# Patent

## Title
Plants having improved growth characteristics and a method for making the same

## Abstract

The present invention relates to a method for improving plant growth by increasing activity of DP protein in shoot tissue. The invention also relates to transgenic plants having improved growth characteristics, due to increased expression of a DP nucleic acid specifically in shoot-tissue. The increased expression of the nucleic acid encoding a DP protein, according to the methods of the present invention, may be mediated by a shoot-tissue-specific promoter.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of European Application No. 04102392.0 filed May 28, 2004 and U. S. Provisional Application Ser. No. 60/576,250 filed Jun. 2, 2004, which are herein incorporated by reference in their entirety.


The present invention concerns a method for improving plant growth characteristics. More specifically, the present invention concerns a method for improving plant growth characteristics by increasing, in a plant, activity of an E2F Dimerisation Partner (DP) protein in shoot tissue. The present invention also concerns plants transformed with a DP gene, controlled by a shoot-preferred control element, which plants have improved growth characteristics relative to corresponding wild-type plants.
Given the ever-increasing world population, it remains a major goal of agricultural research to improve the efficiency of agriculture. Conventional means for crop and horticultural improvements utilise selective breeding techniques to identify plants having desirable characteristics. However, such selective breeding techniques have several drawbacks, namely that these techniques are typically labour intensive and result in plants that often contain heterogenous genetic components that may not always result in the desirable trait being passed on from parent plants. In contrast, advances in molecular biology have allowed mankind to more precisely manipulate the germplasm of plants. Genetic engineering of plants entails the isolation and manipulation of genetic material (typically in the form of DNA or RNA) and the subsequent introduction of that genetic material into a plant. Such technology has led to the development of plants having various improved economic, agronomic or horticultural traits. A trait of particular economic interest is high yield and/or biomass.
The ability to improve one or more plant growth characteristics, would have many applications in areas such as crop enhancement, plant breeding, production of ornamental plants, arboriculture, horticulture, forestry, production of algae or plants (for use as bioreactors for example, for the production of pharmaceuticals, such as antibodies or vaccines, or for the bioconversion of organic waste, or for use as fuel, in the case of high-yielding algae and plants).
It has now been found that increased expression and/or activity of DP in shoot-tissue, gives plants having improved growth characteristics relative to corresponding wild-type plants.
Dp proteins are widely conserved proteins and are involved in the control of the cell cycle (Gutierrez et al. (2002) Current opinion in Plant Biology 5: 480-486). Dp factors act together with E2F factors to form a heterodimer, capable of initiating transcription of S-phase specific genes. The identification of E2F factors, DP factors and E2F-DP-like (DEL) factors has been reported (Magyar et al. 2000, FEBS letters, 486: 79-97). Based on sequence comparison the Arabidopsis genes encoding these proteins were grouped into distinct categories as described in Vandepoele et al. 2002, plant cell 14(4): 903-16, which reference is incorporated herein by reference as if fully set forth. The structural characteristics of typical DP proteins are detailed in Magyar et al., which reference is incorporated herein by reference as if fully set forth. For example in FIG. 3 A and B of Magyar et al. the location of the DNA binding domain and the dimerisation domain in the Arabidopsis DP proteins is presented. FIG. 5 of Vandepoele et al. nicely illustrates that DP proteins are distinct from related proteins such as E2F factors and DEL's by the presence of one DNA binding domain and one dimerisation domain.
WO00/47614 (Pioneer Hi-Bred, filed Feb. 11, 2000) suggests that controlling DP expression using tissue-specific or cell-specific promoters provides a differential growth characteristic. More particularly, it suggests that (i) using a seed-specific promoter will stimulate cell division rate and result in increased seed biomass; (ii) using a strongly-expressed, early, tassel-specific promoter will enhance development of this entire reproductive structure; and (iii) that using a root-specific promoter will result in larger roots and faster growth (i.e. more biomass accumulation). However, plants obtainable by these methods, which plants have such differential growth characteristics, have not yet been illustrated or disclosed.
Even the later filed document WO01/21644 (Consejo Superior De Investigaciones Cientificas, filed Sep. 25, 2000), merely suggests that plant growth may be controlled by expression of a recombinant DP. This document does not show transgenic plants, of which plant growth is controlled. Despite the statement “particularly useful are nucleic acids of which the expression is controlled using a tissue-specific promoter or a chemically-inducible promoter”, this document did not lead to the development of plants with improved growth characteristics.
Despite the above suggestions, no improved transgenic plants have been generated so far, indicating that using DP as suggested above is insufficient to improve plant growth characteristics.
Unexpectedly, it has now been found that plant growth may be effectively improved by increasing activity of DP specifically in the shoot-tissue of a plant.
Accordingly, the present invention provides a method for improving plant growth characteristics relative to corresponding wild-type plants, comprising increasing activity of a DP polypeptide or homologue thereof specifically in shoot tissue.
Advantageously, performance of the method according to the present invention leads to plants having a variety of improved growth characteristics relative to corresponding wild-type plants, especially increased biomass. The improved growth characteristics may be stable and inheritable in further generations.
The term “growth characteristic” as used herein, preferably refers to, but is not limited to, increased biomass or to any other growth characteristic as described hereinafter.
The term “biomass” refers to the amount of produced biological material. Generally, the term “increased biomass” means an increase in biomass in one or more parts of a plant relative to the biomass of corresponding reference plants, for example relative to corresponding wild-type plants. The plants according to the invention are characterised by increased above-ground biomass, which is particularly important for crop plants grown for their vegetative tissues. For silage corn, for example, typical parameters for economic value are the above-ground biomass and energy content of the leaves. For trees and sugarcane, typical parameters of economical value are the above-ground biomass of stems.
Increased biomass as used herein may also encompass increased seed yield.
The term “growth characteristic” as used herein, also encompasses plant architecture. The plants according to the invention exhibit improved architecture, which is manifested in altered shape, because of their increased above-ground biomass. This characteristic may be advantageous for ornamental plant. The term “architecture” as used herein encompasses the appearance or morphology of a plant, including any one or more structural features or combination of structural features thereof. Such structural features include the shape, size, number, position, texture, arrangement, and pattern of cells, tissues, organs or groups of cells, tissues or organs of a plant. The plants of the present invention are characterised by increased number of tillers and increased number of branches. Therefore, the term altered “architecture” as used herein encompasses altered number and size of tillers, branches or leaves.
The abovementioned growth characteristics may advantageously be modified in a variety of plant species.
The term “plant” as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, roots (including tubers), and plant cells, tissues and organs. The term “plant” also therefore encompasses suspension cultures, embryos, meristematic regions, callus tissue, leaves, seeds, roots, shoots, gametophytes, sporophytes, pollen, and microspores. Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Viridiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acacia spp., Acer spp., Actinidia spp., Aesculus spp., Agathis australis, Albizia amara, Alsophila tricolor, Andropogon spp., Arachis spp, Areca catechu, Astelia fragrans, Astragalus cicer, Baikiaea plurijuga, Betula spp., Brassica spp., Bruguiera gymnorrhiza, Burkea africana, Butea frondosa, Cadaba farinosa, Calliandra spp, Camellia sinensis, Canna indica, Capsicum spp., Cassia spp., Centroema pubescens, Chaenomeles spp., Cinnamomum cassia, Coffea arabica, Colophospermum mopane, Coronillia varia, Cotoneaster serotina, Crataegus spp., Cucumis spp., Cupressus spp., Cyathea dealbata, Cydonia oblonga, Cryptomeria japonica, Cymbopogon spp., Cynthea dealbata, Cydonia oblonga, Dalbergia monetaria, Davallia divaricata, Desmodium spp., Dicksonia squarosa, Diheteropogon amplectens, Dioclea spp, Dolichos spp., Dorycnium rectum, Echinochloa pyramidalis, Ehrartia spp., Eleusine coracana, Eragrestis spp., Erythrina spp., Eucalyptus spp., Euclea schimperi, Eulalia villosa, Fagopyrum spp., Feijoa sellowiana, Fragaria spp., Flemingia spp, Freycinetia banksii, Geranium thunbergii, Ginkgo biloba, Glycine javanica, Gliricidia spp, Gossypium hirsutum, Grevillea spp., Guibourtia coleosperma, Hedysarum spp., Hemarthia altissima, Heteropogon contortus, Hordeum vulgare, Hyparrhenia rufa, Hypericum erectum, Hyperthelia dissoluta, Indigo incamata, Iris spp., Leptarrhena pyrolifolia, Lespediza spp., Lettuca spp., Leucaena leucocephala, Loudetia simplex, Lotonus bainesii, Lotus spp., Macrotyloma axillare, Malus spp., Manihot esculenta, Medicago sativa, Metasequoia glyptostroboides, Musa sapientum, Nicotianum spp., Onobrychis spp., Omithopus spp., Oryza spp., Peltophorum africanum, Pennisetum spp., Persea gratissima, Petunia spp., Phaseolus spp., Phoenix canariensis, Phormium cookianum, Photinia spp., Picea glauca, Pinus spp., Pisum sativum, Podocarpus totara, Pogonarthria fleckii, Pogonarthria squarrosa, Populus spp., Prosopis cineraria, Pseudotsuga menziesii, Pterolobium stellatum, Pyrus communis, Quercus spp., Rhaphiolepsis umbellata, Rhopalostylis sapida, Rhus natalensis, Ribes grossularia, Ribes spp., Robinia pseudoacacia, Rosa spp., Rubus spp., Salix spp., Schyzachyrium sanguineum, Sciadopitys verticillata, Sequoia sempervirens, Sequoiadendron giganteum, Sorghum bicolor, Spinacia spp., Sporobolus fimbriatus, Stiburus alopecuroides, Stylosanthos humilis, Tadehagi spp, Taxodium distichum, Themeda triandra, Trifolium spp., Triticum spp., Tsuga heterophylla, Vaccinium spp., Vicia spp. Vitis vinifera, Watsonia pyramidata, Zantedeschia aethiopica, Zea mays, amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, canola, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, rice, soybean, straw, sugar beet, sugar cane, sunflower, tomato, squash tea, trees, grasses (including forage grass) and algae, amongst others.
According to a preferred feature of the present invention, the plant is a crop plant, such as soybean, sunflower, canola, rapeseed, cotton, alfalfa, tomato, potato, tobacco, papaya, squash, poplar, eucalyptus, pine, leguminosa, flax, lupinus and sorghum. According to a further preferred embodiment of the present invention, the plant is a monocotyledonous plant, such as sugarcane, further preferably the plant is a cereal, such as rice, maize (including forage corn), wheat, barley, millet, oats and rye.
Accordingly, the present invention provides any of the methods as described herein, or a transgenic plant as described herein, wherein the plant is a monocotyledonous plant, preferably a cereal, such as rice or corn.
The term “DP” means E2F Dimerisation Partner. The term “DP polypeptide” as used herein means a protein as represented by SEQ ID NO 2 or homologues of SEQ ID NO 2. Specific examples of DP proteins are Arabidopsis thaliana DP proteins as described Magyar et al. (2000, FEBS, 486(1): 79-87), Triticum aestivum DP proteins as described in Ramirez-Parra & Gutierrrez (2000, FEBS, 86(1): 73-8) and Impatiens, soybean and corn DP proteins as described in WO99/53075 (Du Pont).
