polypeptides and encoding nucleic acid are described in more detail above.

Sequence variations, such as mutations and polymorphisms, which reduce or abolish the expression or activity may include a deletion, insertion or substitution of one or more nucleotides, relative to the wild-type nucleotide sequence, a gene amplification or an increase or decrease in methylation, for example hypermethylation. The one or more sequence variations may be in a coding or non-coding region of the nucleic acid sequence. Mutations in the coding region of the gene encoding the component may prevent the translation of full-length active protein i.e. truncating mutations, or allow the translation of full-length but inactive or impaired function protein i.e. mis-sense mutations. Mutations or epigenetic changes, such as methylation, in non-coding regions of the gene encoding the component, for example, in a regulatory element, may prevent transcription of the gene. A nucleic acid comprising one or more sequence variations may encode a variant polypeptide which has

reduced or abolished activity or may encode a wild-type polypeptide which has little or no expression within the cell, for example through the altered activity of a regulatory element. A nucleic acid comprising one or more sequence variations may have one, two, three, four or more mutations or polymorphisms relative to the control sequences .

The presence of one or more sequence variations in a nucleic acid may be determined by detecting the presence of the variant nucleic acid sequence in one or more plant cells or by detecting the presence of the variant polypeptide which is encoded by the nucleic acid sequence. Preferred nucleic acid sequence variation detection techniques include ARMS™-allele specific amplification, OLA, ALEX™, COPS, Taqman, Molecular Beacons, RFLP, and restriction site based PCR and FRET techniques.

Numerous suitable methods for determining the amount of a nucleic acid encoding an SHR polypeptide, or the presence or absence of sequence variation in a nucleic acid encoding an SHR polypeptide, in a plant cell, are available in the art (see for example (see for example Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell (2001) Cold Spring Harbor Laboratory Press NY; Current Protocols in Molecular Biology, Ausubel et al . eds . John Wiley & Sons (1992); DNA Cloning, The Practical Approach Series (1995), series eds. D. Rickwood and B. D. Hames, IRL Press, Oxford,

UK and PCR Protocols: A Guide to Methods and Applications (Innis, et al . 1990. Academic Press, San Diego, Calif.)) . Many current methods for the detection of sequence variation are reviewed by Nollau et al . , Clin. Chem. 43, 1114-1120, 1997; and in standard textbooks, for example "Laboratory Protocols for Mutation Detection", Ed. by U.

Landegren, Oxford University Press, 1996 and "PCR", 2 ^nd Edition by Newton & Graham, BIOS Scientific Publishers Limited, 1997.

Preferred polypeptide sequence variation techniques include immunoassays, which are well known in the art e.g. A Practical Guide to ELISA by D M Kemeny, Pergamon Press 1991; Principles and Practice of Immunoassay, 2 ^nd edition, C P Price & D J Newman, 1997, published by Stockton Press in USA & Canada and by Macmillan Reference in the United Kingdom.

In some embodiments, nucleic acid or an amplified region thereof may be sequenced to identify or determine the presence of polymorphism or mutation therein. A polymorphism or mutation may be identified by comparing the sequence obtained with the known sequence of SHR, for example as set out in sequence databases. Alternatively, it can be compared to the sequence of the corresponding nucleic acid from control cells. In particular, the presence of one or more polymorphisms or mutations that cause reduction but not total abrogation of function may be determined. Sequencing may be performed using any one of a range of standard techniques. Sequencing of an amplified product may, for example, involve precipitation with isopropanol, resuspension and sequencing using a TaqFS+ Dye terminator sequencing kit (e.g. from GE Healthcare UK Ltd UK) . Extension products may be electrophoresed on an ABI 377 DNA sequencer and data analysed using Sequence Navigator software.

A progeny plant identified as having reduced SHR expression may be tested for increased biomass, growth and growth rate relative to controls .

A method of producing a plant having increased growth and/or biomass may comprise: crossing a first and a second plant to produce a population of progeny plants,- determining the expression of an Short Root (SHR) polypeptide in the progeny plants in the population, and

identifying a progeny plant in the population in which expression of the SHR polypeptide is reduced relative to controls but not abolished.

A progeny plant in which expression of the SHR polypeptide is reduced, but not abolished (i.e. expression is not completely eliminated) relative to controls (e.g. other members of the population) may display increased primary and/or secondary growth relative to the controls or increased biomass accumulation. A woody plant may show increased wood density.

The identified progeny plant may be further propagated or crossed, for example with the first or second plant (i.e. backcrossing) or self -crossed to produce inbred lines.

The identified progeny plant may be tested for increased biomass, growth and growth rate relative to controls.

Other aspects of the invention provide the use of an SHORT-ROOT (SHR) polypeptide or encoding nucleic acid as described herein as a marker for the selective breeding of a plant which has increased biomass or growth in its above ground portion, relative to control plants, and a method of selective breeding of a plant which has increased biomass or growth in its above ground portion, relative to control plants, which employs the SHORT-ROOT (SHR) amino acid or encoding nucleic acid sequence.

In some embodiments, plants having reduced expression of the SHORT- ROOT (SHR) polypeptide may be produced by random mutagenesis, followed by screening of mutants for reduced SHR expression.

Suitable techniques are well known in the art and include Targeting Induced Local Lesions IN Genomes (TILLING) . TILLING is a high- throughput screening technique that results in the systematic

identification of non-GMO-derived mutations in specific target genes (Comai and Henikoff , The Plant Journal (2006) 45, 684-694) .

A method of producing a plant having increased growth and/or biomass may comprise: exposing a population of plants to a mutagen, determining the expression of an SHR polypeptide or nucleic acid in one or more plants in said population, and identifying a plant with reduced expression of the SHR polypeptide relative to other members of said population.

Suitable mutagens include ethane methyl sulfonate (EMS) .

Methods for determining the expression of SHR polypeptide or nucleic acid in plants is described in more detail above.

The identified plant may be further tested for increased above ground biomass, growth and/or growth rate relative to controls.

A plant identified as having reduced expression of the SHR polypeptide relative to controls (e.g. other members of the population) may display increased primary and/or secondary growth in its above ground portion relative to the controls or increased biomass accumulation. A woody plant may show increased wood density.

A plant produced or identified as described above may be sexually or asexually propagated or grown to produce off -spring or descendants. Off -spring or descendants of the plant regenerated from the one or more cells may be sexually or asexually propagated or grown. The plant or its off-spring or descendents may be crossed with other plants or with itself.

Another aspect of the invention provides a plant which is produced by a method described herein, wherein said plant shows increased growth and/or biomass relative to control plants.

Also provided is any part or propagule of such a plant, for example seeds, selfed or hybrid progeny and descendants.

A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders Rights.

In addition to a plant produced by a method described herein, the invention encompasses any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part or propagule of any of these, such as cuttings and seed, which may be used in reproduction or propagation, sexual or asexual. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off -spring, clone or descendant.

In other embodiments, expression of the SHORT-ROOT (SHR) polypeptide may be increased in the cells relative to control plants in order to increase the above-ground growth and/or biomass of a plant. Expression of the SHORT-ROOT (SHR) polypeptide may, for example, be increased within cells of said plant by up to 2-fold, 5-fold, 10- fold, or 100-fold of the expression within cells of control plants.

Expression of the SHORT-ROOT (SHR) polypeptide may be increased within cells of said plant by any convenient method. For example, expression in the cells of said plant may be increased by expressing a heterologous nucleic acid encoding the SHORT-ROOT (SHR) polypeptide within cells of said plant.

SHORT-ROOT (SHR) polypeptides are described in more detail above. A nucleic acid encoding a SHR polypeptide may comprise or consist of the nucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 or may be a variant or fragment of any one of these sequences which retains SHR activity.

A variant sequence may be a mutant, homologue, or allele of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 may differ from one of these sequences by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid that make no difference to the encoded amino acid sequence are included. A nucleic acid encoding a SHR polypeptide, which has a nucleotide sequence which is a variant of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 sequence may comprise a sequence having at least 30% sequence identity with the nucleic acid sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15, for example, preferably greater than 40%, greater than 50%, greater than 60%, greater than 65%, greater than 70%, greater than 80%, greater than 90% or greater than 95%. Sequence identity is described above.

A fragment or variant may comprise a sequence which encodes a functional SHR polypeptide i.e. a polypeptide which retains one or more functional characteristics of the polypeptide encoded by the wild-type SHR gene, for example, the ability to stimulate growth in a plant .

In other embodiments, a nucleic acid encoding a SHR polypeptide, which has a nucleotide sequence which is a variant of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13 or 15 sequence may selectively hybridise under stringent conditions with this nucleic acid sequence or the complement thereof .

Stringent conditions include, e.g. for hybridization of sequences that are about 80-90% identical, hybridization overnight at 42°C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55 ⁰ C in 0. IX SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65 ⁰ C in 0.25M Na ₂ HPO ₄ , pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60 ⁰ C in 0. IX SSC, 0.1% SDS.

An alternative, which may be particularly appropriate with plant nucleic acid preparations, is a solution of 5x SSPE (final 0.9 M NaCl, 0.05M sodium phosphate, 0.005M EDTA pH 7.7), 5X Denhardt ' s solution, 0.5% SDS, at 50 ⁰ C or 65°C overnight. Washes may be performed in 0.2x SSC/0.1% SDS at 65°C or at 50-60 ⁰ C in Ix SSC/0.1% SDS, as required.

A nucleic acid encoding a Short Root (SHR) polypeptide or a nucleic acid which suppresses the expression of a Short Root (SHR) polypeptide (i.e. a suppressor RNA molecule) may be operably linked to a regulatory sequence, such as a promoter, for example a constitutive, inducible, tissue-specific or developmental specific promoter .

A regulatory sequence operably linked to a SHR nucleic acid sequence is preferably heterologous or foreign to the SHR nucleic acid sequence (e.g. from a different species, class or type of organism) . Preferably, the regulatory sequence is a plant specific regulatory sequence to provide for efficient expression within a plant cell. A plant specific regulatory sequence or element preferentially directs the expression (i.e. transcription) of a nucleic acid within a plant cell relative to other cell types. For example, expression from such a sequence may be reduced or abolished in non-plant cells, such as bacterial or mammalian cells.

Many suitable regulatory sequences are known in the art and may be used in accordance with the invention. Examples of suitable regulatory sequences may be derived from a plant virus, for example the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, (1990) EMBO J 9: 1677-1684). Leaf specific promoters may also be used (see for example Lagrange et al Plant Cell. 1997 9 (8) : 1469- 1479) . Other suitable constitutive regulatory elements include the cauliflower mosaic virus 19S promoter; the Figwort mosaic virus promoter; and the nopaline synthase (nos) gene promoter (Singer et al., Plant MoI. Biol. 14:433 (1990); An, Plant Physiol. 81:86 (1986) ) .

In some embodiments, an inducible promoter such as the alcohol inducible ale gene-expression system (Roslan et al . , Plant Journal; 2001 Oct; 28 (2) :225-35) may be employed.

Heterologous nucleic acid may be contained on a nucleic acid construct or vector. The construct or vector is preferably suitable for transformation into and/or expression within a plant cell.

A vector is, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form, which may or may not be self transmissible or mobilizable, and which can transform prokaryotic or eukaryotic host, in particular a plant host, either by integration into the cellular genome or exist extrachromasomally (e.g. autonomous replicating plasmid with an origin of replication) .

Specifically included are shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different organisms, which may be selected from actinomyces and related species, bacteria and eukaryotic (e.g. higher plant, mammalia, yeast or fungal) cells.

A construct or vector comprising nucleic acid as described above need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

Constructs and vectors may further comprise selectable genetic markers consisting of genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones, glyphosate and d-amino acids.

Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression, in particular in a plant cell. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press.

Those skilled in the art can construct vectors and design protocols for recombinant gene expression, for example in a microbial or plant cell. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Sambrook et al , 2001, Cold Spring Harbor Laboratory Press and Protocols in Molecular Biology, Second Edition, Ausubel et al . eds . John Wiley & Sons, 1992. Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl . Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux,

(1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.

When introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct that contains effective regulatory elements that will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, the target cell type is preferably such that cells can be regenerated into whole plants.

Techniques well known to those skilled in the art may be used to introduce nucleic acid constructs and vectors into plant cells to produce transgenic plants with the properties described herein.

Agrobacterium transformation is one method widely used by those skilled in the art to transform woody plant species, in particular hardwood species such as poplar. Production of stable, fertile transgenic plants is now routine in the art: (Toriyama, et al . (1988) Bio/Technology 6, 1072-1074; Zhang, et al . (1988) Plant Cell Rep. 7, 379-384; Zhang, et al . (1988) Theor Appl Genet 16, 835-840;

Shimamoto, et al . (1989) Nature 338, 274-276; Datta, et al . (1990) Bio/Technology 8, 736-740; Christou, et al . (1991) Bio/Technology 9, 957-962; Peng, et al . (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al . (1992) Plant CeH Rep. 11, 585-591; Li, et al . (1993) Plant Cell Rep. 12, 250-255; Rathore, et al . (1993) Plant Molecular Biology 21, 871-884; Fromm, et al . (1990) Bio/Technology 8, 833-839; Gordon-Kamm, et al . (1990) Plant Cell 2, 603-618; D'Halluin, et al . (1992) Plant Cell 4, 1495-1505; Walters, et al . (1992) Plant Molecular Biology 18, 189-200; Koziel, et al .

(1993) Biotechnology 11, 194-200; Vasil, I. K. (1994) Plant Molecular Biology 25, 925-937; Weeks, et al . (1993) Plant Physiology 102, 1077-1084; Somers, et al . (1992) Bio/Technology 10 , 1589-1594; WO92/14828; Nilsson, O. et al (1992) Transgenic Research 1, 209- 220) .

Other methods, such as microprojectile or particle bombardment (US 5100792, EP-A-444882, EP-A-434616 ) , electroporation (EP 290395, WO 8706614), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al . (1987) Plant Tissue and Cell Culture, Academic Press), direct DNA uptake (DE 4005152, WO 9012096, US 4684611), liposome mediated DNA uptake (e.g. Freeman et al . Plant Cell Physiol. 29: 1353 (1984)), or the vortexing method (e.g. Kindle, PNAS U.S.A. 87: 1228 (199Od)) may be preferred where Agrobacterium transformation is inefficient or ineffective, for example in some gymnosperm species.

Physical methods for the transformation of plant cells are reviewed in Oard, 1991, Biotech. Adv. 9: 1-11.

Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-A-486234 ) or microprojectile bombardment to induce wounding followed by co- cultivation with Agrobacterium (EP-A-486233 ) .

Following transformation, a plant may be regenerated, e.g. from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al . , Cell Culture and Somatic Cell Genetics of Plants, VoI I, II and III, Laboratory Procedures and Their Applications, Academic Press, 1984, and Weissbach and Weissbach, Methods for Plant Molecular Biology, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

Another aspect of the invention provides a method of producing a plant having increased growth and/or biomass comprising: incorporating a heterologous nucleic acid which alters the expression of a SHORT-ROOT (SHR) polypeptide into a plant cell by means of transformation, and; regenerating the plant from one or more transformed cells.

In some embodiments described above, the heterologous nucleic acid may increase the expression of a SHORT-ROOT (SHR) polypeptide. For example the heterologous nucleic acid may encode a SHORT-ROOT (SHR) polypeptide. The growth and or biomass of the above ground portions of the plant may be increased by the increase in SHORT-ROOT (SHR) polypeptide expression.

In other embodiments described above, the heterologous nucleic acid may reduce but not abolish the expression of a SHORT-ROOT (SHR) polypeptide. For example the heterologous nucleic acid may encode or transcribe a nucleic acid which suppresses the expression of SHORT- ROOT (SHR) polypeptide, for example an RNAi molecule.

A plant produced by such methods may show increased growth and/or biomass relative to control plants. For example, the above ground portions of the plant may show increased growth and/or biomass relative to controls.

Preferably, the nucleic acid recombines with the cell genome nucleic acid, such that it is stably incorporated therein.

The SHR polypeptide, the encoding nucleic acid, and/or the vector comprising the nucleic acid are described in more detail above and may be heterologous (e.g. exogenous or foreign) to the cell transformed therewith.

The regenerated plant shows increased growth and/or biomass relative to controls. For example, the above ground portions of the plant may show increased growth and/or biomass. A regenerated woody plant may show increased wood density. A plant regenerated from a plant cell may be sexually or asexually propagated or grown to produce off- spring or descendants. Off -spring or descendants of the plant regenerated from the one or more cells may be sexually or asexually propagated or grown

Another aspect of the invention provides a plant which is produced by a method described herein, wherein said plant shows increased growth and/or biomass, for example increased above-ground growth and/or biomass, relative to control plants.

For example, plant may be provided which comprises a heterologous nucleic acid encoding an SHORT-ROOT (SHR) polypeptide or a suppressor of the expression of SHORT-ROOT (SHR) polypeptide within one or more of its cells.

Also provided is any part or propagule of such a plant, for example seeds, selfed or hybrid progeny and descendants.

A plant may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders Rights. It

is noted that a plant need not be considered a "plant variety" simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.

In addition to a plant, any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part or propagule of any of these, such as cuttings and seed, may be useful in reproduction or propagation, sexual or asexual. A plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant may also be useful.

A plant may have increased or decreased expression of SHR polypeptide, relative to the wild-type (i.e. 'unmodified') plant. SHR expression may be increased, for example, by expressing a nucleic acid encoding an SHR polypeptide in the cells of the plant or reduced by expressing a nucleic acid which causes anti-sense, sense or RNAi down- regulation of SHR in the cells of the plant, as described above.

Control experiments may be performed as appropriate in the methods described herein. The performance of suitable controls is well within the competence and ability of a skilled person in the field.

Examples of suitable plants for use in accordance with any aspect of the invention described herein include monocotyledons, dicotelydons, gymnosperms and algae, ferns and mosses. Of particular interest are transgenic higher plants, especially agricultural crops, for example cereals, and flowers, which have been engineered to carry a heterologous nucleic acid as described above, including tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, linseed, hemp and rye.

Examples of SHR protein sequences from Arabidopsis, Poplar, Rice, Barley and Medicago are set out herein.

In some preferred embodiments, the plant is a perennial plant, for example a woody perennial plant. A woody perennial plant is a plant which has a life cycle which takes longer than 2 years and involves a long juvenile period in which only vegetative growth occurs. This is contrasted with an annual or herbaceous plant such as Arabidopsis thaliana or Lycopersicon esculentum (tomato) , which have a life cycle which is completed in one year.

A woody perennial plant has hard, lignified tissues and forms a bush or tree. Preferred perennial plants are trees (i.e. plants of tree forming species) . A woody perennial plant may be a gymnosperm (non- flowering plant) or an angiosperm (flowering plant) . Angiosperms are divided into two broad classes and a perennial plant may be a monocotyledonous or dicotyledonous angiosperm.

Examples of woody perennial plants include conifers such as cypress, Douglas fir, fir, sequoia, hemlock, cedar, juniper, larch, pine, redwood, spruce and yew; hardwoods such as acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple and sycamore; fruit bearing plants such as apple, plum, pear, banana, orange, kiwi, lemon, cherry, grapevine and fig; and other commercially significant plants, such as cotton, bamboo and rubber.

Other aspects of the invention relate to the use of the SHR promoter to screen for compounds which alter (i.e. increase or reduce) the expression of SHR polypeptide in a plant.

A method of screening for a compound which increases the above- ground growth and/or biomass of a plant comprising;

providing a nucleic acid construct comprising an SHR promoter operably linked to a reporter gene, in an expression system in which the reporter is expressed, contacting the construct with a test compound, and determining the expression of the reporter, wherein an increase or decrease in the expression of the reporter in the presence relative to the absence of test compound is indicative that the compound increases the above-ground growth and/or biomass of a plant.

A SHR promoter may have the sequence of SEQ ID NO: 22 or 23 or may be a variant or fragment thereof. Variants and fragments of reference SHR sequences, such as SEQ ID NO: 22 and 23, are described above .

Suitable reporter genes are well known in the art and include genes encoding fluorescent proteins such as GFP.

The SHR promoter may also be useful in driving expression of a heterologous gene in the wood- forming tissues of a woody perennial plant. Wood-forming tissues include the vasculature and primary and secondary meristems in the shoot, including the cambial zone.

A method of producing a plant may comprise: incorporating a nucleic acid construct or vector comprising an SHORT-ROOT (SHR) promoter operably linked to a heterologous gene into a plant cell by means of transformation, and; regenerating the plant from one or more transformed cells.

Preferably the plant is a woody perennial plant and the SHORT-ROOT (SHR) promoter drives expression of the heterologous gene in the wood forming tissue of the woody perennial plant.

Suitable heterologous sequences may include sequences encoding polypeptides that alter the growth or composition of cell, tissues, and/or organs of the transformed plant, in particular growth promoting genes and genes which improve wood quality.

The regenerated plant may be crossed or propagated as described above .

Other aspects of the invention provide a nucleic acid construct or vector comprising a SHORT-ROOT (SHR) promoter operably linked to a heterelogous gene, the use of such a nucleic acid construct or vector to specifically express the heterologous gene in the wood- forming tissues of a woody perennial plant, and a method of expressing a heterologous gene in the wood forming-tissues of a woody perennial plant by introducing such a construct or vector into the woody perennial plant.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure. All documents mentioned in this specification are incorporated herein by reference in their entirety.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures described below.

Figure 1 shows an unrooted cladogram of Poplar and Arabidopsis sequences with similarity to AtSHR. The phylogenetic analysis was performed with full length predicted amino acid sequences from Poplar and Arabidopsis members of the SHR branch of the GRAS family. ClustalW was used to align the protein sequences. The Phylip format was used for tree output, using 1000 bootstrap replicates. The cladogram was visualised using TreeViewX (version 0.5.0) .

Figure 2 shows a nucleotide sequence alignment between the predicted coding sequences of AtSHR and the three Poplar SHR- like proteins, PtSHRl ₁ PtSHR2A and PtSHR2B.

Figure 3 shows a comparison of the deduced amino acid sequence of PtSHRl, PtSHR2A, PtSHR2B and AtSHR. Identical, similar and conserved amino acids are shown in black and grey backgrounds, respectively. Amino acid motifs conserved across members of the GRAS family are shown .

Figure 4 shows differential growth of WT T89 and PtSHRl RNAi Line 2B trees. Trees after a growth period of 52 days in the glasshouse and two of the same trees as initial plantlets in tissue culture ( inset) .

Figure 5 shows girth measurements of Poplar stems after 52 days growth in the glasshouse. Independent transformation events of Poplar PtSHRl RNAi lines (2A, 2B and 4A) compared to WT T89. Results are presented as means plus and minus 1 SD. Means are the average width from 10 internodes from 9 trees for each of the RNAi and WT T89 lines, beginning at an internode 200 mm above the soil.

Figure 6 shows transverse sections of WT T89 and PtSHRl RNAi Line 2B. Sections were taken from the middle of internodes 500mm above the soil and are stained with toluidine blue. Double headed arrows indicate xylem (wood cells) .

Figure 7 shows accumulated biomass of Poplar stems after 52 days growth in the glasshouse. Independent transformation events of Poplar PtSHRl RNAi lines (2A, 2B and 4A) compared to WT T89. Results are presented as means plus and minus 1 SD. Means, n = 9, are the average total fresh mass in grams of stems plus leaves from 200 mm above the soil .

Figure 8 shows average internode length of Poplar stems after 52 days growth in the glasshouse. Independent transformation events of Poplar PtSHRl RNAi lines (2A, 2B and 4A) compared to WT T89. Results are presented as means plus and minus 1 SD. Means, n = 9, are the average lengths of 15 fully expanded internodes beginning at 200mm above the soil .

