WO2006040684A2

WO2006040684A2 - Methods for increasing plant growth

Info

Publication number: WO2006040684A2
Application number: PCT/IB2005/003415
Authority: WO
Inventors: Vaughan Hurry; Kjell-Ove Holmstrom; Jean-Marie Mouillon
Original assignee: Swetree Technologies Ab
Priority date: 2004-10-15
Filing date: 2005-10-14
Publication date: 2006-04-20
Also published as: WO2006040684A3; GB0422978D0

Abstract

Methods for Increasing Plant Growth This invention relates to methods for increasing growth and biomass accumulation in woody perennial plants, in particular in trees and other forestry crops, by increasing sucrose-phosphate synthase (SPS) activity in the plant, for example, by expression of a heterologous nucleic acid encoding an SPS polypeptide.

Description

Methods for Increasing Plant Growth

This invention relates to methods for increasing growth and biomass accumulation in plants, in particular in trees and other forestry crops.

The primary objectives of forest-tree engineering and molecular breeding are to improve wood quality and yield (Tzfira, T. et al Trends Biotechnol. 16 439-446 (1998), Merkle, S.A. & Dean, J.F. D. Curr. Opin. Biotech. 11 298-302 (2000), Fenning, T.M. &

Gershenzon, J. Trends Biotechnol. 20 291-296 (2002)) . The global demand for wood products is growing at around 1.7% annually, and this increase in wood consumption is occurring despite the fact that the maximum sustainable rate of harvesting from the worlds forests has already been reached or exceeded (South, D.B. New Forests 17 193-212 (1999)) . There is therefore a need for increases in plantation wood production worldwide (Heilman, P.E. New Forests 17 89-93 (1999) ) . Forestry plantations may also have advantages as a carbon sequestration crop in response to increasing atmospheric CO₂ (Myneni, R.B. et al. Proc. Natl. Acad. Sci. USA 98, 14784-14789 (2001), Norby, R.J. et al Plant Cell Environ. 22, 683-714 (1999)) .

Sucrose represents one of the most important end products from photosynthetic metabolism, and is the most common carbohydrate involved in long-distance transport, making sucrose synthesis the interface between photosynthesis and plant growth and development (Paul, M.J. & Foyer, CH. J. Exp. Bot. 52, 1383-1400 (2001)) . One of the keys points for the regulation of sucrose synthesis is the reaction catalysed by the sucrose-phosphate synthase (SPS; EC 2.3.1.14) (Huber, S.C. & Huber, J.L. Annu. Rev. Plant Physiol. Plant MoI. Biol. 47, 431-444 (1996)) . SPS occupies a strategic site downstream of the point where the pathways for sucrose synthesis and starch mobilisation converge, strongly influencing the partitioning of photosynthetic carbon in leaves (Lunn, J.E. & Hatch, M.D. Aust. J. Plant Physiol. 24, 1-8 (1997)) . The effects of increasing SPS activity in transgenic plants has been studied using Arabidopsis thaliana as an herbaceous plant model (Signora, L. et al J. Exp. Bot. 49, 669-680 (1998)), and tomato {Lycopersicon esculentum var. UC82B) as a fruiting agricultural plant model (Galtier, N., Foyer, CH. et al Plant Physiol. 101, 535-543 (1993); Laporte, M.M. et al . Plants 203, 253-259 (1997), Laporte, M.M. et al. Planta 212, 817-822 (2001); Worrell, A.C. et al Plant Cell 3, 1121-1130 (1991)) . Over- expression of SPS in Arabidopsis thaliana under the control of the promoter for the small subunit of ribulose-1, 5-bisphosphate carboxylase (RbcS) resulted in an increase in the leaf sucrose/starch ratio. Arabidopsis thaliana plants over-expressing SPS had similar maximum photosynthetic rates to untransformed controls grown in air but maintained higher rates when grown under elevated CO₂.

The present inventors have recognised that increasing SPS activity in woody perennial plants results in an unexpected increase in both growth rate and wood density.

One aspect of the invention provides a method of increasing the growth rate of a woody perennial plant comprising; expressing a heterologous nucleic acid encoding a sucrose- phosphate synthase (SPS) polypeptide within cells of said perennial plant.

Expression of the SPS polypeptide within cells of the woody perennial plant may increase the rate of height growth and/or the rate of accumulation of biomass in the leaves, roots and stems of the woody perennial plant.

Expression of the SPS polypeptide is also shown herein to increase wood density in a woody perennial plant. Another aspect of the invention provides a method of increasing the wood density in a woody perennial plant comprising; expressing a heterologous nucleic acid encoding a sucrose- phosphate synthase (SPS) polypeptide within cells of said 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.