The term “DP polypeptide or homologue thereof” as defined herein refers to a polypeptide having in increasing order of preference at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a DP protein, for example, to any one of SEQ ID NO 2, 4, 13, 15, 17, 19, 21 and 23.
DP proteins of Arabidopsis thaliana have been subdivided into two different classes (Vandepoele et al., 2002, Plant Cell., 14(4): 903-16), DPa and DPb. The members of both classes are also encompassed by the term “homologue” as used herein. Advantageously, these different classes DP proteins, or their encoding nucleic acids, may be used in the methods of the present invention. Accordingly, the present invention provides a method as described herein, wherein the DP nucleic acid or DP protein is obtained from a plant, preferably from a dicotyledoneous plant, further preferably from the family Brassicaceae, more preferably from Arabidopsis thaliana. According to a further embodiment, DP polypeptide is a DPb polypeptide. A person skilled in the art will recognize that a “DPb” is a protein being closer related to AtDPb, than to AtDPa. This closer relationship may be determined by calculating percentage of sequence identity, or by comparing the presence of conserved motifs as described hereinafter. The closest relationship between the protein in question and AtDPa and AtDPb may also be identified by making a phylogenetic tree as represented in FIG. 5 and including the protein in question in the tree. A DPb protein should group closer to AtDPb than to AtDPa.
According to a preferred embodiment, such DP polypeptide or homologue has at least one of the conserved DP domains and motifs as described herein. The conserved domains of DP proteins have been illustrated in Magyar et al. and in Vandepoele et al. Typically, a DP protein comprises one DNA binding domain and one dimerisation domain. As an example, the location of these domains is illustrated on the Arabidpsis thaliana DPb sequence as shown in FIG. 3.
Preferred DP polypeptides or homologues, useful in the methods of the present invention have a percentage of sequence identity to for example SEQ ID NO 2, 4, 13, 15, 17, 19, 21 and 23 as mentioned above, which percentage of identity may be calculated over the conserved region which is typically present in all DP proteins. This region, which is highly conserved between DP proteins, starts from about residues CEKVES (e.g. from position 111 of SEQ ID NO 2) to about FVLKTM (e.g. to position 290 of SEQ ID NO 2) see FIG. 3.
Three motifs are particularly conserved in a subclass of DP proteins, which subclass comprises DPb of Arabidopsis thaliana. The consensus sequences for these “DPb” motifs are represented herein by SEQ ID NO 9 (motif 1, LDIXXDDA), SEQ ID NO 10 (motif 2, KKKK/RR) and SEQ ID NO 11 (motif 3, AXGXDK) (see FIG. 3).
Preferably, these motifs are present in the DP polypeptide or homologues used in the methods of the present invention. FIG. 3 shows an alignment of DP proteins with the location of the “DPb” motifs. As can be seen from the alignment, refining the consensus sequences is possible. For example, at position 4 in motif 1, there is a high probability for a Q or an H residue and at position 5, there is a high probability for a G or an A residue. Also in motif 3, at position 2, there is a high probability for a V, T or A residue and at position 4, there is a high probability for a P or an A residue. A person skilled in the art will recognize that a DPb motif may deviate, by for example 1 or 2 mismatches, from the consensus DPb motifs as represented by SEQ ID NO 9, 10 or 11, without losing its functionality.
These newly identified “DPb” motifs may also be used to search databases and to identify homologous DPb polypeptides and encoding sequences.
The identification of protein domains, motifs and boxes, would also be well within the realm of a person skilled in the art by using domain information available in the PRODOM database available through The University of London (UCL), PIR database available through Georgetown University Medical Center (GUMC), PROSITE database available through ExPASy or the pFAM database available through Washington University in St. Louis. Software programs designed for such domain searching include, but are not limited to, MotifScan, MEME, SIGNALSCAN, and GENESCAN. MotifScan is a preferred software program and is available through Stanford University, which program uses the protein domain information of PROSITE and pFAM. A MEME algorithm (Version 3.0) may be found in the GCG package or through the San Diego Supercomputer Center (SDSC). SIGNALSCAN version 4.0 information is available through The University of Minnesota College of Biological Sciences. GENESCAN may be found through Stanford University.
A DP polypeptide or homologue may be found in (public) sequence databases. Methods for the alignment and identification of DP protein homologues in sequence databases are well known in the art. Such methods involve screening sequence databases with the sequences provided by the present invention, for example, SEQ ID NOs: 2, 4, 13, 15, 17, 19, 21 and 23 (or SEQ ID NO: 1). Different search algorithms and software for the alignment and comparison of sequences are well known in the art and include, for example, GAP, BESTFIT, BLAST, FASTA and TFASTA. Preferably, the BLAST software is used, which calculates percent sequence identity and performs a statistical analysis of the similarity between the sequences. The suite of programs referred to as BLAST programs has five different implementations: three designed for nucleotide sequence queries (BLASTN, BLASTX and TBLASTX) and two designed for protein sequence queries (BLASTP and TBLASTN) (Coulson, Trends in Biotechnology: 76-80, 1994; Birren et al., GenomeAnalysis, 1:543, 1997). The software for performing BLAST analysis is publicly available through the National Centre for Biotechnology Information. Useful sequence databases include, but are not limited to, Genbank, available through The National Center for Biotechnology Information (NCBI), the European Molecular Biology Laboratory Nucleic Acid Database (EMBL) or versions thereof, available through The European Bioinformatics Institute (EBI), or the MIPS database, available through the Munich Information Center for Protein Sequences.
Preferred DP polypeptides used in the methods of the present invention have at least 51% sequence identity with any one of SEQ ID NO 2, 4, 13, 15, 17, 19, 21 and 23. The percentage of sequence identity, may be calculated using a pairwise global alignment program implementing the algorithm of Needleman-Wunsch (J. Mol. Biol. 48: 443-453, 1970), which maximizes the number of matches and keeps the number of gaps to a minimum. For calculation of the above-mentioned percentages, the program needle (EMBOSS package) may be used with a gap opening penalty of 10 and gap extension penalty of 0.1. For proteins, the blosum62 matrix with a word length of 3 is preferably used. For nucleic acids, the program needle uses the matrix “DNA-full”, with a word-length of 11, as provided by the EMBOSS package. The Needleman-Wunsch algorithm is best suited for analysing related protein sequences over their full length. Alternatively, analysing related proteins and determining the percentage of sequence identity as mentioned above, may be calculated in the conserved region, domains or motifs as mentioned above.
Examples of polypeptides failing under the definition of “a DP polypeptide or homologue thereof” are Arabidopsis thaliana DPb (SEQ ID NO 2 and corresponding encoding sequence SEQ ID NO 1). Other examples of DP proteins are represented by their Genbank accession number in FIG. 3, and their coding sequences as well as their protein sequences are herein represented by SEQ ID NO 12 to 23. The genome sequences of Arabidopsis thaliana and Oryza sativa are now available in public databases such as Genbank and other genomes are currently being sequenced. Therefore, it is expected that further homologues will readily be identifiable by sequence alignment with any one of SEQ ID NO 1 to 4 or 12 to 23 using the programs BLASTX or BLASTP or other programs.
Despite what may appear to be a relatively low sequence homology (as low as approximately 51%), DP proteins are highly conserved, all of them having a DNA binding domain and a dimerisation domain. It is to be understood that the term DP polypeptide or homologue thereof is not to be limited to the sequences represented by SEQ ID NO 2, 4, 13, 15, 17, 19, 21 and 23, but that any polypeptide meeting the criteria of having at least 51% sequence identity with any one of these SEQ ID NOs and having any of the aforementioned conserved regions, domains or motifs, may be suitable for use in the methods of the invention.
According to a preferred embodiment, such DP polypeptide or homologue retains similar functional and/or biological activity or at least part of the functional and/or biological activity of a DP protein. Typically a DP protein is capable of dimerizing with an E2F transcription factor. This may be tested for example by a Two-Hybrid assay as described in Magyar et al. 2000, FEBS letters, 486: 79-97 or co-immunoprecipitation. Preferably the DP polypeptide or homologue thereof is capable of binding DNA. Biological activity is the activity of the protein when it is in its natural environment. The Biological activity results from its functional activity and results in the modifications in growth characteristics that DP proteins exert as demonstrated in the methods of the present invention.
A DP polypeptide or homologue thereof is encoded by a “DP nucleic acid” or “DP gene”. The terms “DP nucleic acid” or “DP gene” are used interchangeably herein and mean a nucleic acid encoding a DP polypeptide or homologue thereof as described hereinabove. Examples of DP nucleic acids include those represented by any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 or 22. DP nucleic acids and functional variants thereof may be suitable in practicing the methods of the present invention. Functional variants of DP nucleic acids include portions of a DP nucleic acid and/or nucleic acids capable of hybridising with a DP nucleic acid. The term “junctional” in the context of a functional variant refers to a variant which encodes a polypeptide having at least one of the above-mentioned functional domains, conserved region or motifs of a DP protein as described hereinabove and retains part of the functional activity and/or biological activity as described hereinabove.
The term portion as used herein refers to a piece of DNA comprising at least 80 nucleotides and which portion which portion has at least one of the above described domains, conserved regions of motifs of a DP protein. The portion may be prepared, for example, by making one or more deletions to a DP nucleic acid. Preferably, the functional portion is a portion of a nucleic acid as represented by any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 or 22.
Another variant DP nucleic acid is a nucleic acid capable of hybridizing, preferably under stringent conditions, with a DP nucleic acid as hereinbefore defined, which hybridizing sequence encodes a polypeptide having at least one of the abovementioned domains, conserved regions or motifs of a DP protein. The hybridizing sequence is preferably at least 80 nucleotides in length. Preferably, the hybridizing sequence is capable of hybridizing to a nucleic acid as represented by any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 and 22.
The term “hybridising” as used herein means annealing to a substantially homologous complementary nucleotide sequences in a hybridization process. The hybridisation process may occur entirely in solution, i.e. both complementary nucleic acids are in solution. The hybridisation process may also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin. The hybridisation process may furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e.g. photolithography to e.g. a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips). In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids. The stringency of hybridisation is influenced by conditions such as temperature, sodium/salt concentration and hybridisation buffer composition.
Hybridization occurs under reduced stringency conditions, preferably under stringent conditions. The stringency of hybridisation is influenced by conditions such as temperature, salt concentration, ionic strength and hybridisation buffer composition. Hybridisation occurs under reduced stringency conditions, preferably under stringent conditions. Examples of stringency conditions are shown in Table 1 below: stringent conditions are those that are at least as stringent as, for example, conditions A-L; and reduced stringency conditions are at least as stringent as, for example, conditions M-R.