Figure 9 shows average number of leaves (nodes) in Poplar stems after 52 days growth in the glasshouse. Poplar PtSHRl RNAi line 2B compared to WT T89. Results are presented as means plus and minus 1 SD. Means, n = 9, are the average number of leaves produced from 200mm above the soil to the youngest leaf of at least 40mm in length.

Figure 10 shows a comparison of the lengths and widths of tracheids from the middle of fully expanded internodes of independent transformation events of Poplar PtSHRl RNAi lines (2A and 2B) compared to WT T89. Results are presented as means plus and minus 1 SD. Means, n = 30, are the average widths and lengths (arbitrary units) of tracheids. A & C and B & D represent measurements from sections taken from separate internodes of approximately the same length.

Figure 11 shows average leaf area and fresh weight of fully expanded leaves from Poplar stems after 52 days growth in the glasshouse. Poplar PtSHRl RNAi lines (2A, 2B and 4A) compared to WT T89. Results are presented as means plus and minus 1 SD. Means are the average area and weight of 15 fully expanded leaves from 200mm above the soil of 9 separate trees for each of the lines 2A, 2B, 4A and WT T89.

Figure 12 shows a comparison of PtSHRl transcript levels in various organs of Poplar plants. Data is presented as relative expression Iog2 scale with error bars indicating 1 SD. Data represents levels of PtSHRl transcripts relative to a pooled sample of all tissues (eg apex vs pool, leaf vs pool) . Apex = shoot apex; PrimRoot = primary root (tissue culture grown); SecRoot = secondary (lateral) root (tissue culture grown) .

Figure 13 shows germination and early growth of AtSHR-suppressed Arabidopsis RNAi and WT control plants. A. Germination of seeds of transgenic lines 4, 6 and 11 and WT seeds. B. WT and Line 11 seeds after 18 hours at 23oC. C. WT and Line 11 seeds after 40 hours at 23oC. D. WT and Line 11 seedlings after 5.5 days at 23oC. E. Etiolated WT and Line 11 seeds after 5.5 days at 23oC. F. Average cotyledon size of light-grown WT and Line 11 seedlings after 5.5 days at 23oC. G. Average hypocotyl length of etiolated seedlings after 5.5 days at 23oC. H. Average root lengths of WT, shr and transgenic seedlings after 5.5 days at 23oC. Plates for plants grown in the light (A, B, C, D, F and H) contained 1% sucrose. Etiolated seedlings were grown on plates without sucrose. Results for A are presented as percentage germination of samples of 140 seeds over time. All experiments were conducted three times with similar results. The presented results are representative of all three experiments. WT and RNAi seeds for the experiments were grown under the same conditions, at the same time. After harvesting these seeds

they were left to fully mature for at least 2 months prior to conducting the germination and growth experiments. Results for F, G and H are presented as the means plus and minus 1 SD.

Figure 14 shows a comparison of growth in Arabidopsis RNAi transgenic and WT control plants. A. Line 11 plants at stage 1.06 (Boyes et al . , The Plant Cell, Vol. 13, 1499-1510, 2001) at 15 DAG. B. WT plants at the same stage of development (at 18 DAG) . C. Area of fully expanded true leaves 1 and 2. D. Line 11 plants at 18 DAG (compare B and D) . E. Total leaf area at 18 DAG in the RNAi lines and in WT controls. F. Radial longitudinal sections through the apexes of WT control and Line 11 transgenic plants at day 18. Circles in A and B highlight the 5 ^th true leaf. The dotted lines in F represent the diameter of the WT apical dome.

Figure 15 shows a multiple amino acid alignment of PtSHR genes, AtSHR and Rice and Medicago predicted sequences.

Figure 16 shows transcript levels of three independent SHR RNAi suppression lines of Aspen (2A-P in two replicates, 2B-P in two replicates and 4A-P) and five WT T89 lines, determined by RT-PCR.

Experiments

Poplar transformation The CaMV 35S: Inverted repeat DNA construct for PtSHRl was transformed into the pK7GWIWG2(I) binary vector (Karimi et al . , Trends Plant Sci . 2002 May;7(5): 193-195) and subsequently transferred to Agrobacterium tumefaciens and used to transform hybrid aspen (Populus tremula x Populus tremuloides, clone T89) stem segments as described previously (Nilsson et al . , 1992 Transgenic Research 1: 209-220). Kanamycin resistant transformants were regenerated as described previously (Nilsson et al . supra) . The primers used for cloning the PtSHRl sequence used for the inverted repeat construct were 5' -AGAAAGCTGGGTAACCACCACCATCATCACTATC^'

(contains an ATTB2 site) and 5' -AAAAGCAGGCTGCTTTCACCTTCAAATGCTTCC-3 ' (contains an ATTBl site) . The template used for the PCR amplification of the sequence was EST clone UB21CPG06. Cloning into the binary vector was carried out according to the manufacturers recommended protocol (Gateway, Invitrogen, USA) . The primer sequences used in the RT-PCR analysis of remnant transcript levels in the transgenic lines were: 5' -CATCACCTGACCTTCACTCC-3' and 5'- GTTCGGATTGTTGTTGGAGAC- 3' . Primers used for determining transcript levels of close homologs of PtSHRl (PtSHR2A and PtSHR2B) in PtSHRl RNAi -suppressed lines were 5'-AGCAACAACAACAACAATCAG-S' and 5'-

GCACACTCACTAAGAAGCC- 3 ' . The analysis showed no down regulation of the transcript levels of these two genes.

Twenty four independent lines were generated. Such a group of transgenic trees produced using the short root construct is hereafter called "construction group" . Each transgenic line within the construction group was a different transformation event and therefore most probably had the recombinant DNA inserted into different locations in the plant genome. This may make the different lines within one construction group partly different. For example, it is known that different transformation events will produce plants with different levels of gene down-regulation when using RNAi constructs of the type used here.

Poplar Plant growth

After initiation and establishment of rooting in tissue culture, transgenic Poplar lines were grown together with their wild type control (WT T89) trees, in a greenhouse under a photoperiod of 18 h and a temperature of 22°C/15°C (day/night) . The humidity was RH 70 %. The plants were fertilized weekly using the fertiliser "Weibulls Rika S NPK 7-1-5" diluted 1 to 100 ( final concentrations NO3 , 55g/l; NH4 , 29g/l; P, 12g/l; K, 56g/l; Mg 7,2g/l; S, 7,2g/l; B, 0,18g/l; Cu, 0,02g/l; Fe, 0,84g/l; Mn, 0,42g/l; Mo, 0,03g/l; Zn, 0,13g/L). The plants were grown for 7-9 weeks before harvest. During

this time their positions within the glasshouse were altered every 2-3 days and their heights and diameters were measured as described in the results. A number of wild type trees (typically 8-25 trees) and a number of transgenic trees comprising the construction group were grown in parallel in the greenhouse under the same above conditions. All comparisons between the wild type trees and the construction group are made within each growth group.

Poplar Growth Measurements Under the above defined growth conditions, Poplar plants exhibited an exponential growth pattern (plant height) up to an approximate height of 80 cm or a maximum of up to day 40 in the greenhouse. Height measurements were taken at the times stipulated in the results. Under the above defined growth conditions, stem width exhibited a comparatively linear increase over time.

A height growth rate measure (here named "Maximum height growth rate") was defined as the slope of a linear function fitted over four consecutive height data points. A height growth rate value was calculated for data point 1 - 4, data point 2 - 5 etc. in a stepwise manner, se Figure 4 for an example. A maximum growth rate defined as the maximum value, produced from step-wise linear regression analysis, for each plant was computed. The primary data for high Maximum height growth rate values from individual transformants in a construction group were checked so they were not based on bad values.

Immunocytochemistry and Confocal Laser Scanning Microscopy- Tissues were prepared as butyl -methylmethacrylate resin-embedded, semithin sectioned material. The resin-embedded material was prefixed with 100 μM m-male- imidobenzoyl N-hydroxysuccinimide ester (Sigma) in 25 mM Pipes buffer (Sigma), pH 6.9. It then was fixed in 3.7% formaldehyde with 0.2% (v/v) glutaraldehyde in 25 mM Pipes buffer, pH 6.9, embedded in a butyl -methylmethacrylate resin

mixture, polymerized under UV light, and sectioned at 7 μm. After resin removal with acetone, sections were incubated in a blocking solution containing 5% skim milk powder in PBS containing 137 mM NaCl, 2.7 mM KCl, 2 mM Na ₂ HPO ₄ , 2 mM KH ₂ PO ₄ , pH 7.2 to 7.4, and 1% Tween 20 for ~45 min before application of the primary antibody.

Sections were incubated in primary antibody solution for 2 h at room temperature or overnight at 4 ⁰ C before washing in 0.1% Tween 20 in PBS and application of the secondary antibody conjugated to fluorescein isothiocyanate (FITC) . Sections were then incubated for 1 h at room temperature, washed extensively as described above, and stained with 0.01% toluidine blue O for 1 min to minimize tissue autofluorescence . The sections were mounted in Vectashield (Vector Laboratories) and examined by confocal laser scanning microscopy using the Zeiss LSM 510 instrument, with 488- and 568-nm argon- krypton lasers, generating the FITC signal (detected at 505 to 550 nm) and the autofluorescence signal (detected at >585 nm) . These signals were detected in separate channels.

In the second method of tissue preparation, fresh material was sectioned with a razor blade and fixed in 4% paraformaldehyde in 50 mM Pipes buffer, pH 7, containing 5 mM MgSO ₄ and 5 mM EGTA for 30 min. After fixation, the material was treated basically as the methylmethacrylate -embedded material, except that autofluorescence quenching was not needed. Because the autofluorescence of the walls was low, the FITC signal was projected on a transmitted light image for anatomical detail.

Antibodies

Monoclonal antibodies raised against recombinant protein PtSHRl in rabbit were used to localize PtSHRl. Secondary antibodies were anti- rabbit FITC conjugates (Jackson ImmunoResearch Laboratories , West Grove, PA) used at a dilution of 1:100. Control and experimental preparations were processed in parallel and viewed at the same confocal laser scanning microscopy settings. To test for tissue autofluorescence , material was processed without the secondary

antibodies. Primary antibodies were also saturated with the antigen at 10 nmol/mL to determine if the signal from experimental sections was attributable to the antibodies reacting with the target antigen or some other antibodies . These controls gave minimal background signals that were considered nonspecific.

Confocal Scanning Laser Microscopy

CLSM was carried out with a Leica TCS SP2 AOBS scanning system mounted on a Leica DM IRE2 inverted microscope employing Leica TCS software. An oil-corrected 633objective NA = 1.4 (HCX PL APO lbd.BL 63.0xl.40 01, Leica) and a water-corrected 633 objective NA = 1.2 (HCX PL APO 63.0xl.20WBD UV, Leica) were used. Excitation wavelengths were 488 ran (argon laser) for GFP and FITC and 340-380 nm for the counterstain, calcofluor white. Emission was detected between 505 and 520 nm for GFP, 520 and 550 nm for FITC and 420 and 440 nm for calcofluor white. Images were overlaid with Adobe Photoshop 7.0.