Preferably, an SPS polypeptide is expressed in photosynthetic tissue of the woody perennial plant, such as leaves and needles.

An SPS polypeptide is a polypeptide which catalyses the conversion of UDPglucose and fructose 6-phosphate to UDP and sucrose 6' -phosphate. A suitable SPS polypeptide may be obtained from a plant, in particular a monocotyledon such as maize {Zea mays) .

In some preferred embodiments, a SPS polypeptide may have the amino acid sequence of Zea mays SPS (Database accession no. AAA33513.1, GI: 168626, SEQ ID NO: 1) or may be a fragment or variant of this sequence which retains SPS activity (EC 2.3.1.14) .

A SPS polypeptide which is a variant of Zea mays SPS may comprise an amino acid sequence which shares greater than 30% sequence identity with the amino acid sequence of SEQ ID NO: 1, 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%.

In other embodiments, a SPS polypeptide which is a variant of Zea mays SPS may share greater than 30% sequence similarity with the amino acid sequence of SEQ ID NO: 1, preferably 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%.

Similarity allows for "conservative variation", i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Particular amino acid sequence variants may differ from a known SPS 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.

A SPS polypeptide has activity in promoting plant growth and/or wood density.

Preferred SPS polypeptides for use in the present methods are not susceptible to post-translational modifications in tree cells which reduce activity (Lunn et al Current Opinion in Plant Biology 2003, 6:208-214) . Post-translational modification and down-regulation of a particular polypeptide may be readily determined using standard techniques.

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.

A nucleic acid encoding a SPS polypeptide may comprise or consist of the nucleotide sequence of Zea mays SPS (Ace No: M97550.1: SEQ ID NO:2) or may be a variant or fragment of this sequence.

A variant sequence may be a mutant, homologue, or allele of the Zea mays SPS (Ace No: M97550.1) sequence and may differ from this sequence 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 SPS polypeptide, which has a nucleotide sequence which is a variant of the Zea mays SPS (Ace No: M97550.1) sequence may comprise a sequence having at least 30% sequence identity with the nucleic acid sequence of Zea mays SPS (SEQ ID N0:2, Ace No: M97550.1), 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 SPS polypeptide i.e. a polypeptide which retains one or more functional characteristics of the polypeptide encoded by the wild-type SPS gene, for example, the ability to stimulate growth a woody perennial plant.

In other embodiments, a nucleic acid encoding a SPS polypeptide, which has a nucleotide sequence which is a variant of SEQ ID NO:2 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 Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 55⁰C in 0. IX SSC, 0.1% SDS. For detection of sequences that are greater than about 90% identical, suitable conditions include hybridization overnight at 65⁰C in 0.25M Na₂HPO₄, pH 7.2, 6.5% SDS, 10% dextran sulfate and a final wash at 60⁰C in 0.1X 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 5O⁰C or 65°C overnight. Washes may be performed in 0.2x SSC/0.1% SDS at 65⁰C or at 50-60⁰C in Ix SSC/0.1% SDS, as required.

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.

A regulatory sequence operably linked to a SPS nucleic acid sequence is preferably heterologous or foreign to the SPS 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.

"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.

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)) .

Preferred regulatory elements may be preferentially active in sucrose biosynthesis tissue ( 'source tissue' ) relative to sucrose metabolising tissue ( ^Λsink tissue' ) . In other words, the regulatory element may show reduced or abrogated activity in the sink tissue relative to the source tissue. In some embodiments, a regulatory element may show increased activity in photosynthetic tissue relative to stem tissue. For example, a regulatory element may show preferential activity in leaf tissue relative to other tissue. In some preferred embodiments, a regulatory element may show preferential activity in mesophyll cells relative to other cells.

Suitable promoters for use in accordance with the present methods include the ribulose-1, 5-bisphosphate carboxylase (RbcS) promoter (Holmstrom KO et al J Exp Bot. 2000 Feb; 51(343) : 177- 85) .

In some embodiments, a regulatory sequence operatively linked to the nucleic acid sequence may be inducible. Inducible promoters are well known in the art and include, for example the HSP promoter (Severin K. and Schόffl F. 1990. Plant MoI. Biol. 15: 827-833) . In essence, expression under the control of an inducible promoter is "switched on" or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously) . The nature of the stimulus varies between promoters. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus. The preferable situation is where the level of expression increases in the presence of the relevant stimulus by an amount effective to alter a phenotypic characteristic i.e. to increase growth. Thus, an inducible (or "switchable") promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about the desired phenotype (and may in fact be zero) . Upon application of the stimulus, expression is increased (or switched on) to a level that causes alterations in the plant phenotype.