TABLE 1







Examples of stringency conditions














Hybridization
Wash


Stringency
Polynucleotide
Hybrid Length
Temperature
Temperature


Condition
Hybrid±
(bp)‡
and Buffer†
and Buffer†





A
DNA:DNA
> or equal to 50
65° C.; 1 × SSC−
65° C.; 0.3 × SSC





or −42° C.;





1 × SSC, 50%





formamide


B
DNA:DNA
<50
Tb*; 1 × SSC
Tb*; 1 × SSC


C
DNA:RNA
> or equal to 50
67° C.; 1 × SSC−
67° C.; 0.3 × SSC





or −45° C.;





1 × SSC, 50%





formamide


D
DNA:RNA
<50
Td*; 1 × SSC
Td*; 1 × SSC


E
RNA:RNA
> or equal to 50
70° C.; 1 × SSC−
70° C.; 0.3 × SSC





or −50° C.;





1 × SSC, 50%





formamide


F
RNA:RNA
<50
Tf*; 1 × SSC
Tf*; 1 × SSC


G
DNA:DNA
> or equal to 50
65° C.; 4 × SSC−
65° C.; 1 × SSC





or −45° C.;





4 × SSC, 50%





formamide


H
DNA:DNA
<50
Th*; 4° SSC
Th*; 4 × SSC


I
DNA:RNA
> or equal to 50
67° C.; 4 × SSC−
67° C.; 1 × SSC





or −45° C.;





4 × SSC, 50%





formamide


J
DNA:RNA
<50
Tj*; 4 × SSC
Tj*; 4 × SSC


K
RNA:RNA
> or equal to 50
70° C.; 4 × SSC−
67° C.; 1 × SSC





or −40° C.;





6 × SSC, 50%





formamide


L
RNA:RNA
<50
Tl*; 2 × SSC
Tl*; 2 × SSC


M
DNA:DNA
> or equal to 50
50° C.; 4 × SSC−
50° C.; 2 × SSC





or −40° C.;





6 × SSC, 50%





formamide


N
DNA:DNA
<50
Tn*; 6 × SSC
Tn*; 6 × SSC


O
DNA:RNA
> or equal to 50
55° C.; 4 × SSC−
55 xC.; 2 × SSC





or −42° C.;





6 × SSC, 50%





formamide


P
DNA:RNA
<50
Tp*; 6 × SSC
Tp*; 6 × SSC


Q
RNA:RNA
> or equal to 50
60° C.; 4 × SSC−
60° C.; 2 × SSC





or −45° C.;





6 × SSC, 50%





formamide


R
RNA:RNA
<50
Tr*; 4 × SSC
Tr*; 4 × SSC





‡The “hybrid length” is the anticipated length for the hybridising nucleic acid. When nucleic acids of known sequence are hybridised, the hybrid length may be determined by aligning the sequences and identifying the conserved regions described herein.


†SSPE (1 × SSPE is 0.15 M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) may be substituted for SSC (1 × SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridisation and wash buffers; washes are performed for 15 minutes after hybridisation is complete. The hybridisations and washes may additionally include 5 × Denhardt's reagent, .5-1.0% SDS, 100 μg/ml denatured, fragmented salmon sperm DNA, 0.5% sodium pyrophosphate, and up to 50% formamide.