Arabidopsis transformation and growth

The pK7GWIWG2D(II) binary vector (Karimi et al . , Trends Plant Sci . 2002 May;7(5): 193-195) was used to transfer an inverted repeat DNA construct for AtSHR into Columbia (CoIO) WT Arabidopsis plants using the floral dip method as previously described (Clough and Bent, 1998, Plant J 16:735-43) . In addition, the following seed stocks were used: shr2 (homozygotic stock) in ColO background, tpd::GUS line Al, homozygotic, in Columbia (Col) background (gift from Malcolm Bennett) . Seeds germinated on plates were surface sterilized with 70% ethanol for 5 min, incubated in Bayrochlor (Bayrol, Planegg, Germany; one tablet per 400 ml H20) for 30 min, and washed three times with sterile, distilled water (dH2O) . Sterilised seeds were plated on agar plates containing 1 X MS medium (Duchefa Biochemie, Haarlem, The Netherlands), 1% plant agar (Duchefa) , 1% sucrose (or 0% sucrose for etiolated growth) , buffered to pH 5.7 with 1 M morpholinoethane-sulfonic acid (Sigma, Steinheim, Germany) . Sterilised, plated seeds were vernalised at 4°C in

darkness for minimum three days and subsequently grown in continuous light or 16 hours light/8 hours dark at the photon flux density of 1780 μE m ^"2 s ^"1 , 70% relative humidity and a constant temperature of 23°C.

For detecting the glucuronidase (GUS) activity, fresh tissue sections were incubated for 16-18 hrs at 37 ⁰ C with gentle shaking in a substrate solution consisting of: 1 mM 5-bromo-4 -chloro-3- indoyl-beta-D-glucuronide , cyclohexylammonium salt (BioVectra, PEI, Canada), 50 mM sodium phosphate buffer, pH=7.2, 0.1% Triton X-100, 1 mM K ₃ [Fe(CN) ₆ ] and 1 mM K ₄ [Fe(CN) ₆ ]. The sections were then fixed for 10 min. in a fixing solution (5% formaldehyde, 5% acetic acid and 50% ethanol), washed few minutes in 50% and 100% ethanol and cleared by incubation in 0.24M HCl in 20% methanol at 57 C for 15 min., followed by 15 min in 7% NaOH in 60% EtOH at room temperature. Then the sections were rehydrated in a graded ethanol series (40, 20 and 10% EtOH), water and mounted in 50% glycerol. Sections were examined using an Axioplan 2 microscope and images were recorded with an AxioVision camera (both from Zeiss) .

RT-PCT

Quantification of mRNA expression in transgenic hybrid Aspen lines was done according to the manufacturers protocol (Biorad) . The realtime RT-PCR was run on a MyiQ PCR machine (Bio-Rad) using SYBR Green Supermix kit (Bio-Rad) . Primer sequences for the RT-PCR analysis of PtSHR transcript levels in WT and RNAi lines were, 5'- CCATCACCTGACCTTCACTCC -3' and 5'- TGTTCGGATTGTTGTTGGAGAC -3' .

Results Three Poplar genes code for SHR-like proteins

The GRAS protein family that includes SHR can be divided into a number of distinct subgroups based on sequence and function ( (Bolle C, Planta. 2004 Mar ; 218 (5 ) : 683- 92 ) ) . A phylogenetic analysis of the most closely related Poplar and Arabidopsis homologs to the well-

characterized AtSHR protein (Populus tremula: PtSHRl (eugene3.01860017) , PtSHR2A (eugene3.00070144 ), PtSHR2B (eυgene3.00640143) , PtSCL35b (eugene3.00050544 ), PtSCL53b (eugene3.00640007) , PtSCL62 (fgenesh4_pm . C_LG_III000210) , PtSCL69b (eugene3.00070272) , PtSCL92b (eugene3.00030248) , PtSCL97b

(eugene3.00011016) ; Arabidopsis thaliana: AtSHR (At4g37650) , AtSCL29 (At3gl3840) , AtSCL32 (At3g49950) (Fig.l) indicated that there are three SHR- like genes in Poplar and that, of the three, PtSHRl is the most closely related to AtSHR.

The entire poplar genome is publicly available on-line from the Joint Genome Institute website (currently at http://genome.jgi- psf . org/Poptrl_l/Poptrl_l .home .html) and is described in Tuscan et al. (2006) Science 313 (5793), 1596.

Multiple sequence alignments indicated strong sequence similarities between the Poplar nucleotide (Fig.2) and amino acid sequences (Fig.3) and the corresponding AtSHR sequences. PtSHRl shares 67% nucleotide similarity with AtSHR ₁ and the predicted 540 amino acid PtSHRl sequence shares 73.52% similarity with the Arabidopsis sequence. The most obvious difference between the proteins is the approximately 110 N-terminal amino acid residues that are present in PtSHRl and AtSHR, but are absent from PtSHR2A and PtSHR2B (Fig.3). The predicted 411 amino acid sequences of PtSHR2A and PtSHR2B share 93% amino acid identity over the length of the sequences and 71.14% and 68.78% amino acid similarity with AtSHR, respectively. All three Poplar SHR- like sequences contain variations on the distinguishing GRAS family-specific VHIID motif and its requisite surrounding leucine-rich regions (Fig.3) . They also all contain the conserved GRAS C-terminal SAW motif, but only PtSHR2A and PtSHR2B contain variations on the RVER motif present in many GRAS protein family members. The genomic sequences of all three genes are similar, with no intron-encoding sequences present.

Partial suppression of PtSHRl leads to an acceleration of growth The in planta functions of the Poplar SHR- like genes were investigated by suppressing the expression of PtSHRl. In contrast to our expectations, of the 24 independent transformation lines generated, none showed reduced growth, but many showed a marked increase in both primary (height) (Fig.4, Table 1) and secondary (girth) (Figs. 5 and 6) growth. The combined effect was a substantial increase in total above-ground biomass accumulation after 52 days growth in the glasshouse (Fig.7). The lines that showed an increase in growth were found to have a partial suppression of PtSHRl transcripts. Three lines, representing independent transformation events, Line 2A, Line 2B and Line 4A, had 50-80% of the levels of PtSHRl transcripts found in WT trees. Growth was measured in trees that began as uniform three leaf Poplar cuttings (insert Fig.4) . After 4 weeks in tissue culture, the trees were transplanted into soil and moved to the glasshouse. From the first measurement (16 days after transplantation, DAT) , the transgenic lines were significantly taller than the control, T89, plants (Table 1) . By 52 DAT, RNAi Line 2B plants were on average 27% taller than WT T89 plants. This was despite the average internode length of the transgenic plants being significantly smaller than the WT plants (Fig.8) . The increased height resulted from an accelerated rate of node (leaf) production in the transgenic lines (Fig.9).

The increase in girth resulted from a proportional increase in the width of the various components of the stem (Fig.6) . There was no consistently significant difference in the length and width of tracheids between the transgenic and WT plants (Fig.10), indicating that the increased growth was predominantly the result of an increase in cell number. There were also no significant differences in leaf shape or average fully expanded leaf surface area and fresh weight between the transgenic lines and WT trees (Figs HA and HB) . Poplar cuttings establish through the production of adventitious roots. We were unable to observe any differences in root

establishment or early root growth between the RNAi and WT T89 plants .

mRNA expression was quantified in transgenic hybrid aspen lines (Populus tremula x Populus tremuloides, clone T89) using RT-PCR. The partial suppression 20-80% of WT levels of steady-state PtSHRl transcript levels seen in figure 16 and Table 6 resulted in a dramatic acceleration of both primary and secondary growth compared to wild-type (WT) T89 trees (Figures 4, 5 and 6) .

Arabidopsis shr mutant shoots have altered secondary vascular development

In stark contrast to the phenotype of the Poplar knockdown lines, the shoots of Arabidopsis shr knockout lines are clearly dwarfed. Despite this, we examined shr hypocotyls and floral stems in order to reveal discrepancies or similarities that would shed light on the Poplar phenotype. Secondary growth was clearly reduced in the mutant. Transverse sections through the hypocotyl of flowering plants revealed a reduction in the thickness of shr hypocotyls compared to the WT. The vascular bundles in the mutant hypocotyls were small and irregular and lacked the typical WT radial alignment of vessels. The hypocotyls of flowering WT plants had a considerable amount of secondary xylem produced by the activity of the vascular cambium and a thick periderm produced by cork cambium activity. In contrast, the hypocotyls of flowering shr plants exhibited very little vascular cambium activity, producing 3-9 layers of secondary xylem. There was also no apparent cork cambium.

Similar expression patterns for AtSHR and PtSHRl provide further evidence that they are functionally equivalent

Expression data from the BASE Poplar microarray database showed that PtSHRl transcripts were present in a wide variety of organs (Fig.12). We used native promoter : reporter constructs and immunohisto- localisation to more precisely determine PtSHRl and

AtSHR expression patterns. PtSHRl promoter-driven GFP expression was similar to the published root profile for AtSHR expression (Nakajima et al., Nature. 2001 Sep 20 ; 413 (6853 ): 307-11) . In roots, a 2.5 kbp sequence upstream of the PtSHRl coding sequence drove GFP expression in the stele and during the initiation and early development of lateral roots. In the shoots, GFP was observed in the vasculature of leaves, branches and the main stem. In transverse and radial longitudinal sections of fully expanded internodes in actively growing two month old poplar stem, GFP was observed throughout the cambial zone (CZ) . The CZ is the zone of cell division where stem cells and the transit amplifying (mother) cells undergo divisions, forming new cells for continued xylem (wood) and phloem formation and the consequent increase in girth. GFP was also observed in the phloem parenchyma and in the ray parenchyma and initials. GFP was observed in the ray files through the cambium to the phloem and deep into the maturing xylem (sapwood). Immunofluorescence labeling with a monoclonal anti-PtSHRl antisera also indicated the presence of PtSHRl epitopes in these cells. Immunofluorescent labelling was completely inhibited by a competitor PtSHRl polypeptide.

In the shoot apex, GFP was observed in a group of cells in the corpus, immediately below the tunica in the apical dome. There was also strong GFP expression in the procambial tissues and leaf primordia. Immunofluorescence labelling of radial longitudinal sections through the shoot apex demonstrated the occurrence of

PtSHRl epitopes throughout the apical dome and flanking tissues, providing indication that the protein acts, at least in part, non- cell -autonomously in the shoot apex. Transverse sections immediately below the apex showed GFP expression isolated to the fascicular procambium, however, by internode 4 the expression had extended to the interfascicular procambium and could be observed wherever there was developing vasculature, for example in leaf traces in the stem.

In Arabidopsis, native promoter-driven GUS activity indicated that, similarly to PtSHRl in Poplar, AtSHR is expressed throughout the vasculature of roots and shoots. GUS activity was observed in the vasculature of newly emerged seedlings and in fully developed cotyledons and rosette leaves. It was also observed in the vasculature of the various parts of the flower. When interfascicular cambial activity was present in the inflorescence stems, GUS was found in both the fascicular and interfascicular cambial zones. In the hypocotyl exhibiting secondary growth, activity was associated with the cambial zone and the phloem side of this zone, similarly to the GFP expression in the promPtSHRl :GFP lines. In addition, some GUS labelling was scattered in the secondary xylem of both stems and hypocotyls where it was associated with paratracheal parenchyma.

PtSHRl complements the shr mutant phenotype

In order to test whether the Poplar PtSHR- like proteins are capable of functioning similarly to AtSHR, at least in Arabidopsis, we individually expressed the coding sequences for PtSHRl, PtSHR2A and PtSHR2B, driven by a 2.5kbp 5' upstream sequence of the native AtSHR promoter, in the shr2 (CoIO) loss-of -function mutant. Polymerase chain reaction verification showed that there was expression of the constructs in the respective complemented lines. The primer sets for PtSHR2A and PtSHR2b amplified sequences in both PtSHR2 complemented lines because of the almost identical nucleotide sequences of the two genes. All three PtSHR genes were able to partially complement the shr2 mutant phenotype. The dwarf root and shoot growth phenotype was fully complemented, but the floral stems of the complemented lines remained agravitropic . Interestingly, complementation of the shr mutant with an AtSHR: GFP fusion protein driven by a similar AtSHR promoter sequence also failed to complement the agravitropic shoot phenotype. Also, ectopic CaMV 35S promoter-driven expression of AtSHR in WT (CoIO) Arabidopsis plants resulted in a loss of floral shoot gravitropism. Together, these results provide

indication that a precise spatio/temporal control of AtSHR expression is necessary for this aspect of AtSHR function.