The 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 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 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 woody 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. I₁ 379-384; Zhang, et al. (1988) Theor Appl Genet 76, 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 Cell 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 woody perennial plant comprising; incorporating a heterologous nucleic acid encoding a SPS polypeptide, or a vector comprising such a nucleic acid, into a woody perennial plant cell by means of transformation and; regenerating the woody perennial plant from one or more transformed cells.

A perennial plant produced by such a method may show increased growth and/or wood density relative to control plants.

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

The SPS 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.

A woody perennial plant regenerated from said plant cell be sexually or asexually propagated or grown to produce off-spring or descendants.

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

For example, a woody perennial plant is provided which comprises a heterologous nucleic acid encoding an SPS 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.

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.

The disclosures of all documents mentioned herein are incorporated herein by reference.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

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

Figure 1 shows the construct of the plasmid DNA for the creation of SPS transgenic hybrid aspen lines. LB, left border; 3'ocs, octopine synthase terminator; NPTII, neomycin phosphotransferase; pNOS, nopaline synthase promoter; pRbcSl, ribulose-1, 5- bisphosphate carboxylase small subunit Sl promoter; polyA, polyadenylation site; RB, right border.

Figure 2 shows SPS activities in wild type and transgenic hybrid aspen lines. Each value for the enzyme activity represents the mean (SD) of four different leaves for four different plants.

Figure 3 shows the effects of elevated SPS activity on leaf carbohydrate content. (A) leaf sucrose content (B) stem sucrose content (c) Total carbohydrate content of leaf (D) Total carbohydrate content of stem. Each value represents the mean (SD) of four different leaves for four different plants. Statistically significant differences are indicated at the 5% (*) and 1% (**) probability level (Dunnett Multiple Comparison Test) .

Figure 4 shows the effect of SPS expression on plant height growth.

Figure 5 shows biomass production in transgenic hybrid aspen lines over-expressing SPS activity. The number of plants used for the measurements were as follows: 9 controls; 7 of line 26; 9 of line 27; 5 of line 28; 6 of line 29; 12 of line 30 and 39 for all transgenics. For all statistical analyses ANOVA was used to compare the plants with respect to genotype. Statistically significant differences are indicated at the 5% (*) and the 1% (**) probability levels (Dunnett Multiple Comparison Test) .

Table 1 shows biomass characterisation of wild type and transgenic hybrid aspen expressing the maize SPS gene. The number of plants used for the measurements in Exp 1 were as follows: 9 wild type; 7 of line 26; 9 of line 27; 5 of line 28; 6 of line 29; 12 of line 30 and 39 for all transgenics; for Exp 2: 6 wild type; 4 of line 26; 5 of line 27; 5 of line 28; 7 of line 29; 6 of line 30 and 27 for all transgenics. Values represent mean ± SEM. ANOVA was used to compare the plants with respect to genotype. Statistically significant differences are indicated at the 5% (*) and the 1% (**) probability levels (Dunnett Multiple Comparison Test) . All transgenics were compared to WT using a non-paired two-tailed T-test and statistical significance is denoted as: ^tP=0.05, ^ftP=0.005, ^t+t P=O.0005, *P=0.002, ** P=O.007

Experimental

Materials and Methods

Plant vector construction

For expression of an active Zea mays SPS enzyme (GenBank accession no. M97550) in hybrid aspen leaves, a transcriptional fusion was constructed between the 17-kb Arabidopsis thaliana pRbcS (ribulose 1, 5-bisphosphate carboxylase small subunit promoter) promoter and the 3.5-kb maize SPS cDNA sequence. The 3' region contains poly(A) addition sites at position 5459. This fusion construct was inserted into the binary vector pKOH-200³⁹, creating pKOH-200- pftocS-SPS (Fig. 1), which was used for hybrid aspen transformation via Arobacterium tumefaciens.

Plant transformation and growth conditions

Hybrid aspen, Populus tremula L. x P. tremuloides Minch. Clone T89 was transformed and regenerated as described previously (Nilsson, 0. Transgen. Res. 1 209-220 (1992)) . Eleven independent hybrid aspen lines over-expressing the maize SPS were generated and used for further analysis. Individual trees in each iine were multiplied by cuttings, and rooted in vitro on half-strength Murashige-Skoog (MS) medium containing minerals and vitamins. Wild- type control plants were taken through tissue culture in parallel. Plants were grown under long day (16 h) controlled light (250 μmol photons iif² s^"1) and temperature (23⁰C) . Plants were watered daily, and re-potted and fertilised with a complete nutrient solution (SuperbaS, Supra Hydro AB, Landskrona, Sweden) when needed. For biomass determinations the plants were grown in greenhouse under natural light.