*Tb-Tr: The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5-10° C. less than the melting temperature Tm of the hybrids there Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (° C.) = 2 (# of A + T bases) + 4 (# of G + C bases). For hybrids between 18 # and 49 base pairs in length, Tm (° C.) = 81.5 + 16.6 (log.sub.10[Na+]) + 0.41 (% G + C) − (600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([NA+] for 1 × SSC = .165 M).


±The present invention encompasses the substitution of any one, or more DNA or RNA hybrid partners with either a PNA, or a modified nucleic acid.






Other variant DP nucleic acids useful in the methods of the present invention are allelic variants of a DP nucleic acid, splice variants, variants due to the degeneracy of the genetic code, family members of a DP nucleic acid and variants interrupted by one or more intervening sequences, such as introns, spacer sequences or transposons.
DP nucleic acids or functional variants thereof may be in the form of DNA, or a complement DNA, RNA, cDNA, genomic DNA, synthetic DNA as a whole or a part, double-stranded or single-stranded nucleic acid.
The methods according to the present invention may also be practised using one of the above-mentioned DP variants, for example using an alternative splice variant of SEQ ID NO 1. One example of an alternative splice variant of SEQ ID NO 1 is herein represented by SEQ ID NO 3. Other examples of splice variants are found in Oryza sativa, where two DPb proteins each have two different splice forms: AAO72709.1 and AY224589 are two splice variants of the same genomic DNA, and AAO72671.1 and AY224551 are two splice forms of the same genomic DNA encoding the other DPb protein. The term “alternative splice variant” as used herein encompasses variants of a nucleic acid in which selected introns and/or exons have been excised, replaced or added. Suitable splice variants will be the ones in which the functional and/or biological function of the protein is retained, which may be achieved by selectively retaining functional segments of the protein. Such splice variants may be found in nature or may be manmade. Methods for making such splice variants are well known in the art.
Another variant DP nucleic acid useful in practising the method of the present invention is an allelic variant of a DP gene, for example, an allelic variant of SEQ ID NO 1. Allelic variants exist in nature and encompassed within the methods of the present invention is the use of these natural alleles. Allelic variants also encompass Single Nucleotide Polymorphisms (SNPs) as well as Small Insertion/Deletion Polymorphisms (INDELs). The size of INDELs is usually less than 100 bp. SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms.
The DP nucleic acid or variant thereof may be derived from any natural or artificial source. The nucleic acid/gene or variant thereof may be isolated from a microbial source, such as bacteria, yeast or fungi, or from a plant, algae or animal (including human) source. This nucleic acid may be modified from its native form in composition and/or genomic environment through deliberate human manipulation. The nucleic acid is preferably of plant origin, whether from the same plant species (for example to the one in which it is to be introduced) or whether from a different plant species. The nucleic acid may be isolated from a dicotyledonous species, preferably from the family Brassicaceae, further preferably from Arabidopsis thaliana. More preferably, the DP isolated from Arabidopsis thaliana is represented as by SEQ ID NO 1 or 3, and the DP amino acid sequence is as represented by SEQ ID NO 2 or 4. Other preferred sequences are as represented by SEQ ID NO 12, 14, 16, 18, 20 and 22 and the corresponding amino acid sequence as represented by SEQ ID NO 13, 15, 17, 19, 21 or 23.
The DP nucleic acid sequence useful in the methods of the present invention may have at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a DP nucleic acid, for example, to any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 or 22.
“Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having an amino acid substitution, deletion and/or insertion relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived. To produce such homologues, amino acids of the protein may be replaced by other amino acids having similar properties (such as similar hydrophobicity, hydrophilicity, antigenicity, propensity to form or break α-helical structures or β-sheet structures). Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins. W.H. Freeman and Company).
Homologues of a particular DP protein may exist in nature and may be found in the same or different species or organism from which the particular DP protein is derived. Two special forms of homologues, orthologues and paralogues, are evolutionary concepts used to describe ancestral relationships of genes. The term “orthologues” relates to genes in different organisms that are homologous due to ancestral relationship. The term “paralogues” relates to gene-duplications within the genome of a species leading to paralogous genes. The term “homologues” as used herein also encompasses paralogues and orthologues of a DP protein, which are also useful in practising the methods of the present invention.
Orthologues of a DP protein in other plant species may easily be found by performing a reciprocal Blast search. This method comprises searching one or more sequence databases with a query gene or protein (for example, any one of SEQ ID NO 1 to 4 or 12 to 23), using for example, the BLAST program. The highest-ranking subject genes that result from this search are then used as a query sequence in a similar BLAST search. Only those genes that have as a highest match again the original query sequence are considered to be orthologous genes. For example, to find a rice orthologue of an Arabidopsis thaliana gene, one may perform a BLASTN or TBLASTX analysis on a rice database such as the Oryza sativa Nipponbare database available at the NCBI website (<www.ncbi.nlm.nih.gov>. In a next step, the highest ranking rice sequences are used in a reverse BLAST search on an Arabidopsis thaliana sequence database. The method may be used to identify orthologues from many different species, for example, from corn.
Paralogues of a DP protein in the same species may easily be found by performing a Blast search on sequences of the same species from which the DP protein is derived. From the sequences that are selected by the Blast search, the true paralogues may be identified by looking for the highest sequence identity. Preferably a DP paralogue comprises the conserved DP region as described hereinabove. Further preferably, a DP paralogue comprises the DPb motifs as described hereinafter.
Some of the DP variants or homologues as mentioned hereinabove may occur in nature and may be isolated from nature. Once the sequence of a homologue is known, and its corresponding encoding sequence, the person skilled in the art will be able to isolate the corresponding DP nucleic acid from biological material such as genomic libraries, for example, by the technique of PCR. One example of such an experiment is outlined in Example 1. Alternatively, when the sequence is not known, new DP proteins may be isolated from biological material via hybridization techniques based on probes from known DP proteins.
Alternatively and/or additionally, some DP variants or homologues as mentioned above may be manmade via techniques involving, for example, mutation (substitution, insertion or deletion) or derivation. These variants are herein referred to as “derivatives”, which derivatives are also useful in the methods of the present invention. Derivatives of a protein may readily be made using peptide synthesis techniques well known in the art, such as solid phase peptide synthesis and the like, or by protein engineering via recombinant DNA manipulations. The manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art. For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, Ohio), QuickChange Site Directed mutagenesis (Stratagene, San Diego, Calif.), PCR-mediated site-directed mutagenesis or other site-directed mutagenesis protocols.
Accordingly, a homologue may be in the format of a substitutional variant. The term “substitutional variants” of a DP protein refers to those variants in which at least one residue in an amino acid sequence has been removed and a different amino acid inserted in its place. Amino acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide; insertions usually are of the order of about 1-10 amino acids, and deletions can range from about 1-20 amino acids. Preferably, amino acid substitutions comprise conservative amino acid substitutions.
Homologues may also be in the form of an “insertional variants” of a protein in which one or more amino acids are introduced into a predetermined site in the DP protein. Insertions may comprise amino-terminal and/or carboxy-terminal fusion as well as intra-sequence insertion of single or multiple amino acids. Generally, insertions within the amino acid sequence are of the order of about 1 to 10 amino acids. Examples of amino- or carboxy-terminal fusions include fusion of the binding domain or activation domain of a transcriptional activator as used in the yeast two-hybrid system, phage coat proteins, (histidine)6-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag-100 epitope, c-myc epitope, FLAG-epitope, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope.
Homologues in the form of “deletion variants”, are characterised by the removal of one or more amino acids from the protein.
The DP polypeptide of homologue thereof may be a derivative in the form of peptides, oligopeptides, polypeptides, proteins or enzymes, characterised by substitutions, and/or deletions and/or additions of naturally and non-naturally occurring amino acids compared to the amino acids of a naturally-occurring DP protein. A derivative may also comprise one or more non-amino acid substituents compared to the amino acid sequence from which it is derived. Such non-amino acid substituents include for example, non-naturally occurring amino acids, a reporter molecule or other ligand, covalently or non-covalently bound to the amino acid sequence. Such a reporter molecule may be bound to facilitate the detection of the DP protein.