Partial suppression of AtSHR in Arabidopsis leads to a similar phenotype to that observed in the Poplar PtSHRl RNAi lines.

Another possibility for the discrepancy between the loss of function phenotype in the shr mutant and the knockdown phenotype in the Poplar lines is that there may be fundamental differences between a reduction and total absence of the protein. As all of the shr mutant alleles are null, we used an RNAi strategy in Arabidopsis similar to that used in the Poplar transgenics in order to obtain lines that retained a remnant expression of AtSHR. We generated 26 individual RNAi AtSHR suppression lines. Three lines were chosen, Lines 4, 6 and 11, that had between 40 and 75% of the normal levels of AtSHR transcripts found in WT plants. These lines all showed a substantial acceleration of several aspects of vegetative development. After 3 days stratification at 4oC, populations of WT (CoIO) seeds took from 18 to 27 hours to fully germinate (radical emergence) in the light at 23oC on agar plates containing 1 x MS and 1% sucrose (Fig.13A) . The seed samples from the three RNAi suppressed lines commenced germinating several hours before the WT seeds and had fully germinated three hours before the WT population reached the same level of germination (Figs.l3A and 13B) . Forty hours after placing the seeds at 23oC, in contrast to the WT seedlings, most of the transgenic seedlings had fully emerged from the seed coat and were rapidly elongating (Fig.13C). At 5.5 days, the cotyledons (Figs. 13D and 13F) and roots (Fig.13H) of RNAi seedlings grown in the light were substantially bigger than those of the WT growing on the same plates. In contrast, at 5.5 days there were no significant differences between WT and RNAi line hypocotyl lengths and cotyledon sizes of etiolated seedlings grown on 0% sucrose plates (Figs.l3E and 13G) .

Boyes et al . (Boyes et al . , The Plant Cell, Vol. 13, 1499-1510, 2001) published a model for the growth stages of Arabidopsis plants. In the transgenic lines, growth stage 1.06 (six rosette leaves > than lmm in length) was reached by 15 days after sowing (Fig. 14A) . WT seedlings did not reach this stage until day 18 (Fig.14B) . By day 18, immature leaves 1 and 2 were fully expanded in both the WT and the transgenic lines. At this stage, there was no significant difference between the WT and RNAi leaf areas (Fig.14C) . In contrast, the combined area of all of the emerged leaves on each plant was significantly greater in the RNAi lines (Fig.l4E and compare Figs. 14B & 14D) than in the WT. This was due to both the larger size of the expanding leaves and the emergence of more leaves in the RNAi lines. In other words, the RNAi plants were more developmentally advanced than the WT. At 18 days, radial longitudinal sections indicated that the apical domes of the Line 11 plants were substantially larger than the WT (Fig. 14F) .

Height Growth rate of Poplars

Figure 4 and Table 1 show an example of height comparisons of Poplar RNAi and T89 WT plants. From Table 1, it can be seen that the height growth advantage is greatest during the early phase of growth.

Because growth phases have different timing in different plants and there is some noise added, the method described above is useful in calculating the maximum growth speed in these conditions for the different individual trees. These results (using fewer biological replicates for each independent transformation event) can be seen in

Tables 4 and 5.

Diameter Growth rate Under the above defined growth conditions, stem width exhibit a comparatively linear increase over time. Linear regression on diameter data was used for estimating diameter growth. d(t) = c*t +d ₀

where d ₀ is the initial width and c is the rate of diameter growth (slope) .

These height and width growth measurements were compared between WT T89 and the transgenic RNAi plants. For these results, the WT T89 plants were collectively called the wild-type population and the transgenic trees were collectively called the construction group.

Table 4 shows growth data for the KR462 (PtSHRl RNAi) construction group and corresponding wild-type group. Table rows contain height and diameter measurements of individuals of the KR462 (PtSHRl RNAi) group and control wildtype group. This data is summarised in Table 5. The KR462 (PtSHRl RNAi) construct was found to increase the height growth rate by approximately 12% and the diameter growth rate by approximately 18%.

Partial suppression of SHR accelerates morphogenesis in determinate and indeterminate species.

In Arabidopsis, the complete absence of the AtSHR protein leads to the collapse of the root meristem, the dwarfing of the shoots and a reduction in secondary growth. In contrast, we have observed that in both Poplar and Arabidopsis the effect of the partial suppression of PtSHRl and AtSHi?, respectively, leads to an acceleration of the rate of growth of the shoots. The increased growth rate appears to be due to an increased rate of mitotic divisions in the VC and the shoot apical meristem (SAM) .

Many plants have evolved mechanisms for avoiding shading from neighbouring plants by increasing height through increased elongation of internodal cells. In the Poplar transgenic lines, the internodes were shorter than the WT T89 lines and there was no significant difference in the lengths of tracheal elements between the WT T89 plants and the transgenic plants. Increased height, without a commensurate increase in internode or cell length

indicates that, similarly to the increase in girth, the accelerated height growth was the result of additional cell divisions. The combination of taller plants, shorter internodes and no reduction in cell length indicates that the plastochron, the timing of the production of new organs in the SAM, is decreased in the transgenic lines. A number of mutants have been identified where the plastochron is affected. For example, the maize terminal earl, tel (VeIt et al . , 1998. Nature 393, 166-168) and Arabidopsis altered meristem program I ₁ ampl (Chaudhury et al . , 1993. Plant Journal 4, 907-916; Helliwell et al . , 2001. Plant Cell 13, 2115-2125). In addition to an altered plastochron these two mutants have altered phyllotaxy (the fraction of the circumference that seperates two successive leaves on a stem) . Phyllotaxy did not differ between the Poplar WT T89 and RNAi transgenic plants nor between the Arabidopsis WT and RNAi lines. One mutant has recently been identified in rice that has a reduced plastochron (Miyoshi et al . , PNAS Vol. 101 No. 3 2004, 875-880). The PLASTOCHRON 1 gene encodes a cytochrome P450 that is expressed in leaf primordia. Similarly to the PtSHRl and AtSHR RNAi lines, the plastochron 1 mutant is affected in the timing of the production of new leaves, but is not affected in spacing

(phyllotaxy) of new leaf initiation (Miyoshi et al . , PNAS Vol. 101 No. 3 2004, 875-880) .

Several lines of evidence provide further indication of conserved function between PtSHRl and AtSHR

Given that the mis-regulation of PtSHRl and AtSHR both affected VC activity, we considered that there may be conserved patterns of transcript accumulation in the two species. The BASE global transcript microarray data indicated that PtSHRl was expressed in various organs throughout the root and shoot (Fig.12). Native PtSHRl promoter-driven GFP expression also indicated that the gene is expressed throughout the shoot and that this expression is associated mostly with vascular development. In the shoot apex, GFP was observed in a group of cells in the corpus, immediately below

the tunica in the apical dome. The combination of a lack of a GFP signal and the presence of immunofluorescent labeling in zones flanking the central zone of the SAM suggests that PtSHRl has the ability to move from cell to cell. In Arabidopsis, cell-to-cell movement of AtSHR is critical for the function of the protein and proper root development (Nakajima et al . Nature. 2001 Sep 20;413 (6853) : 307-11; Benfey et al . , Benfey et al . , Development. 1993 Sep; 119 (1) : 57-70) . Shoot tip growth is ultimately derived from a population of stem cells in the centre of the SAM (Fletcher JC. , 2002 Annual Review of Plant Biology, 53, 45-66) . Another mechanism by which PtSHRl epitopes could be present in cells where the gene is not expressed is that the gene could be expressed in the stem cells and could then be retained in these cells and daughter cells as they move out from the apex during cell division and apical development. GUS staining in the Arabidopsis promAtSHR:GUS lines indicated that

AtSHR is expressed in similar zones throughout the Arabidopsis plant providing further evidence that the two proteins carry out the same functions .

Further evidence in support of this hypothesis is that all three

Poplar PtSHR- like proteins were able to individually complement the shr2 loss-of -function mutant phenotype when driven by the driven by a 2.5kbp 5' upstream sequence of the native AtSHR promoter (and by the constitutive CaMV 35S promoter) .

Partial suppression of AtSHR in Arabidopsis leads to a similar phenotype to that observed in the Poplar PtSHRl RNAi lines. The phenotype of the AtSHR knockdown lines provided further strong evidence in support of the hypothesis that PtSHRl and AtSHR have similar functions. Vegetative development is accelerated in the

PtSHRl RNAi lines. In Arabidopsis, already by germination, the AtSHR RNAi lines were developmentalIy about 3 hours advanced on the WT seedlings. Boyes et al . (Boyes et al . , The Plant Cell, Vol. 13, 1499-1510, 2001) published analyses of Arabidopsis that defined a

series of growth stages for use in the analysis of transgenic and mutant lines. Their growth stage 1.06 was reached by 15 days after sowing in the transgenic line (Line 11) (Fig.14) . This stage was not reached in the WT seedlings until day 18. Combined with the data showing that there was no difference in the final fully expanded size of leaves 1 and 2, this indicates that, similarly to the PtSHRl RNAi lines, the affect of the suppression of AtSHR expression is to increase the rate of new organ initiation, but not the final size of those organs. This data suggests strongly that there is a fundamental difference between reduced expression and a total lack of the AtSHR protein.

shr mutant shoots have altered morphology, but the plastochron remains unchanged. The sum total of our data provides strong evidence that AtSHR and PtSHRl are functionally equivalent. In both Poplar and Arabidopsis transgenic lines, a decrease in the levels of SHR transcripts led to an increased growth rate. This suggests that the proteins act as negative regulators of cell division and growth. In the shr mutants, where there is a total absence of the protein, the root apical meristems collapse, the shoots are dwarfed and secondary vascular development is greatly reduced. Clearly, some level of AtSHR is essential for normal cell division in both the roots and shoots. The rate of vegetative development and the timing of vegetative to floral transition are not delayed in the shr mutants, however. At flowering they have, on average, the same number of leaves as WT plants. This provides indication that, whereas SHR regulates cell division and vegetative development in the shoot, the decreased plastochron in the both the Arabidopsis and Poplar RNAi lines is a secondary effect of the suppression of SHR gene expression. The primary effect is a stimulation of cell division in the SAM.

Lines

Days after RNAi RNAi RNAi WT planting Line 2A Line 2B Line 4A T89

16 days 216* ^♦ 1 (17) 239*** (25) 205* (33 ) 175 (23)

28 days 429* * * (42) 508*** 50) 412*** ( 35) 336 (42)

40 days 933* * * (84) 1053*** (64) ₉₁ 3*** ( 80) 778 (65)

52 days 1364 * * * (95) 1497*** (57) 1316** ( 105) 1173 (75)

Table 1

Means (n = 9) of heights of trees (SDs within parentheses) for glasshouse growth comparison of PtSHRl RNAi lines 2A, 2B and 4A with control T89 trees

Stars indicate the significance value of the mean difference between each line and the Wild Type T89 (tested with Dunett's post hoc test to compare all samples with a control) *p < .05, **p < .01, ***p < .001

Table 2

Table 3

Height (cm) Diameter (mm)