SDS-PAGE and Immunoblotting Soluble proteins were extracted from hybrid aspen leaves according to Goulas et al (Goulas, E. et al . Ann. Bot. 88, 789- 795 (2001)) . Protein separation was performed using 12% polyacrylamide Bis-Tris NuPAGE Gels (Novex, San Diego, USA) with MOPS running buffer. Proteins were transferred electrophoretically to a PVDF membrane (Bio-Rad, US) and detection was with ECL chemiluminescent kit (Amersham Pharmacia Biotech, UK) . For immunodetection of SPS, polyclonal antibodies were raised against a BSA-conjugated synthetic peptide corresponding to a conserved amino acid sequence deduced from alignments of SPS protein sequences (RGENMELGRDSDTGGQVKYVVC) . The conjugated synthetic peptide was injected into rabbits intramuscularly and subcutaneously (AgriSera, Vannas, Sweden) . Total proteins were determined according to Lowry et al (Lowry, O.H. et al J. Biol. Chem. 193, 265-275 (1951)) .

Amino acids

In the middle of the photoperiod, leaf material was harvested into liquid nitrogen. Amino acids were extracted and quantitatively analysed by high-performance liquid chromatography (Nasholm, T. et al J. Chromatogr. 396, 225-236 (1987)) .

Sucrose-phosphate synthase activity

Leaf material was chilled to the temperature of liquid N₂. The frozen material was ground to a fine powder at the temperature of liquid N₂ in a mortar. Enzymes were extracted and sucrose-phosphate synthase measured (Strand, A. et al Plant Cell and Environment (2002) ) .

Soluble sugars and starch In the middle of the photoperiod, leaf material was harvested into liquid N_z. Carbohydrates were extracted in 80% ethanol containing 4 mM Hepes-KOH (pH 7.5) at 80⁰C for 30 min. Samples were then centrifuged for 15 min at 2000Og, the supernatant decanted and stored on ice and the pellet re-suspended in 80% ethanol-Hepes (pH 7.5) and again put on the heat block for 30 min. This hot extraction was repeated twice more, once with 50% ethanol-Hepes (pH 7.5) and once with only 4 mM Hepes (pH 7.5) and the supernatants combined and assayed for soluble sugars (Stitt, M. et al in Methods in Enzymology: Biomembranes, Vol. 174. (eds. S. Fleischer & B. Fleischer) 518-552 (Academic Press, Amsterdam; 1989)) . For starch extraction 0.5 ml of 0.2 M KOH was added to re-suspend the pellet, and the samples are healed at 90⁰C for 2 h. For each cleavage, a 350 μl aliquot of the heated suspension was added to 150 μl of Na-acetate incubation buffer (pH 4.8) containing 28 U amyloglucosidase and the samples were incubated at 37⁰C for 16h. The samples were then centrifuged for 15 min at 20000 g and the supernatant assayed for glucose.

Growth measurements and sampling At seven weeks of age, the plants were randomly positioned

(Random Block Design) to start measurement of height and stem diameter every third to fourth day. Plants grown under long days were harvested after 45 days, using 9 control plants (wild type genotype) and 39 plants representing five transgenic independent lines. Leaf tissues were frozen in liquid N₂ immediately after sampling for SPS activity determination and western-blot analysis. All parts remaining after sampling were separated into leaf, stem, and root fractions, and used for fresh-weight biomass determinations. Dry weight was determined after drying the samples at 80°C for 4 days.

Results

Creation of SPS transgenic hybrid aspen lines

Eleven transgenic hybrid aspen lines, isolated from independent explants in two different transformations, were generated over- expressing Zea mays SPS under the control of the Arabidopsis RbcS promoter, and five of these were selected for further experimental analysis (Fig. 1) .

Western-blot analyses on leaf protein extracts from these transgenic lines showed increases in the amount of SPS protein that correlated with increases in SPS activity (Fig. 2) .