Another type of DP polypeptide useful in the methods of the present invention is an active fragment of a DP protein. “Active fragments” of a DP protein encompass at least 80 contiguous amino acid residues of a DP protein, which residues retain similar biological and/or functional activity to a naturally occurring protein or a part thereof. Suitable fragments include fragments of a DP protein starting at the second or third or further internal methionine residues. These fragments originate from protein translation, starting at internal ATG codons, whilst retaining its functionality in the methods of the present invention. Suitable functional fragments of a DP protein, or suitable portions of nucleic acids that correspond to such fragments, useful in the methods of the present invention, may have one or more of the conserved region, domain or motifs as described herein above, whilst retaining its functionality in the methods of the present invention. One particular example of a functional fragment is the fragment corresponding to the conserved region common to all DP proteins, as marked in FIG. 3 and further described hereinabove.
The activity of a DP polypeptide or a homologue thereof may be increased by introducing a genetic modification (preferably in the locus of an DP gene). The locus of a gene as defined herein is taken to mean a genomic region, which includes the gene of interest and 10 KB up- or down stream of the coding region.
The genetic modification may be introduced, for example, by any one (or more) of the following methods: TDNA activation, tilling, site-directed mutagenesis, homologous recombination or by introducing and expressing in a plant a nucleic acid encoding an DP polypeptide or a homologue thereof. Following introduction of the genetic modification there follows a step of selecting for increased activity of a DP polypeptide, which increase in activity gives plants having improved growth characteristics.
T-DNA activation tagging (Hayashi et al. Science (1992) 1350-1353) involves insertion of T-DNA usually containing a promoter (may also be a translation enhancer or an intron), in the genomic region of the gene of interest or 10 KB up- or down stream of the coding region of a gene in a configuration such that such promoter directs expression of the targeted gene. Typically, regulation of expression of the targeted gene by its natural promoter is disrupted and the gene falls under the control of the newly introduced promoter. The promoter is typically embedded in a T-DNA. This T-DNA is randomly inserted into the plant genome, for example, through Agrobacterium infection and leads to overexpression of genes near to the inserted T-DNA. The resulting transgenic plants show dominant phenotypes due to overexpression of genes close to the introduced promoter. The promoter to be introduced may be any promoter capable of directing expression of a gene in the desired organism, in this case a plant. For example, constitutive, tissue-preferred, cell type-preferred and inducible promoters are all suitable for use in T-DNA activation.
A genetic modification may also be introduced in the locus of a DP gene using the technique of TILLING (Targeted Induced Local Lesions IN Genomes). This is a mutagenesis technology useful to generate and/or identify, and to eventually isolate mutagenised variants of a DP nucleic acid capable of exhibiting DP activity. TILLING also allows selection of plants carrying such mutant variants. These mutant variants may even exhibit higher DP activity than that exhibited by the gene in its natural form. TILLNG combines high-density mutagenesis with high-throughput screening methods. The steps typically followed in TILLING are: (a) EMS mutagenesis (Redei and Koncz, 1992; Feldmann et al., 1994; Lightner and Caspar, 1998); (b) DNA preparation and pooling of individuals; (c) PCR amplification of a region of interest; (d) denaturation and annealing to allow formation of heteroduplexes; (e) DHPLC, where the presence of a heteroduplex in a pool is detected as an extra peak in the chromatogram; (f) identification of the mutant individual; and (g) sequencing of the mutant PCR product.
Methods for TILLING are well known in the art (McCallum Nat Biotechnol. 2000 April; 18(4):455-7, reviewed by Stemple 2004 (TILLING a high-throughput harvest for functional genomics. Nat Rev Genet. 2004 5(2):145-50).
Site directed mutagenesis may be used to generate variants of DP nucleic acids or portions thereof that retain activity. Several methods are available to achieve site directed mutagenesis, the most common being PCR based methods (current protocols in molecular biology. Wiley Eds. <www.4ulr.com/products/currentprotocols/index.html>.
TDNA activation, TILLING and site-directed mutagenesis are examples of technologies that enable the generation novel alleles and DP variants that retain DP function and which are therefore useful in the methods of the invention.
Homologous recombination allows introduction in a genome of a selected nucleic acid at a defined selected position. Homologous recombination is a standard technology used routinely in biological sciences for lower organism such as yeast or the moss physcomitrella. Methods for performing homologous recombination in plants have been described not only for model plants (Offringa et al. Extrachromosomal homologous recombination and gene targeting in plant cells after Agrobacterium-mediated transformation. 1990 EMBO J. 1990 October; 9(10): 3077-84) but also for crop plants, for example rice (Terada R et al. Nat. Biotechnol. 2002 Efficient gene targeting by homologous recombination in rice; Lida and Terada Curr Opin Biotechnol. 2004 15(2): 132-8: A tale of two integrations, transgene and T-DNA: gene targeting by homologous recombination in rice). The nucleic acid to be targeted (which may be a DP nucleic acid or variant thereof as hereinbefore defined) need not be targeted to the locus of a DP gene, but may be introduced in, for example, regions of high expression. The nucleic acid to be targeted may be an improved allele used to replace the endogenous gene or may be introduced in addition or the endogenous gene.
According to a preferred embodiment of the invention, plant growth characteristics may be improved by introducing in a plant and expressing a nucleic acid encoding a DP polypeptide or a homologue thereof. According to a preferred embodiment of the present invention, the expression is preferably in the shoot tissue of the plant.
A preferred method for introducing a genetic modification (which in this case need not be in the locus of an DP gene) is to introduce and express in a plant a nucleic acid encoding a DP polypeptide or a homologue thereof. A DP polypeptide or a homologue thereof as mentioned above is one having in increasing order of preference, at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to a DP protein, for example, to any one of SEQ ID NO 2, 4, 13, 15, 17, 19, 21 and 23. Preferably said DP polypeptide comprises at least one of the aforementioned conserved region, domains or motifs.
According to a preferred aspect of the present invention, enhanced or increased expression of the DP nucleic acid or variant thereof is envisaged. Methods for obtaining enhanced or increased expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to upregulate expression of an DP nucleic acid or variant thereof. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, U.S. Pat. No. 5,565,350; Zarling et al., PCT/US93/03868), or isolated promoters may be introduced into a plant cell in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
According to the methods of the present invention, the activity of DP is increase specifically in shoot tissue, and preferably this is mediated by increased expression of a DP nucleic acid specifically in shoot tissue. The term “shoot” as used herein encompasses all aerial parts of the plants. Typical shoot-tissues include but are not limited to tissues of stems, branches, leaves, buds, flowers, reproductive organs, seeds, and shoot-derived structures such as stolons, corms, bulbs or tubers. Preferably in the methods of the present invention the DP gene is preferentially expressed in young shoot tissue.
In a preferred method of the present invention, the shoot-tissue-specific expression of the DP gene is mediated by a shoot-tissue-specific promoter operable linked to the introduced DP gene. Therefore, according to a preferred embodiment of the invention there is provided a method for improving plant growth characteristics relative to corresponding wild-type plants, comprising the introduction into a plant of a nucleic acid encoding a DP protein, and comprising the expression of said nucleic acid specifically in shoot-tissue.
The term “shoot-tissue specific promoter” means a promoter that is at least 5 times stronger in shoot than in other plant organs, such as roots. The shoot-tissue-specific promoter is a tissue-specific promoter, characterized by the fact that it preferentially, but not exclusively expressed in aerial parts of the plant. The term “tissue-specific” promoter may be used interchangeably herein with a “tissue-preferred” promoter.
Alternatively, the shoot-tissue-specific expression of the DP gene is mediated by selective transformation techniques, where for example ballistics are used to transform the aerial tissues.
Alternatively, the shoot-tissue-specific expression of the DP gene is mediated by T-DNA tagging, a technique well known by a person skilled in the art. For example, one can introduce a promoter randomly in the plant and select those plants in which the DP expression is increased specifically in the shoot tissues.
If polypeptide expression is desired, it is generally desirable to include a polyadenylation region at the 3′-end of a polynucleotide coding region. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from T-DNA. The 3′ end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or less preferably from any other eukaryotic gene.
An intron sequence may also be added to the 5′ untranslated region or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in both plant and animal expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold, Buchman and Berg, Mol. Cell biol. 8:4395-4405 (1988); Callis et al., Genes Dev. 1:1183-1200 (1987). Such intron enhancement of gene expression is typically greatest when placed near the 5′ end of the transcription unit. Use of the maize introns Adh1-S intron 1, 2, and 6, the Bronze-1 intron are known in the art. See generally, The Maize Handbook, Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).
The invention also provides genetic constructs and vectors to facilitate introduction and/or expression of the nucleotide sequences useful in the methods according to the invention.
Therefore, according to a further embodiment of the present invention, there is provided a genetic construct comprising:

    
    
        (a) a DP nucleic acid or a variant thereof;
        (b) one or more control sequences capable of preferentially expressing the nucleic acid of (a) in shoot tissue; and optionally
        (c) a transcription termination sequence.
    