Days In greenhouse 18 26 33 39 47 53 65 33 39 47 53 59 65

KR462 (PtSHRI) 2A-A 28 44 62 100 120 143 193 49 66 77 91 85 93

KR462(PtSHR1)-2A-B 26 42 63 99 116 137 181 46 58 58 65 69 75

KR462(PtSHR1)-2B-A 18 28 38 65 80 99 140 35 53 61 69 78 79

KR462 (PISHR 1 )-4A-A 20 40 57 95 112 133 168 46 64 74 85 87 99

KR462 (PtSHR 1)-5A 17 27 39 67 83 100 140 37 51 62 71 75 83

T89-133 23 37 51 84 103 121 156 47 67 77 84 91 97

T89-134 25 38 50 80 95 112 149 44 62 71 76 76 84

T89135 17 29 43 71 86 104 140 48 66 77 86 91 91

T89-136 19 31 45 72 85 103 138 46 53 60 64 73 78

T89-137 22 37 52 82 96 112 151 50 65 73 78 N/A 100

T89-138 21 33 47 78 94 109 140 49 60 70 78 84 90

T89-139 24 39 56 92 108 124 159 48 57 63 70 76 84

T89-140 25 41 56 88 101 115 148 49 60 60 68 70 78

T89-141 17 30 45 77 87 104 132 50 53 62 64 69 72

T89-142 24 38 54 85 99 116 146 53 59 63 66 76 83

T89-143 24 40 56 89 107 122 155 47 57 63 68 73 84

T89-144 23 37 52 76 89 107 139 45 59 65 69 71 79

T89-146 27 43 58 84 99 116 153 46 60 67 74 78 85

T89-147 26 44 62 95 111 130 167 54 65 69 74 80 87

T89-148 21 32 47 77 91 109 146 42 54 61 66 69 78

Table 4

AMHGR MMHGR

Average ADGR Maximum

AFH AFD height Average MFH MFD height MDC

Average Average growth Diameter Maximum Maximum growth Maximum

Final Final rate Coef f ici Final Final rate Diamete

Height Diameter (KRmean ent Height Diameter Maxima Coef f ic

Constructi (KRmean/ (KRmean/ / (KRmean/ (KRmax/ (KRmax/ KRmax/ t (KRma on group WTmean) WTmean) WTmean) WTmean) WTmax) WTmax) WTmax WTmax)

KR462 1, 11 1,01 1, 12 1, 18 1, 16 0, 99 1, 10 1,05

Table 5

Table 6

Sequences

PtSHRl eugene3.01860017 (SEQ ID NO : 1 ) GGGAGAAGACTGGGCTAGCTAGCTAGATAAAAGAATATCCCAAACCCCACCCAATTTGAT TCACATCGAC CAAGATCCTCTCTTTTATCATTCTCTACCCCAATATTCCCACAGTTAAAAACACAAACAC CCATCCCCTT ACCTTCACTCCAAGCCATCCCCAACAAACTCCATCCGAATTCTATTGATGTGGGTCTCTG GATTTTGGTT AAATGGATACCTTGTTTAGGCTAGTTAGTCTCCAACAACAATCTGAACAATCTTTCAACT CTACTAGCAG AACCTCTAGTAGCTCTAGATCATCAAGACAAAACAACAACCACCACCATCATCACTATCA ACAAGAAGAC GAAGAATGCTTCAACTTTTTCATGGATGAGGAAGACTTCTCTTCATCTTCTTCTAAGCAC TACTATCCTC

CAACACTAGCACCCCTTCTACTCACCATGTCCTTGATTCCGCTGACTTCTCTTTCTC CCCTTCTCATGAC AAAACAGCGCTCGTCTCCAGCAATTGATGTGGATGCTTAATGAGCTTGGTTCACCTTATG GTGACACAGA

AAGAGGTGAGTCCTTGGACTACTTTTGGTCACGTATCTTGTAATGGCGCAATTATGG AAGCATTTGAAGG

CTAGCAACTCGCACTGATGAGACACCACACTTGAAGTTAACCACCGTAGTGGCTAGC AAAAGTAGTGGTA AATGGAAAAATTTGCCAGGCTTATGGGAGTCCCATTTAAGTTTAATGTTATCCACCATGC TGGTGATTTA

TACACTCAATCACTCCAGCTTCTCGTCGCCGAGATTATGTTATATCTAGTTTTAGAA CATTGCAACCAAG

TTAAGATGGTTTAGGGTTTACTTTGAATCATTGGATGAGAGCTTTCCAAGAACCAGT AACGAACAGTTGA TGCTTGAAAGAGCAGCAGGCCGCGCTATCGTTGACTTAGTGGCATGTCCTCCATCTGATT CGATCGAAAG

GAGGTTTGTGATGATGTACGCGCCTTATTGAGGAGGTATAAGGAGGGTTGGTCAATG ACACAGTGCGGGG ATTGGTGATGGTTTTCACTTTTCACTTTTCTTTTCTTTCTTTTTTCTTTTGTTTTTGCTT TTCTCCTTGT TTAAGGATTGGAATAGTAAATGAAGTTAATCAATTTTCATTGATCTTTGTTTAATTATCA TTTGCGGTTA

AACAAATTAGTTGAGCAAGATTTATCTTTGGGGTAAATAAATGATTTAATTTAGGAA AGTTATTCGATAA TTA

PtSHRl eugene3.01860017 (SEQ ID NO : 2 )

MDTLFRLVSLQQQSEQSFNSTSRTSSSSRSSRQNNNHHHHHYQQEDEECFNFFMDEE DFSSSSSKHYYPPYHHNQ QQQHQHQTTTTTPTTTTTNTSTPSTHHVLDSADFSFSPSHDLNFEFSGKWVTDILLESAH AIADKNSARLQQLMW MLNELGSPYGDTEQKLASYFLQALFSRMNDSGERCYRTLASASEKTCSFDSTRKMVLKFQ EVSPWTTFGHVSCNG AIMEAFEGESKLHIIDISNTYCTQWPTLLEALATRTDETPHLKLTTWASKSSGNNIGLTS TGGLASVHKVMKEI GNRMEKFARLMGVPFKFNVIHHAGDLCDLNLAELDVKDDEALAINCVGALHSITPASRRR DYVISSFRTLQPRII TWEEEADLDGLDFVKGFQECLRWFRVYFESLDESFPRTSNEQLMLERAAGRAIVDLVACP PSDSIERRETATRW SGRLHSCGFSPIIFSDEVCDDVRALLRRYKEGWSMTQCGDAGIFLCWKEQPWWASAWRP PtSHR2A eugene3.00070144 (SEQ ID NO : 3 )

AAGTCCTCGCCACTTATATGTGTTTATTGAGAAAAAAATATATATTATACATGTAGT ACGTACGTATGA TATGCAATATCGAACAAGTATATACTAGCTAGGGCTTTGAGGTTATTGCAATTATGGTTG CCAAATCCAA CCACTACACTAGCAATTAGGTATTTTTCATCTCGAGATCACAATATCATTAATTTGAAGC TAATAATACA CATAAAAATAGAAATTCATATTAAGAAAAAATATATATATATAGCATGACTATGACCCCC AAACCCCAAA ATATAATACTACGGATTTCTATATCATAGCTTTGTACTTGGCCAATTTTTTTGAATTAGC AGAGGTACTG CCTCCAGAGTCAAAGGGGGCCTTCCCTAGCTATCACCCACGGACATAACCATCTTCGGTC CAAAAGACAG ACTCACACACTTATTTCCTTCCCACCAACAAAGCAATGATCAAATAGAAGCTAGCTATAG ACATGCAAAG CAATAGCAGCAACAACAACAACAATCAGCCTCAGACTAGTCATACATCAACAAGCCGTTC TTCGGACTCC GGTGAGGCTTGTGGGGCAGGAAACAAATGGGCATCAAGGCTTCTTAGTGAGTGTGCAAGA GCAATCTCAG AGAAGGACTCTAGCAAGATCCATAACCTTCTATGGATGTTAAATGAGCTTGCCTCTCCTT ATGGAGATTG TGAACAGAAATTGGCATCTCATTTCTTGCAAGCTCTCTTTTGTAAGGCTACCGACTCCGG CCAACGGTGC

TTCAAAACCCTAACAACAGTAGCTGAAAAGAGCCACTCCTTTGATTCAGCTAGGAAA TTGATACTAAAAT TCCAAGAGGTAAGCCCATGGACTACTTTTGGTCATGTGGCTTCAAACGGTGCAATTTTGG AGGCCTTAGA

GCTTTAGCTACAAGAAATGATGAGACGCCGCATTTAAAGCTCACCGTTGTGGTAACT GCTAGCATTGTAA

TAATGTAATTAGTGGGCTAAACCATTTAGGAGAGCTCACAAAGGACAGGCTAGGAGT TCAAGAAGATGAA GCTGTCGCGATTAATTGCAATGGGGCATTGAGAAGAGTTGGAGTAGAGGAAAGAAATTCT GTGATCCAGA TGTTTCAATCACTTAACCCTCGAGTTGTGACAATTGTTGAAGAAGAAGCTGATTTTACTA GCTCAAGATA TGACTTTGTCAAGTGCTTTGAAGAGTGCCTTAGGTATTATACACTATATTTTGAGATGCT AGAGGAGAGC TTTGTCCCAACTAGTAATGAGAGATTGATGTTGGAGAGGGAATGTTCAAGGAACATAGTT AGAGTTTTGG CTTGTGATGAAGGAAATGATGGAGGAGAGTGTGAAAGAAGGGAGAGGGGAAGCCAATGGT TTGAAAGGCT

TACCGAGCTGGGTGGGCACTAGTGCTACCTCAAGGAGATCATGACTCAGGAATTTAC TTAACATGGAAAG ACACTCAAAGTCAAGGCTTAGCATGGCAATTTATTCACAATTTGGGAAGCACTGCCATGA ACACAACATA TGTCTTCTGTACTCCAACTTTTCTTTTCTTATGCT

PtSHR2A eugene3.00070144 (SEQ ID NO : 4) MQSNSSNNNNNQPQTSHTSTSRSSDSGEACGAGNKWASRLLSECARAISEKDSSKIHNLL WMLNELASPYGDCEQ KLASHFLQALFCKATDSGQRCFKTLTTVAEKSHSFDSARKLILKFQEVSPWTTFGHVASN GAILEALDGASKLHI IDISHTLCTQWPTLLEALATRNDETPHLKLTVWTASIVRSVMKEIGQRMEKFARLMGVPF ELNVISGLNHLGEL TKDRLGVQEDEAVAINCNGALRRVGVEERNSVIQMFQSLNPRWTIVEEEADFTSSRYDFV KCFEECLRYYTLYF EMLEESFVPTSNERLMLERECSRNIVRVLACDEGNDGGECERRERGSQWFERLREAFSPV GFSDDVVDDVKALLK RYRAGWALVLPQGDHDSGIYLTWKEEPWWASAWKP

PtSHR 2B eugene3.00640143 (SEQ ID NO : 5 )

ACATATAAGAATATTTCAAATATAATACACAAGAAATTATTCACATTAACAAAATAG GATGACTATGACC

CCCCACCCCAAGATATAATTATACTATGGATATCTATATCAATATAGCTTTGTACTT GGCCATGTCTGAA CTCAAATGAAATTTGAGTTTGGCAAAGGTTCTGCCTCTAGAGTCAAAGGGGGCCTCGCCG ATCACCAATG

GACATAACTCTCTTCAGTCCAAAAGACACACTGACACACTTCTTTCCTTCCCACCAG CAAAGCAATGATC AAGTAGCAGCTATAGACATGCAAAGCAATAGCAGCAACAACAACAATAATCAGCCTCAAA CCAGCCATAC ATCAACAAGCCGGTCTTCGGACTCCGGTGAGGCCTGTGGAGGAGGAAACAAGTGGGCATC AAAGCTTCTT AGTGAGTGTGCAAGAGCAATCTCAGAGAAGGACTCTAGCAAGATCCACCACCTTCTATGG ATGTTAAATG AGCTTGCCTCTCCTTATGGAGATTGTGATCAGAAATTGGCATCTTATTTCTTGCAAGCTC TATTCTGTAA

GGCTACCGAGTCTGGTCAACGGTGTTTCAAAACCCTAACAACAGTAGCTGAAAAGAG CCACTCCTTTGAT TCAGCTAGGAAATTGATACTAAAATTCCAAGAGGTAAGCCCGTGGACTACTTTCGGTCAT GTAGCTTCAA ATGGTGCAATTTTGGAGGCCTTAGATGGGGAAAGCAAACTTCACATAATTGATATCAGCA ATACCCTTTG CACACAGTGGCCTACTTTGCTAGAAGCTTTAGCCACAAGAAATGATGAGACGCCGCGATT AAAGCTCACC GTTGTGGTAACTGCTAGCATTGTAAGATCAGTCATGAAAGAAATTGGCCAAAGAATGGAG AAGTTTGCTA