Carbohydrates leaf content and partitioning Increasing SPS activity in the transgenic lines significantly increased mesophyll sucrose concentrations in 4 of the 5 transgenic lines tested (Fig. 3a) . No significant changes were found in the mesophyll concentrations of the other main soluble sugars (glucose & fructose) or in leaf starch content. These data indicate that the increased sucrose was not being made at the expense of other sugars and that the increase in SPS activity has resulted in a net increase in leaf sugar production. This conclusion is also supported by data showing a significant increase the size of the total carbohydrate pool in three of the five transgenic lines (Fig. 3C). The increase in mesophyll sucrose contents was supported by an average 12% increase in photosynthetic C0_z fixation in the transgenic lines under ambient condition (35 Pa C0_z, 250 μmol photons m^"2 s^"1) and by an average 30% increase in light-and light and CO₂-saturated photosynthesis. These increases in leaf mesophyll carbohydrate concentrations also translated into significant increases in stem sucrose (Fig. 3B) and into consistent but non-significant increases in total stem carbohydrates (Fig. 3D) , indicating that the increase in mesophyll sucrose was resulting not only in increased sucrose production but also increased translocation of sucrose to stem sinks.

Amino acids

Amino acids produced by mesophyll cell metabolism are transported to sink tissues and storage organs, and consequently amino acids are one of the major constituents of the phloem sap. All transgenic lines showed increases in total amino acid contents (from 16.0 ± 4.2 μmol g^"1 DWT in WT leaves to an average of 22.4 ± 3.5 μmol g^"1 DWT in all transgenics; p<0.02) . The increase in total amino acids in all transgenic lines was mainly due to increases in aspartate, glutamate and glycine. Aspartate and glutamate are two amino acids that act as N-donors and play an important role in cell transamination reactions and N-storage. Aspartate also plays a central role in the biosynthesis of several aspartate-derived amino acids. Thus, in addition to carbohydrate synthesis elevated SPS activity also appears to enhance amino acid synthesis in hybrid aspen leaves.

Biomass production

Two separate biomass experiments were carried out on tissue (leaf, stem and root) from 90-day-old aspen trees to see whether elevated sucrose synthesis and elevated mesophyll and stem sucrose content could enhance biomass production by transgenic hybrid aspen trees (Tab. 1) . In the first experiment, lines 27 & 28 were significantly- taller than WT plants by day 90 (Fig. 4) and in the second experiment, lines 26, 27, 28 and 29 and all pooled transgenic plants were also significantly taller than WT by 90 days (Tab. 1) . Lines 27, 28 & 29 and all pooled transgenic plants showed significantly greater stem diameter 15 cm above soil height (Tab.

1).

Biomass measurements of 5 independent transgenic lines showed that an increase in SPS activity results in a significant (All transgenics; p<0,005) 30 - 40 % increase in dry weight accumulation (Tab. 1 & Fig. 5), with 3 of the 5 lines (Line 26, 27 & 29) showing independent significant increases in total dry weight accumulation (Tab. 1) . These increases in total biomass production were also associated with significant increases in stem density (Tab. 1), suggesting that the increased carbohydrate supply altered cell wall thickness or other cell wall properties, However, the change (s) underlying this increase in dry matter and stem density remain to be determined.

In both WT and the transgenic lines, the leaves accounted for the largest component of total plant biomass (approx 45%) at this growth stage. The bulk of the increase in biomass production in these transgenic aspen was therefore found in the leaves, and to a less extent in the roots. Leaf dry weights were significantly higher in lines 26, 29 and 30, and root dry weight was significantly higher in line 27. There was also a general increase in stem dry weights but this was not statistically significant

(Tab. 1) . Nevertheless, these data show that the increase in plant biomass was distributed throughout the different components of the plant (leaf, stem and roots) and therefore the increase in mesophyll sucrose content in the transgenic aspen (Fig 3) was being converted into increased growth in distant parts of the plant, and hence into increased export of photoassimilates. These results show the net benefit of elevated SPS activity on hybrid aspen growth, indicating that increased sucrose synthesis in photosynthetic tissues leads increased biomass production throughout the plant.

Transgenic hybrid aspen (Populus tremulaxP. tremuloides) trees with increased SPS activity and enhanced sucrose synthesis were shown to produce higher concentrations of sucrose and amino acids in the mesophyll cells of source leaves and significantly faster growth rates relative to wild type. This increase in growth was shown in a comparison of 5 independent transgenic lines both through increased height growth and by 30-40% increased biomass accumulation in leaves, roots and stems.

These transgenic hybrid aspen demonstrate that it is possible to significantly enhance the growth of fast growing plantation tree species by increasing the sucrose and amino acid concentrations in the mesophyll of source leaves. The transgenic hybrid aspen show higher biomass production and offer a valuable perspective for the global need for wood products.