    


Constructs useful in the methods according to the present invention may be constructed using recombinant DNA technology well known to persons skilled in the art. The gene constructs may be inserted into vectors, which may be commercially available, suitable for transforming into plants and suitable for maintenance and expression of the gene of interest in the transformed cells. Preferably, the genetic construct according to the present invention is a plant expression vector, suitable for introduction and/or maintenance and/or expression of a nucleic acid in a plant cell, tissue, organ or whole plant.
One example of a genetic construct according to the present invention is herein represented by SEQ ID NO 8 and encompasses a DP gene under the control of a rice beta-expansin promoter and followed by a double transcription termination sequence (see FIG. 2).
Accordingly, the present invention provides genetic constructs as described above wherein the control sequence of (b) is a shoot-tissue preferred promoter, such as a beta-expansin promoter or a promoter having a comparable expression profile to the beta-expansin promoter.
The nucleic acid according to (a) is advantageously any of the nucleic acids described hereinbefore. A preferred nucleic acid is a nucleic acid represented by SEQ ID NO 1, 2, 12, 14, 16, 18, 20 or 22 or a functional variant thereof as described hereinabove, or is a nucleic acid encoding a protein as represented by SEQ ID NO 2, 4, 13, 15, 17, 19, 21 or 23 or a variant thereof as described hereinabove.
Plants are transformed with a vector comprising the sequence of interest (i.e. a DP nucleic acid or a variant thereof). The sequence of interest is operably linked to one or more control sequences, preferably a promoter described as above. With the term “promoter” it meant a transcription control sequence. The promoter of (b) is operable in a plant, and suitable promoters are preferably derived from a plant sequence. The terms “transcription control sequence” or “promoter” are used interchangeably herein and are to be taken in a broad context to refer to regulatory nucleic acids capable of effecting expression of the sequences to which they are operably linked. Encompassed by the aforementioned terms are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i.e. upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner. Also included within the term is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a −35 box sequence and/or −10 box transcriptional regulatory sequences. The term “regulatory element” also encompasses a synthetic fusion molecule or derivative, which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ.
The term “operably linked” as used herein refers to a functional linkage between the promoter sequence and the gene of interest, such that the promoter sequence is able to initiate transcription of the gene of interest. Preferably, the gene of interest is operably linked in the sense orientation to the promoter.
Advantageously, any promoter may be used for the methods of the invention, provided that it has a shoot-tissue-specific expression pattern. These promoters have, when compared to a strong constitutive promoter (such as the strong constitutive/ubiquitous CaMV35S promoter), a lower expression level in roots.
One example of such a promoter the rice beta-expansin promoter EXPB9, represented herein by SEQ ID NO 7. This promoter may be isolated from the Oryza sativa (japonica cultivar-group) chromosome 10, BAC OSJNBa0082M15, where it is located upstream of EXPB9 gene encoding the mRNA as represented by the Genbank accession number AF261277. The term “shoot-tissue-specific promoter” as used herein therefore also means a promoter that has the same or similar activity, as the rice beta-expansin promoter EXPB9 in Oryza sativa. Similar activity in this context means an activity that is at most 20-fold higher or lower than the beta-expansin promoter EXPB9, preferably at most 10-fold higher or lower or 5-fold higher or lower or 3-fold higher or lower.
One method to measure the promoter strength is through the use of promoter-beta-glucuronidase fusions. The promoter if hereby fused to the Escherichia coli UidA gene encoding beta-glucuronidase and the chimeric construct is transformed into a plant. Proteins are extracted from the plant material and GUS activity is measured (Jefferson et al., 1987, EMBO J. 20; 6(13):3901-7). Promoter activity is then calculated as the optical density in units per mg of extracted protein.
Preferably, the shoot-tissue-preferred promoter is expressed preferably during vegetative growth of the plant or in young shoot-tissue. Therefore, in the context of this invention, GUS activity is preferably measured from tissues after germination. Preferably, these measurements are performed during vegetative growth of the plant, for example after 2, preferably after 4 weeks post germination.
Another example of a shoot-tissue-preferred promoter is a protochlorophyl reductase promoter.
Optionally, in the genetic construct according to the invention, one or more terminator sequences may also be incorporated. The term “transcription termination sequence” encompasses a control sequence at the end of a transcriptional unit, which signals 3′ processing and polyadenylation of a primary transcript and termination of transcription. Additional regulatory elements, such as transcriptional or translational enhancers, may be incorporated in the genetic construct. Those skilled in the art will be aware of terminator and enhancer sequences, which may be suitable for use in performing the invention. Such sequences would be known or may readily be obtained by a person skilled in the art.
The genetic constructs of the invention may further include an origin of replication, which is required for maintenance and/or replication in a specific cell type. One example is when a genetic construct is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid). Preferred origins of replication include, but are not limited to, the f1-ori and colE1.
The genetic construct may optionally comprise a selectable marker gene. As used herein, the term “selectable marker gene” includes any gene, which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells, which are transfected or transformed with a genetic construct of the invention. Suitable markers may be selected from markers that confer antibiotic or herbicide resistance. Cells containing the recombinant DNA will thus be able to survive in the presence of antibiotic or herbicide concentrations that kill untransformed cells. Examples of selectable marker genes include genes conferring resistance to antibiotics (such as nptil encoding neomycin phosphotransferase capable of phosphorylating neomycin and kanamycin, or hpt encoding hygromycin phosphotransferase capable of phosphorylating hygromycin), to herbicides (for example, bar which provides resistance to Basta; aroA or gox providing resistance against glyphosate), or genes that provide a metabolic trait (such as manA that allows plants to use mannose as sole carbon source). Visual marker genes result in the formation of colour (for example, beta-glucuronidase, GUS), luminescence (such as luciferase) or fluorescence (Green Fluorescent Protein, GFP, and derivatives thereof). Further examples of suitable selectable marker genes include the ampicillin resistance gene (Ampr), tetracycline resistance gene (Tcr), bacterial kanamycin resistance gene (Kanr), phosphinothricin resistance gene, and the chloramphenicol acetyltransferase (CAT) gene, amongst others
The present invention also encompasses plants obtainable by the methods according to the present invention. The present invention therefore provides plants obtainable by the method according to the present invention, which plants have introduced therein an DP nucleic acid or variant thereof.
The invention also provides a method for the production of transgenic plants having improved growth characteristics, comprising introduction and expression in a plant of a DP nucleic acid or a variant thereof.
Accordingly, there is provided a method for the production of a transgenic plant comprising:

    
    
        (a) introducing into a plant cell a DP nucleic acid or a variant thereof, preferably introducing a genetic construct as described hereinabove;
        (b) cultivating said plant cell under conditions promoting plant growth.
    