GGTTAATGGGAGTGCCCTTTGAGTTTAAAGTAATTAGTGTGCTAAATCATATAGGAG AGCTCACAAAGGA AGGACTGGGTGTTCAAGAAGATGAAGCAGTCGCGATTAATTGCATTGGGGCATTGAGAAG AGTTGAAGTA GATGAAAGAAGTTCTGTAATCCAGTTGTTCCGATCACTTAACCCTCGAGTTGTGACAATT GTAGAGGAAG AAGCTGATTTTACTAGCTCAAGATATGACTTCGTCAAGTGCTTTGAAGAGTGCCTGAGGT ATTATACACT ATATTTTGAGATGCTAGAGGAGAGCTTTGTCCCAACTAGTAATGAGAGATTGATGTTGGA GAGGGAATGT

TCAAGGAACATAGTTAGGGTTTTGGCTTGTGATGAAGAAACTGGTGGAGGAGAGTGT GAAAGAAGAGAGC GGGGTGTCCAATGGTCTGAAAGGCTAAGGGAGGCATTTTCCCCTGTTGGATTCAGTGATG ATGTTGTCGA TGATGTCAAGGCATTGCTTAAGAGATACAAAGCTGGGTGGGCACTTGTGCTACCTCAAGG AGATCATGAG TCAGGAATTTACCTAACATGGAAAGAGGAACCTGTAGTATGGGCTTCTGCATGGAAACCC TAAAAGGTTG TGGCCAGAACACCATCTCTATGCTCAAATTCAAGGCTCAGCATGGCAATTTATTCACAAG ATGGGAACAG

CGCGCCATGAACACATATTGATAATTAATTATAGAGTACTACTACTTTCACTTTTCA TTTCTTCTTCTTA TTCATGCATATTTGTATCTTATGCACTCCAACTTTTCTTTTCTTATGTTATAT

PtSHR2B eugene3 . 00640143 ( SEQ ID NO : 6 )

MQSNSSNNNNNQPQTSHTSTSRSSDSGEACGGGNKWAS KLLSECARAISEKDSSKIHHLLWMLNELASPYGDCDQ KLASYFLQALFCKATESGQRCFKTLTTVAEKSHSFDSAR KLILKFQEVS PWTTFGHVASNGAILEALDGESKLH I IDISNTLCTQWPTLLEALATRNDETPRLKLTVWTASIVRSVMKEIGQRMEKFARLMGVPF EFKVISVLNHIGEL TKEGLGVQEDEA VAINCIGALRRVE VDERSSVIQLFRSLNPRWTIVEEEADFTSSRYDFVKCFEECLRYYTLYF EMLEESFVPTSNERLMLERECSRNI VR VLACDEETGGGECERRERGVQWSERLREAFS PVGFSDDWDDVKALLK RYKAGWALVLPQGDHESGIYLTWKEEPWWASAWKP AtSHR At4 g37650 ( SEQ ID NO : 7 )

ATGGATACTC TCTTTAGACT AGTCAGTCTC CAACAACAAC AACAATCCGA TAGTATCATT ACAAATCAAT CTTCGTTAAG CAGAACTTCC ACCACCACTA CTGGCTCTCC ACAAACTGCT TATCACTACA ACTTTCCACA AAACGACGTC GTCGAAGAAT GCTTCAACTT TTTCATGGAT GAAGAAGACC TTTCCTCTTC TTCTTCTCAC CACAACCATC ACAACCACAA CAATCCTAAT ACTTACTACT CTCCTTTCAC TACTCCCACC CAATACCATC CCGCCACATC ATCAACCCCT TCCTCCACCG CCGCAGCCGC AGCTTTAGCC TCGCCTTACT CCTCCTCCGG CCACCATAAT GACCCTTCCG CGTTCTCCAT ACCTCAAACT CCTCCGTCCT TCGACTTCTC AGCCAATGCC AAGTGGGCAG ACTCGGTCCT TCTTGAAGCG GCACGTGCCT TCTCCGACAA AGACACTGCA CGTGCGCAAC AAATCCTATG GACGCTCAAC GAGCTCTCTT CTCCGTACGG AGACACCGAG CAAAAACTGG CTTCTTACTT CCTCCAAGCT CTCTTCAACC GCATGACCGG TTCAGGCGAA CGATGCTACC GAACCATGGT AACAGCTGCA GCCACAGAGA AGACTTGCTC CTTCGAGTCA ACGCGAAAAA CTGTACTAAA GTTCCAAGAA GTTAGCCCCT GGGCCACGTT TGGACACGTG GCGGCAAACG GAGCAATCTT GGAAGCAGTA GACGGAGAGG CAAAGATCCA CATCGTTGAC ATAAGCTCCA CGTTTTGCAC TCAATGGCCG ACTCTTCTAG AAGCTTTAGC CACAAGATCA GACGACACGC CTCACCTAAG GCTAACCACA GTTGTCGTGG CCAACAAGTT TGTCAACGAT

CAAACGGCGT CGCATCGGAT GATGAAAGAG ATCGGAAACC GAATGGAGAA ATTCGCTAGG

CTTATGGGAG TTCCTTTCAA ATTTAACATT ATTCATCACG TTGGAGATTT ATCTGAGTTT

GATCTCAACG AACTCGACGT TAAACCAGAC GAAGTCTTGG CCATTAACTG CGTAGGCGCG

ATGCATGGGA TCGCTTCACG TGGAAGCCCT AGAGACGCTG TGATATCGAG TTTCCGACGG TTAAGACCGA GGATTGTGAC GGTCGTAGAA GAAGAAGCTG ATCTTGTCGG AGAAGAAGAA

GGTGGCTTTG ATGATGAGTT CTTGAGAGGG TTTGGAGAAT GTTTACGATG GTTTAGGGTT TGCTTCGAGT CATGGGAAGA GAGTTTTCCA AGGACGAGCA ACGAGAGGTT GATGCTAGAG CGTGCAGCGG GACGTGCGAT CGTTGATCTT GTGGCTTGTG AGCCGTCGGA TTCCACGGAG AGGCGAGAGA CAGCGAGGAA GTGGTCGAGG AGGATGAGGA ATAGTGGGTT TGGAGCGGTG GGGTATAGTG ATGAGGTGGC GGATGATGTC AGAGCTTTGT TGAGGAGATA TAAAGAAGGT

GTTTGGTCGA TGGTACAGTG TCCTGATGCC GCCGGAATAT TCCTTTGTTG GAGAGATCAG CCGGTGGTTT GGGCTAGTGC GTGGCGGCCA ACGTAA

AtSHR At4g37650 ( SEQ ID NO : 8 ) MDTLFRLVSLQQQQQSDS I ITNQSSLSRTSTTTTGSPQTAYHYNFPQNDWEECFNFFMDEEDLSSSSSHHNHHN

HNNPNTYYSPFTTPTQYHPATSSTPSSTAAAAALASPYSSSGHHNDPSAFSIPQTPP SFDFSANAKWADSVLLEA ARAFSDKDTARAQQILWTLNELSSPYGDTEQKLASYFLQALFNRMTGSGERCYRTMVTAA ATEKTCSFESTRKTV LKFQEVSPWATFGHVAANGAILEAVDGEAKIHIVDISSTFCTQWPLLEALATRSDDTPHL RLTTVWANKFVNDQ TASHRMMKEIGNRMEKFARLMGVPFKFNIIHHVGDLSEFDLNELDVKPDEVLAINCVGAM HGIASRGSPRDAVIS

ACEPSDSTERRETARKWSRRMRNSGFGAVGYSDEVADDVRALLRRYKEGVWSMVQCP DAAGIFLCWRDQPWWAS AWRPT

Rice SHR nucleotide sequence (SEQ ID NO: 9)

CTACTAATAGTTTATTTGTTTTACCGGTT

Rice SHR AAS07303.1 GI: 41469537 (SEQ ID NO: 10) MDTLFRLVSL HHHHHHQHAA SPSPPDQPHK SYPSSRGSTS SPSSHHTHNH TYYHHSHSHY NNNSNTNYYY QGGGGGGGGY YYAEEQQPAA YLEECGNGHQ FYMDEDFSSS SSSRQFHSGT GAPSSAPVPP PPSATTSSAG GHGLFEAADF SFPQVDISLD FGGSPAVPSS SGAGAGAGAA

PSSSGRWAAQ LLMECARAVA GRDSQRVQQL MWMLNELASP YGDVDQKLAS YFLQGLFARL

TTSGPRTLRT LATASDRNAS FDSTRRTALK FQELSPWTPF GHVAANGAIL ESFLEAAAAG AAASSSSSSS SSTPPTRLHI LDLSNTFCTQ WPTLLEALAT RSSDDTPHLS ITTWPTAAP

SAAAQRVMRE IGQRLEKFAR LMGVPFSFRA VHHSGDLADL DLAALDLREG GATAALAVNC

VNALRGVARG RDAFVASLRR LEPRWTWE EEADLAAPEA DASSEADTDA AFVKVFGEGL

RFFSAYMDSL EESFPKTSNE RLSLERAVGR AIVDLVSCPA SQSAERRETA ASWARRMRSA

GFSPAAFSED VADDVRSLLR RYKEGWSMRD AGGATDDAAG AAAAGAFLAW KEQPWWASA WKP

Rice SHR nucleotide sequence (SEQ ID NO: 11)

AAATTTTACGTGCATGGTTTTTATCATGCGTACCCGCAA Rice SHR BAD30442.1 GI: 50509213 (SEQ ID NO: 12)

MEKASKAKAE AAKGAHTHCR CLLLLPPMDT LFRLVSLQAA SEQQQQQQQS ASYNSRSTTS SGSRSSSHQT NASYSYYHHS SNSGGGGGGG GGYYYGGQQP PPSQYYYLEP YQEECGNAPH HQLYMDEDFS SSSSSRHFHH GARVQQQQPP ASSTPTGTAP TPPLSTSSTA AGAGHGLFEA ADLSFPPDLN LDFSSPASSS GGGTASSGAV GGGGGGRWAS QLLLECARSV AARDSQRVQQ LMWMLNELAS PYGDVEQKLA SYFLQGLFAR LTASGPRTLR TLAAASDRNT SFDSTRRTAL RFQELSPWSS FGHVAANGAI LESFLEVAAA ASSETQRFHI LDLSNTFCTQ WPTLLEALAT RSADETPHLS ITTWSAAPS APTAAVQRVM REIGQRMEKF ARLMGVPFRF RAVHHSGDLA ELDLDALDLR EGGATTALAV NCVNSLRGW PGRARRRDAF AASLRRLDPR WTWEEEAD LVASDPDASS ATEEGGDTEA AFLKVFGEGL RFFSAYMDSL EESFPKTSNE RLALERGAGR AIVDLVSCPA SESMERRETA ASWARRMRSA GFSPVAFSED VADDVRSLLR RYREGWSMRE AGTDDSAAGA GVFLAWKEQP LVWASAWRP

Barley SHR nucleotide sequence (SEQ ID NO: 13)

GCGTGATGGGTGTAAGCACTATGATTGTTGTTACAGT

Barley SHR HvGI TC147542 (SEQ ID NO: 14) MADTPTSRMI HPFSNMQRQ.N PKQFQFQYPD NPQHPCHPYQ PSPDTHWPQ HHYSLKSHSS

DASYENHVAQ MKHTLVDSSA AAGCMRHDSP SSHSFTPPSI RSGSGSPSSH DDSHSDSTDG

SPVSASCVTV TTEDPNDLKQ KLKDLEAEML GPDAAEIVNS LESSVAKQLS LEPEKWAQMM

DFPRGNLKEL LLACARAVEE KNMYAVDVMV PELRKMVSVS GTPLERLGAY MVEGLVARLA

SSGHSIYKAL RCKEPKSSDL LSYMHFLYEA CPYFKFGYMS ANGAIAEAVK GEDRIHIIDF HIAQGAQWIS LLQALAARPG GPPTVRITGI DDSVSAYARG GGLDLVGRRL SHIAGLCKVP