Sequences

SEQ ID NO:1 (Zea Mays SPS)

1 magnewingy leaildshts srgagggggg gdprsptkaa sprgahmnfn pshyfveevv 61 kgvdesdlhr twikvvatrn arerstrlen mcwriwhlar kkkqlelegi qrisarrkeq 121 eqvrreated laedlsegek gdtigelapv ettkkkfqrn fsdltvwsdd nkekklyivl 181 isvhglvrge nmelgrdsdt ggqvkyvvel aramsmmpgv yrvdlftrqv sspdvdwsyg 241 eptemlcags ndgegmgesg gayivripcg prdkylkkea lwpylqefvd galahilnms 301 kalgeqvgng rpvlpyvihg hyadagdvaa llsgalnvpm vltghslgrn kleqllkqgr 361 mskeeidsty kimrriegee laldaselvi tstrqeideq wglydgfdvk lekvlrarar 421 rgvschgrym prmvvippgm dfsnvvvhed idgdgdvkdd ivglegaspk smppiwaevm 481 rfltnphkpm ilalsrpdpk knittlvkaf gecrplrela nltlimgnrd diddmsagna 541 svlttvlkli dkydlygsva fpkhhnqadv peiyrlaakm kgvfinpalv epfgltliea 601 aahglpivat knggpvditn alnngllvdp hdqnaiadal lklvadknlw qecrrnglrn 661 ihlyswpehc rtyltrvagc rlrnprwlkd tpadagadee efledsmdaq dlslrlsidg 721 eksslntndp lwfdpqdqvq kimnnikqss alppsmssva aegtgstmnk ypllrrrrrl 781 fviavdcyqd dgraskkmlq viqevfravr sdsqmfkisg ftlstampls etlqllqlgk 841 ipatdfdali cgsgsevyyp gtancmdaeg klrpdqdylm hishrwshdg arqtiaklmg 901 aqdgsgdave qdvassnahc vaflikdpqk vktvdemrer lrmrglrchi mycrnstrlq 961 vvpllasrsq alrylsvrwg vsvgnmylit gehgdtdlee mlsglhktvi vrgvtekgse 1021 alvrspgsyk rddvvpsetp laayttgelk adeimralkq vsktssgm

SEQ ID NO:2 (Zea Mays SPS) i gaattccggc gtgggcgctg ggctagtgct cccgcagcga gcgatctgag agaacggtag