    


The produced transgenic plants are characterised by improved plant growth characteristics relative to corresponding wild-type plants.
“Introducing” the DP nucleic acid or the genetic construct into the plant cell is preferably achieved by transformation. The term “transformation” as used herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention. The choice of tissue depends on the particular plant species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypqcotyls, megagametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. Preferably, the DP nucleic acid is stably integrated in the genome of the plant cell, which may be achieved, for example, by using a plant transformation vector or a plant expression vector having T-DNA borders, which flank the nucleic acid to be introduced into the genome.
Transformation of a plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F. A. et al., 1882, Nature 296, 72-74; Negrutiu I. et al., June 1987, Plant Mol. Biol. 8, 363-373); electroporation of protoplasts (Shillito R. D. et al., 1985 Bio/Technol 3, 1099-1102); microinjection into plant material (Crossway A. et al., 1986, Mol. Gen Genet 202, 179-185); DNA or RNA-coated particle bombardment (Klein T. M. et al., 1987, Nature 327, 70) infection with (non-integrative) viruses and the like. A preferred method for the production of transgenic plants according to the invention is an Agrobacterium-mediated transformation method.
Transgenic rice plants are preferably produced via Agrobacterium-mediated transformation using any of the well-known methods for rice transformation, such as the ones described in any of the following: published European patent application EP1198985, Aldemita and Hodges (Planta, 1996, 199: 612-617,); Chan et al. (Plant Mol. Biol., 1993, 22 (3): 491-506,); Hiei et al. (Plant J., 1994, 6 (2): 271-282,); which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotechnol., 1996, 14(6): 745-50) or Frame et al. (Plant Physiol., 2002, 129(1): 13-22), which disclosures are incorporated by reference herein as if fully set forth.
Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers, which are co-transformed with the DP gene.
The resulting transformed plant cell, cell grouping, or plant tissue, may then be used to regenerate a whole transformed plant via regeneration techniques well known to persons skilled in the art. Therefore, cultivating the plant cell under conditions promoting plant growth may encompass the steps of selecting and/or regenerating and/or growing to reach maturity.
Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation. Alternatively or additionally, expression levels of the newly introduced DNA may be monitored using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art.
The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques. For example, a first generation (or T1) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques.
The generated transformed organisms may take a variety of forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (e.g., all cells transformed to contain the expression cassette); grafts of transformed and untransformed tissues (e.g., in plants, a transformed rootstock grafted to an untransformed scion).
The invention also includes host cells containing an isolated nucleic acid molecule encoding a DP or a genetic construct as mentioned hereinbefore. Preferred host cells according to the invention are plant cells. Accordingly, there is provided plant cells, tissues, organs and whole plants that have been transformed with a genetic construct of the invention.
The present invention clearly extends to plants obtainable by any of the methods as described hereinbefore. The present invention extends to plants, which have increased expression levels of a DP nucleic acid and/or increased level and/or activity of a DP protein preferentially in shoot-tissue. The present invention extends to plants containing a genetic construct as described hereinabove. The plants according to the present invention have improved growth characteristics.
The present invention clearly also extends to any plant cell of the present invention and to all plant parts and propagules thereof. The present invention extends further to encompass the progeny of a primary transformed cell, tissue, organ or whole plant of the present invention, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characteristic(s) as those of the parent plants, for example the plants produced by the methods according to the invention.
The invention also extends to any part of the plant according to the invention, preferably a harvestable part of a plant, such as, but not limited to, a seed, leaf, fruit, flower, stem culture, stem, rhizome, root, tuber, bulb and cotton fiber.
The present invention also relates to use of a nucleic acid encoding a DP protein or a variant thereof, under control of a shoot-tissue-preferred promoter for improving plant growth, preferably for increasing biomass.
The present invention also relates to a method for the production of plant biomass, comprising the step of growing a plant according to the present invention as described hereinabove.
DP nucleic acids or variants thereof or DP polypeptides or homologues thereof may find use in breeding programmes in which a DNA marker is identified which may be genetically linked to an DP gene or variant thereof. The DP or variants thereof or DP or homologues thereof may be used to define a molecular marker. This DNA or protein marker may then be used in breeding programs to select plants having altered growth characteristics. The DP gene or variant thereof may, for example, be a nucleic acid as represented by any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 or 22.
Allelic variants of a DP may also find use in marker-assisted breeding programmes. Such breeding programmes sometimes require introduction of allelic variation by mutagenic treatment of the plants, using for example EMS mutagenesis; alternatively, the programme may start with a collection of allelic variants of so called “natural” origin caused unintentionally. Identification of allelic variants then takes place by, for example, PCR. This is followed by a selection step for selection of superior allelic variants of the sequence in question and which give rise improved growth characteristics in a plant. Selection is typically carried out by monitoring growth performance of plants containing different allelic variants of the sequence in question, for example, different allelic variants of any one of SEQ ID NO 1, 3, 12, 14, 16, 18, 20 or 22. Growth performance may be monitored in a greenhouse or in the field. Further optional steps include crossing plants, in which the superior allelic variant was identified, with another plant. This could be used, for example, to make a combination of interesting phenotypic features.
A DP nucleic acid or variant thereof may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. Such use of DP nucleic acids or variants thereof requires only a nucleic acid sequence of at least 15 nucleotides in length. The DP nucleic acids or variants thereof may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the DP nucleic acids or variants thereof. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et al. (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acids may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the DP nucleic acid or variant thereof in the genetic map previously obtained using this population (Botstein et al. (1980) Am. J. Hum. Genet. 32:314-331).
The production and use of plant gene-derived probes for use in genetic mapping is described in Bematzky and Tanksley (1986) Plant Mol. Biol. Reporter 4:37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.
The nucleic acid probes may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel et al. In: Non mammalian Genomic Analysis: A Practical Guide, Academic press 1996, pp. 319-346, and references cited therein).
In another embodiment, the nucleic acid probes may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask (1991) Trends Genet. 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan et al. (1995) Genome Res. 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.
A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the nucleic acids. Examples include allele-specific amplification (Kazazian (1989) J. Lab. Clin. Med 11:95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov (1990) Nucleic Acid Res. 18:3671), Radiation Hybrid Mapping (Walter et al. (1997) Nat. Genet. 7:22-28) and Happy Mapping (Dear and Cook (1989) Nucleic Acid Res. 17:6795-6807). For these methods, the sequence of a nucleic acid is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.
DP nucleic acids or variants thereof or DP polypeptides or homologues thereof may also find use as growth regulators. Since these molecules have been shown to be useful in improving the growth characteristics of plants, they would also be useful growth regulators, such as herbicides or growth stimulators. The present invention therefore provides a composition comprising a DP or variant thereof or a DP polypeptide or homologue thereof, together with a suitable carrier, diluent or excipient, for use as a growth regulator.
The methods according to the present invention result in plants having improved growth characteristics, as described hereinbefore. These advantageous growth characteristics may also be combined with other economically advantageous traits, such as further yield-enhancing traits, tolerance to various stresses, traits modifying various architectural features and/or biochemical and/or physiological features.



The present invention will now be described with reference to the following figures in which:
FIG. 1 is a map of the binary vector pEXP::AtDPb for expression in Oryza sativa of the Arabidopsis thaliana DPb gene (internal reference CDS006) under the control of the rice beta-expansin promoter (beta-EXPB9 promoter with internal reference PRO0061). The AtDPb expression cassette further comprises a T-zein and T-rbcS-deltaGA double transcription termination sequence. This expression cassette is located within the left border (LB repeat, LB Ti C58) and the right border (RB repeat, RB Ti C58) of the nopaline Ti plasmid. Within the T-DNA there is further provided a selectable and a screenable marker, both under control of a constitutive promoter and followed by polyA or a T-NOS transcription terminator sequence. This vector further comprises an origin of replication (pBR322 ori+bom) for bacterial replication and a bacterial selectable marker (Spe/SmeR) for bacterial selection.
FIG. 2 presents of all the SEQ ID NO's used in the description of the present invention. In SEQ ID NO 2, the region which is typically conserved in DP proteins is underlined.
FIG. 3 shows an alignment of DP proteins with the location of the conserved consensus DPb motifs herein represented as SEQ ID NO 9 (motif 1), 10 (motif 2) and 11 (motif 3). Also the DNA binding domain of AtDPb and the dimerisation domain of AtDPb are indicated. The location of the highly conserved region, common to all DP proteins, is indicated with dashed brackets. Multiple sequence alignment across the entire sequences was done using CLUSTAL W (Higgins et al., (1994) Nucleic Acids Res. 22:4673-4680), with the BLOSSUM 62 matrix and with the parameters GAPOPEN 10, GAPEXT 0.05 and GAPDIST 8. The sequences are presented by their Genbank accession number.
FIG. 4 shows the cladogram corresponding to the multiple alignment of FIG. 3. The cladogram view was generated by the program ClustalW. The sequences are presented by their. Genbank accession number.
FIG. 5 shows a phylogram view of DP proteins. The phylogram gives the length of the branches and the distance between the nodes in proportion to the evolutionary distance between the sequences. The cladogram view was generated by the program ClustalW. Two groups of DP proteins may be distinguished based on the presence or absence of the KKKK/RR (SEQ ID NO: 10) motif.