FEFRSVAMAG EEVEEGHLGV VPGEALAVNF TLELHHIPDE TVSTANHRDR ILRLVKGLRP

KVLTLVEQES NTNTAPFPQR FAETLDYYTA IFESIDLTLP RDDRERVNME QHCLAREWN

LIACEGAERV ERHEVFGKWK ARLTMAGFRP SPLSSLVNAT ISKLLQSYSD NYKLAERDGA

LYLGWKKRPL WSSAWH

Barley SHR nucleotide sequence

Barley SHR AAL66734.1 GI : 18254373 (SEQ ID NO:16)

MKREYQDGGG SGGGGDEMGS SRDKMMVSSS EAGEGEEVDE LLAALGYKVR ASDMADVAQK

LEQLEMAMGM GGPAPDDGFA THLATDTVHY NPTDLSSWVE SMLSELNAPP PPLPPAPPQL

NASTSSTVTG GGGYFDLPPS VDSSSSTYAL RPIISPPVAP ADLSADSVRD PKRMRTGGSS TSSSSSSSSS LGGGAARSSV VEAAPPVAAA AAAPALPVW VDTQEAGIRL VHALLACAEA

VQQENLSAAE ALVKQIPLLA ASQGGAMRKV APYFGEALAR RVFRFRPQPD SSLLDAAFAD

LLHAHFYESC PYLKFAHFTA NQAILEAFAG CRRVHWDFG IKQGMQWPAL LQALALRPGG

PPSFRLTGVG PPQPDETDAL QQVGWKLAQF AHTIRVDFQY RGLVAATLAD LEPFMLQPEG

EEDPNEEPEV IAVNSVFEMH RLLAQPGALE KVLGTVRAVR PRIVTWEQE ANHNSGSFLD RFTESLHYYS TMFDSLEGGS SGGPSEVSSG GAAPAAAAGT DQVMSEVYLG RQICNWACE

GTERTERHET LGQWRNRLGN AGFETVHLGS NAYKQASTLL ALFAGGDGYK VEEKEGCLTL GWHTRPLIAT SAWRLAAP

Residues 1 to 120 of PtSHRl (SEQ ID NO: 17)

MDTLFRLVSLQQQSEQSFNSTSRTSSSSRSSRQNNNHHHHHYQQEDEECFNFFMDEE DFSSSSSKHYYPPYHHNQ QQQHQHQTTTTTPTTTTTNTSTPSTHHVLDSADFSFSPSHDLNFE

SEQ ID NO: 18 VMKEIG (N/Q) RMEKFARLMGVPF (K/E) (F/L) (N/K)VI

SEQ ID NO: 19 LNEL (G/A) SPYGD (T/C) (E/D) QKLAS (Y/H) FLQALF

SEQ ID NO: 20 LKFQEVSPW (T/A) TFGHV SEQ ID NO: 21 CTQWPTLLEALATR

AtSHR promoter (SEQ ID NO: 22) agatttcgagaaattacataagaaactgaattgcaacactcgataggtttcgaagaaagg gacaaagaagcagag cgtggggtttcttctaataattgtagaagaaactgatcatgagaacatttgatctaccag agatggtgatgactc ataagatgtaaatatctactgcattatgtctagcctaggctataatgtagatttgatcac tttcttcattaatta gtttggaattttagcatgatatagcatatatctaaatatgtccgaaactttcctacatac tagaaaatatggaga gttatgtaatgtaggtttgcttgttaatatacaaaataacatcatcatttagtttttaga ttttttattttattt tttataatggtgctacgtacgtggcgatcaaattattccaattttgagacttcgggattt taaacgaaattaaac aatgggcatgagctcggggggatagacaagattaatgctttgtatcgagacaaacgagaa aatcatgatgagcct atgcattaagtgccgttggttaattagaggttcgcatatacataaaccagtagacatatg gataaatatgaacac acacaccaaaaaagtgggaaatctaaataagtgtagagaataataagtcctcaggtggga gattcaaagagagga caatgaagggtatatagactctaaacaaaaatggcatgacttagtggagagggttttaaa ttgaaacaagtagga ttgaagaacaagaaaacaaagaagcatgccctagatttctgagataataattacacattg ctgtttatataaggt aagagaatatgacacattggttggtttcttacgggtaaatgtgaagaaaaaaaaatagta atatttgagaaaatc taaaatagtaaagaggtatatatggagaagaagagagaaaagggaaaaatagtggcagag aatggagagaggtta ggaggcaaaggcaaatgtggagctttgatgatgttgatgcacgccgtcagcttttcttca cgcctgctcccactc actcacacctatgaacattctctctctattttataattatattcacatgtctctatgtta ctatgtaaatggtga ccacttaagtatttatatatcatgtatatatcttataggtatcatacaaaatggtcatga aacttttgcaatttc aatctacttgttcattgtagatgctagcttttcacatgttttgaaaattagtctggatct gaaattctttaatta gcattgttttgttggtcaacgtttaatttcttgattattgatgtcaaaaattcagagcgt tcagaactcttacac taatttcttaaaaataatcgattaagagaaaatagagttttcatgcaccagtgttgatag taacgtagtcgcgga atgtctaaaacgattatgagtttggtgttttgattggttagaattggtattagtaggaca ttctaacttttttgt tagtctgttgatttaggatgcgtaaagagtctttttattttacaccagttgagacttggg atcgatagtacttga aacacttggttggtttcatgtatttggcctatatataaacaaacatcgtaattatatacg gatttttttcggaat tttacgccatatctgtaagtatatataacatgcatgtcgttttcaaattcatatgatgaa cgatccacgtaagtg ctactactcctacaatattgcatgagagagatatgtatttataaattttattttgaagaa gaaataagagggaag gttacttgggtggatcgatgtgaaaacaaaagaagaaaaagcgaaacccactaagccatt acatgatatcgacct tcttatctttttcctctttattttatttttctcaggacttttttctacttaatgaaacct ccaaactatctaact aatacactcccatgtagaataaagaaaattatataagatattgttgatattttgtaacta gaaaatatatttgct ctgtaatttttcgtaagttaaatcaacatttttcagtagaaacaaatattactgcaaaaa gtaggatcattattt ttgtccaaaatctcagttagctatagggttgtagtaaaaacaaaacacattcttgatttg ccccaaaaaataaag

agagagaagaatattgttcaaaagtggtctcttctctctctaattatgttttcacta aacccaattagattcaaa cagtctacaaagtccaaaagataaacatgggacaacaattcgatgcaaaaaatcctcttt tcatgctcttttttt attctctagtcttttaaattactaataaaaactcacaaatccaccaaacccattctctac aactcaccttcatct agatttacccactcccaccgagaaacacaagaaaaaaaatatacatatataaatatacaa gacaacacatgatgc tgatgcaatatacacaacaaagtattaaatcttagatattgtgggtctccctttcttcta ttcattttcttattc attaaaaaaaaaaa

PtSHR promoter (SEQ ID NO: 23)

AAATCAATAAAAAAACAAAATAAATAACAATCAAAAGAATGACCAAATTAGATATAA AAGAACAAATAAA ATGAAACATTTATATTTTGGCAAGGAGAAAAGGAGAAAAATAGGGAGGAGAAGAAATAAA TATTCAATCG GAGCAAATCATTACACTGTCATGTACACGCGTCAGACTATTGAATAGAGGATGGCAGGAT GCTTCCATCT TTGCCATGAAAACCGGTGTTTGGAGGGTAGATAATGCCTTAAACATTGCCTAAAAGGCAG GGGTATCGTC CAACGCCCGCACCAACCACTTTTTATTTTTAACTAATATCTAAATTTATCAAAATACCAA ATCGCTTCCA AGTCAACTTGATAATTACTAAAAACATATTATGAAATGACCAAAAAGCACTTGAATAATA GTTTGAAAAT

TCAATATTTTTAGAAAATTTGTTTTTAAGGGTAATAAAGTTTTTAAAATATGCATTT AAAAGGTTAAAAG ACCAAATTATTCCATAAGAAAAAAACCGTTTTACTTCCAATAGCAAGTTAATTGATTTTT TTGTTGGAAG

CTCAATTCTGATAAAAAAAGTTAGTATTTTCTACTTTTAAAAATATTATAAGTTTTT ATTTCAATTATGA ATGTTATAATAATTAAAAAATATATATGATATAGGTGTAATGCTATTATAGAAAAAATAT AAAATAAATT

ACCTACCTTTTTATCTTCTACTCACAAAAAAAAATTAAAATAGTTTTTTTTAAATAG ATTTATGCTTGTT

ACCTTTATTCAAATAATAAGACCGGGTCACCGGGTCATATATATATATATATATATA TATATATATATAT ATCTACACGAGACAGAATCCAGACGTGATAGTTTTCCTAATGATTGATGGAGAAAACCAA GGACGAGCAG

TGATTAGGAACAAGGAAAATTTCAACAGACAAAAGAAACTCATATATATATATATAG ACACACACTCTCA CCTCTTGCGTATGAAAATGTAGAATAGCAATAGAGCATAATGTTGCTGACTATAAAAGCA AAGAGTAGAC CAACCGTGACATACATTAAAATCCAAATATTTGATCTGCGTTGAGGTTTAGCAAGCTGAC ACAGAAAGAA ATAAATGTTTGTGCCTAGCTATATGTAGGCCAACCAAATATAGATATAGTTTCTAGAGAG AATTAAGGTT CAGCTTGAGGACAACAAGGTTAAGACAATAAAAGGCGAAGTAGTACGTGGTTGTGGTGTA ATCATAATGA

ACCGGCTTGGCTATGAATTTTGACTAAAAAAAGAGCAAGTCACTAGCGGGAGTAAGA TTTGAGGTTGTGA CATGGAAGGAGAGCCTAGCTATATATGGTTGGGCCATCTTGAGAAATGGCCTAGCTAGCT AGTTTGGTGC ATATATGGTGTATGGAATGAATGGATTTATAATAAGTGTTTGCTATATATATATATATAT ATATATATAT ATATATATATATATATATAAACTAAACTATCATGTGGAAGTGGGGAAAAAAGTGAAAGAG CACGCCCTAG ATTTTTATATTACAAAAGAGCATTGGTTATAATTAAATGGAAGTTGATTTATGTTACACA GTGTAGAAAA

GGCAAAAGCGGGGTGGAATTTTGGCAGAGATTGAAGAGGGATTAGGAGGCAAGGAAT GTGAATAGGATGG GTTGGAGGGATGATAGTGGGTTTGATAATGATGATGCACGCCGGCAGCTTTACTCATTTA TCTTCCCAAA ATCCAATTGGCACGCCTGCCCCCACTAAAACCTATAACAATATTCTCTTCTCTCTTTCAA TCTTTCTCAT AGACAATGAAAAGCTAATCCACTAACCCATTTGGTGATCTAGCTAGACCTTGTTATCTTT CTCTTTTTTA ATCTTTTTGTCAGATAACATTTCTATTTCTCAACACCAGTAGCTCTTTCATGCCAACTTC CCACGTTCTT

CCAGGGAAAACGTATATTAGTCATATCTCTCATGAGTTGATTAAAAAAGAAAGGAAA AAAGGGAGAAGAC TGGGCTAGCTAGCTAGATAAAAGAATATCCCAAACCCCACCCAATTTGATTCACATCGAC TACAGAAAAA GAAAAGCACATTTAAACATGTATGTATGTATATATATGTATATATATAAATATCCAACTT CAAGATCCTC TCTTTTATCATTCTCTACCCCAATATTCCCACAGTTAAAAACACAAACACCCATCCCCTT ACCTTCACTC CAAGCCATCCCCAACAAACTCCATCCGAA