61 agttccggcc gggcgcgcgg gagaggagga gggtcgggcg gggaggatcc gatggccggg

121 aacgagtgga tcaatgggta cctggaggcg atcctcgaca gccacacctc gtcgcggggt

181 gccggcggcg gcggcggcgg gggggacccc aggtcgccga cgaaggcggc gagcccccgc

241 ggcgcgcaca tgaacttcaa cccctcgcac tacttcgtcg aggaggtggt caagggcgtc

301 gacgagagcg acctccaccg gacgtggatc aaggtcgtcg ccacccgcaa cgcccgcgag

361 cgcagcacca ggctcgagaa catgtgctgg cggatctggc acctcgcgcg caagaagaag

421 cagctggagc tggagggcat ccagagaatc tcggcaagaa ggaaggaaca ggagcaggtg

481 cgtcgtgagg cgacggagga cctggccgag gatctgtcag aaggcgagaa gggagacacc

541 atcggcgagc ttgcgccggt tgagacgacc aagaagaagt tccagaggaa cttctctgac

601 cttaccgtct ggtctgacga caataaggag aagaagcttt acattgtgct catcagcgtg

661 catggtcttg ttcgtggaga aaacatggaa ctaggtcgtg attctgatac aggtggccag

721 gtgaaatatg tggtcgaact tgcaagagcg atgtcaatga tgcctggagt gtacagggtg

781 gacctcttca ctcgtcaagt gtcatctcct gacgtggact ggagctacgg tgagccaacc

841 gagatgttat gcgccggttc caatgatgga gaggggatgg gtgagagtgg cggagcctac

901 attgtgcgca taccgtgtgg gccgcgggat aaatacctca agaaggaagc gttgtggcct

961 tacctccaag agtttgtcga tggagccctt gcgcatatcc tgaacatgtc caaggctctg

1021 ggagagcagg ttggaaatgg gaggccagta ctgccttacg tgatacatgg gcactatgcc

1081 gatgctggag atgttgctgc tctcctttct ggtgcgctga atgtgccaat ggtgctcact

1141 ggccactcac ttgggaggaa caagctggaa caactgctga agcaagggcg catgtccaag

1201 gaggagatcg attcgacata caagatcatg aggcgtatcg agggtgagga gctggccctg

1261 gatgcgtcag agcttgtaat cacgagcaca aggcaggaga ttgatgagca gtggggattg

1321 tacgatggat ttgatgtcaa gcttgagaaa gtgctgaggg cacgggcgag gcgcggggtt

1381 agctgccatg gtcgttacat gcctaggatg gtggtgattc ctccgggaat ggatttcagc

1441 aatgttgtag ttcatgaaga cattgatggg gatggtgacg tcaaagatga tatcgttggt

1501 ttggagggtg cctcacccaa gtcaatgccc ccaatttggg ccgaagtgat gcggttcctg

1561 accaaccctc acaagccgat gatcctggcg ttatcaagac cagacccgaa gaagaacatc

1621 actaccctcg tcaaagcgtt tggagagtgt cgtccactca gggaacttgc aaaccttact

1681 ctgatcatgg gtaacagaga tgacatcgac gacatgtctg ctggcaatgc cagtgtcctc

1741 accacagttc tgaagctgat tgacaagtat gatctgtacg gaagcgtggc gttccctaag

1801 catcacaatc aggctgacgt cccggagatc tatcgcctcg cggccaaaat gaagggcgtc

1861 ttcatcaacc ctgctctcgt tgagccgttt ggtctcaccc tgatcgaggc tgcggcacac

1921 ggactcccga tagtcgctac caagaatggt ggtccggtcg acattacaaa tgcattaaac

1981 aacggactgc tcgttgaccc acacgaccag aacgccatcg ctgatgcact gctgaagctt

2041 gtggcagaca agaacctgtg gcaggaatgc cggagaaacg ggctgcgcaa catccacctc

2101 tactcatggc cggagcactg ccgcacttac ctcaccaggg tggccgggtg ccggttaagg 2161 aacccgaggt ggctgaagga cacaccagca gatgccggag ccgatgagga ggagttcctg 2221 gaggattcca tggacgctca ggacctgtca ctccgtctgt ccatcgacgg tgagaagagc 2281 tcgctgaaca ctaacgatcc actgtggttc gacccccagg atcaagtgca gaagatcatg 2341 aacaacatca agcagtcgtc agcgcttcct ccgtccatgt cctcagtcgc agccgagggc 2401 acaggcagca ccatgaacaa atacccactc ctgcgccggc gccggcgctt gttcgtcata 2461 gctgtggact gctaccagga cgatggccgt gctagcaaga agatgctgca ggtgatccag 2521 gaagttttca gagcagtccg atcggactcc cagatgttca agatctcagg gttcacgctg 2581 tcgactgcca tgccgttgtc cgagacactc cagcttctgc agctcggcaa gatcccagcg 2641 accgacttcg acgccctcat ctgtggcagc ggcagcgagg tgtactatcc tggcacggcg 2701 aactgcatgg acgctgaagg aaagctgcgc ccagatcagg actatctgat gcacatcagc 2761 caccgctggt cccatgacgg cgcgaggcag accatagcga agctcatggg cgctcaggac 2821 ggttcaggcg acgctgtcga gcaggacgtg gcgtccagta atgcacactg tgtcgcgttc 2881 ctcatcaaag acccccaaaa ggtgaaaacg gtcgatgaga tgagggagcg gctgaggatg 2941 cgtggtctcc gctgccacat catgtactgc aggaactcga caaggcttca ggttgtccct 3001 ctgctagcat caaggtcaca ggcactcagg tatctttccg tgcgctgggg cgtatctgtg 3061 gggaacatgt atctgatcac cggggaacat ggcgacaccg atctagagga gatgctatcc 3121 gggctacaca agaccgtgat cgtccgtggc gtcaccgaga agggttcgga agcactggtg 3181 aggagcccag gaagctacaa gagggacgat gtcgtcccgt ctgagacccc cttggctgcg 3241 tacacgactg gtgagctgaa ggccgacgag atcatgcggg ctctgaagca agtctccaag 3301 acttccagcg gcatgtgaat ttgatgcttc ttttacattt tgtccttttc ttcactgcta 3361 tataaaataa gttgtgaaca gtaccgcggg tgtgtatata tatattgcag tgacaaataa 3421 aacaggacac tgctaactat actggtgaat atacgactgt caagattgta tgctaagtac 3481 tccatttctc aatgtatcaa tcggaattc

Genotype

Wild type Line 26 Line 27 Line 28 Line 29 Line 30 All Transgenj

Exp. 1 Dry weight (g)

Leaf 21.9 ± 1.6 32.1 ± 1.4" 26.2 ± 1.3 20.8 ± 1.6 35.5 ± 1.8^** 31.6 + 0.9^** 29.7 ± 0.9^m

Stem 18.0 ± 1.5 19.3 + 1.5 21.8 + 1.6 19.9 ± 1.8 21.1 + 1.6 16.3 + 0.7 19.4 + 0.6

Root 12.5 ± 1.9 16.6 + 1.6 19.4 ± 2.1^* 15.8 ± 3.0 18.5 ± 2.2 14.2 ± 1.5 16.7 ± 0.9^*

Total 52.3 ± 4.7 68.0 + 3.9^* 67.4 + 4.4^* 56.4 ± 6.1 75.2 ± 4.4^** 62.3 + 2.6 65.7 ± 1.9"

Exp. 2

Stem height (cm) 188 ± 8 209 ± 5* 206 ± 2* 208 ± 2* 207 ± 4* 191 + 3 204 ± 2**

Stem diameter (mm) 12.8 + 0.3 13.9 + 0.4 14.4 + 0.2* 14.7 ± 0.3** 14.5 + 0.3* 13.8 + 0.6 14.3 ± 0.2*

Stem density (g cm^"3) 0.37 + 0.006 0.39 : ± 0.011 0.40 i : 0.004* 0.39 ± 0.01 0.40 + 0 .008* 0.39 ± 0.07 0.40 : ± 0.004¹

IS)

Table 1 *-.

Claims

Claims :

1. A method of increasing the growth and/or wood density of a woody perennial plant comprising; expressing a heterologous nucleic acid encoding a sucrose- phosphate synthase (SPS) polypeptide within cells of said perennial plant.

2. A method according to claim 1 or claim 2 wherein the SPS polypeptide comprises an amino acid sequence which shares greater than 30% sequence identity with the amino acid sequence of SEQ ID NO: 1.

3. A method according to claim 3 wherein the SPS polypeptide comprises an amino acid sequence which has the amino acid sequence of SEQ ID NO: 1.

4. A method according to claim 2 or claim 3 wherein the nucleic acid comprises a sequence having at least 30% sequence identity with the nucleic acid sequence of SEQ ID NO: 2.

5. A method according to claim 4 wherein the nucleic acid has the nucleic acid sequence of SEQ ID NO: 2.

6. A method according to any one of the preceding claims wherein nucleic acid is operably linked to a promoter.

7. A method according to claim 6 wherein the promoter is a photosynthetic tissue specific promoter.

8. A method according to claim 7 wherein the promoter is a ribulose-1, 5-bisphosphate carboxylase (RbcS) promoter.

9. A method according to any one of the preceding claims wherein the nucleic acid is comprised in a vector.

10. A method according to any one of the preceding clais comprising sexually or asexually propagating or growing off¬ spring or descendants of the plant expressing the heterologous nucleic acid.

11. A method of producing a woody perennial plant comprising incorporating a heterologous nucleic acid encoding a SPS polypeptide into a perennial plant cell species by means of transformation, and; regenerating the perennial plant from one or more transformed cells.

12. A method according to claim 11 wherein said plant shows increased growth and/or wood density relative to controls.

13. A method according to claim 11 or claim 12 comprising sexually or asexually propagating or growing off-spring or descendants of the plant regenerated from the one or more cells.

14. A method according to any one of the preceding claims wherein the woody perennial plant is a hardwood plant

15. A method according to claim 14 wherein the hardwood plant is selected from the group consisting of acacia, eucalyptus, hornbeam, beech, mahogany, walnut, oak, ash, willow, hickory, birch, chestnut, poplar, alder, maple and sycamore.

16. A method according to claim 14 or claim 15 wherein the hardwood plant is a plant of the Populus or Salicaceae groups.

17. A method according to claim 16 wherein the hardwood plant is a plant of the Populus group.

18. A method according to any one of claims 1 to 13 wherein the woody perennial plant is a conifer.

19. A method according to claim 18 wherein the conifer is selected from the group consisting of cypress, Douglas fir, fir, sequoia, hemlock, cedar, juniper, larch, pine, redwood, spruce and yew.

20. A method according to any one of claims 1 to 13 wherein the woody perennial plant is a fruit bearing plant.

21. A method according to claim 20 wherein the fruit bearing plant is selected from the group consisting of apple, plum, pear, banana, orange, kiwi, lemon, cherry, grapevine and fig.

22. A method according to any one of claims 1 to 13 wherein the woody plant is selected from the group consisting of cotton, bamboo and rubber plants.

23. A woody perennial plant produced by a method according to claim 11, wherein said plant shows increased growth or wood density relative to controls.

24. A woody perennial plant comprising a heterologous nucleic acid encoding a sucrose-phosphate synthase (SPS) polypeptide within one or more of its cells.