EXAMPLES
The present invention will now be described with reference to the following examples, which are by way of illustration alone.
DNA Manipulation
Unless otherwise stated, recombinant DNA techniques are performed according to standard protocols described in Sambrook (2001) Molecular Cloning: a laboratory manual, 3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York or in Volumes 1 and 2 of Ausubel et al. (1998), Current Protocols in Molecular Biology. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfase (1993) by R. D. D. Croy, published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).
Example 1
Cloning of Arabidopsis thaliana DPb
The Arabidopsis DPb gene (internal reference CDS006) was amplified by PCR using as template an Arabidopsis thaliana seedling cDNA library (Invitrogen, Paisley, UK). After reverse transcription of RNA extracted from seedlings, the cDNA fragments were cloned into pCMV Sport 6.0. Average insert size of the cDNA library was 1.5 kb, and original number of clones was about 1.59×107 cfu. The original titer of 9.6×105 cfu/ml was brought to 6×1011 cfu/ml after amplification of the library. After plasmid extraction of the clones, 200 ng of plasmid template was used in a 50 μl PCR mix. The primers used for PCR amplification, prm0319 with the sequence 5′ GGGGACMGTTTXTGTACAAAAAAGCAGGCTTCACAATGACAACTACTGGG TCTAATTCT 3′ (SEQ ID NO 5) and prm0320 with the sequence 5′ GGGGACCACTTTGTAC AAGAAAGCTGGGTTCAATTCTCCGGCTTCAT 3′ (SEQ ID NO 6), comprise an AttB site for Gateway recombination cloning (italics). PCR was performed using Hifi Taq DNA polymerase in standard conditions. A PCR fragment of the expected length was amplified and purified also using standard methods. The first step of the Gateway procedure, the BP reaction, was then performed, during which the PCR fragment recombines in vivo with the pDONR201 plasmid to produce the “entry clone”, p0424. Plasmid pDONR201 was purchased from Invitrogen, as part of the Gateway® technology.
Example 2
Vector Construction (pEXP::AtDPb)
The entry clone p0424 was subsequently used in an LR reaction with p3169, a destination vector used for Oryza sativa transformation. This vector contains as functional elements within the T-DNA borders, a plant selectable marker, a screenable marker and a Gateway cassette intended for LR in vivo recombination with the sequence of interest already cloned in the entry clone. Upstream of this Gateway cassette lies the rice beta-expansin promoter (internal reference PRO061) for shoot-tissue-preferred expression of the gene of interest. After the LR recombination step, the resulting expression vector pEXP::AtDPb (FIG. 1) was transformed into Agrobacterium strain LBA4044 and subsequently into Oryza sativa var. Nipponbare plants. Transformed rice plants were allowed to grown and were examined for various growth characteristics as described in Example 3.
Example 3
Evaluation of T0, T1 and T2 Rice Plants Transformed with pEXP::AtDPb
Approximately 15 to 20 independent T0 transformants were generated. The primary transformants were transferred from tissue culture chambers to a greenhouse for growing and harvest of T1 seed. Six events of which the T1 progeny segregated 3/1 for presence/absence of the transgene were retained. “Null plants” or “Null segregants” or “Nullizygotes” are the plants treated in the same way as a transgenic plant, but from which the transgene has segregated. Null plants can also be described as the homozygous negative transformants. For each of these events, approximately 10 T1 seedlings containing the transgene (hetero- and homo-zygotes), and approximately 10 T1 seedlings lacking the transgene (nullizygotes), were selected by PCR.
Based on the results of the T1 evaluation, three events, which showed improved growth characteristics at the T1 level, were chosen for further characterisation in the T2 and further generations. To this extent, seed batches from the positive T1 plants (both hetero- and homozygotes), were screened by monitoring marker expression. For each chosen event, the heterozygote seed batches were then selected for T2 evaluation. An equal number of positive and negative within each seed batch were transplanted for evaluation in the greenhouse (i.e., for each event 40 plants, of which 20 positives for the transgene and 20 negative for the transgene, were grown). For the three events therefore, a total amount of 120 plants was evaluated in the T2 generation.
T1 and T2 plants were transferred to a greenhouse and were evaluated for vegetative growth parameters, as described hereunder.
(I) Statistical Analysis of Numeric Data
A two factor ANOVA (analyses of variance) corrected for the unbalanced design was used as statistical evaluation model for the numeric values of the observed plant phenotypic characteristics. The numerical values are submitted to a t-test and an F test. The p-value is obtained by comparing the t value to the t distribution or alternatively, by comparing the F value to the F distribution. The p-value stands the probability that the null hypothesis (null hypothesis being “there is no effect of the transgene”) is correct.
A t-test was performed on all the values of all plants of one event. Such a t-test was repeated for each event and for each growth characteristic. The t-test was carried out to check for an effect of the gene within one transformation event, also named herein a “line-specific effect”. In the t-test, the threshold for a significant line-specific effect is set at 10% probability level. Therefore, data with a p-value of the t test under 10% mean that the phenotype observed in the transgenic plants of that line is caused by the presence of the gene. Within one population of transformation events, some events may be under or below this threshold. This difference may be due to the difference in position of the transgene in the genome. It is not uncommon that a gene might only have an effect in certain positions of the genome. Therefore, the above-mentioned “line-specific effect” is also referred to as “position-dependent effect”.
An F-test was carried out on all the values measured for all plants of all events. An F-test was repeated for each growth characteristic. The F-test was carried out to check for an effect of the gene over all the transformation events and to verify an overall effect of the gene, also named herein “gene effect”. In the F-test, the threshold for a significant global gene effect is set at 5% probability level. Therefore, data with a p-value of the F test under 5% mean that the observed phenotype is caused by more than just the presence of the gene and or the position of the transgene in the genome. A “gene effect” is an indication for the wide applicability of the gene in transgenic plants.
(II) Vegetative Growth Measurements
The selected plants were grown in a greenhouse. Each plant received a unique barcode label to link unambiguously the phenotyping data to the corresponding plant. The selected plants were grown on soil in 10 cm diameter pots under the following environmental settings: photoperiod=11.5 h, daylight intensity=30,000 lux or more, daytime temperature=28° C. or higher, night time temperature=22° C., relative humidity=60-70%. Transgenic plants and the corresponding nullizygotes were grown side-by-side at random positions. From the stage of sowing until the stage of maturity (which is the stage were there is no more increase in biomass) the plants were passed weekly through a digital imaging cabinet. At each time point digital images (2048×1536 pixels, 16 million colours) were taken of each plant from at least 6 different angles. The parameters described below were derived in an automated way from the digital images using image analysis software.
Aboveground Area
Plant above-ground area was determined by counting the total number of pixels from above-ground plant parts discriminated from the background. This value was averaged for the pictures taken on the same time point from the different angles and was converted to a physical surface value expressed in square mm by calibration. Experiments show that the above-ground plant area, which corresponds to the total maximum area, measured this way correlates with the biomass of plant parts above-ground.
On average, pEXP::DPb transgenic plants in T1 generation showed an increase in above-ground area of 8% with a p-value of 0.08. The best of the 3 positive T1 lines showed an increase in above-ground area of 30% with a p-value of 0.01. In the T2 generation this line showed 18% increase in above-ground area with a p-value of 0.03.
Example 4
GUS Expression Driven by Beta Expansin Promoter
The beta-expansin promoter was cloned into the pDONR201 entry plasmid of the GatewayTM system (Life Technologies) using the “BP recombination reaction”. The identity and base pair composition of the cloned insert was confirmed by sequencing and additionally, the resulting plasmid was tested via restriction digests.
In order to clone the promoter in front of a reporter gene, each entry clone was subsequently used in an “LR recombination reaction” (Gateway TM) with a destination vector. This destination vector was designed to operably link the promoter to the Escherichia coli beta-glucuronidase (GUS) gene via the substitution of the Gateway recombination cassette in front of the GUS gene. The resulting reporter vectors, comprising the promoter operably linked to GUS were subsequently transformed into Agrobacterium strain LBA4044 and subsequently into rice plants using standard transformation techniques.
Transgenic rice plants were generated from transformed cells. Plant growth was performed under normal conditions.
The plants or plant parts to be tested were covered with 90% ice-cold acetone and incubated for 30 min at 4° C. After 3 washes of 5 min with Tris buffer [15.76 g Trizma HCl (Sigma T3253)+2,922 g NaCl in 1 litre bi-distilled water, adjusted to pH 7.0 with NaOH], the material was covered by a Tris/ferricyanate/X-Gluc solution [9.8 ml Tris buffer+0.2 ml ferricyanate stock (0.33 g Potassium ferricyanate (Sigma P3667) in 10 ml Tris buffer)+0.2 ml X-Gluc stock (26.1 mg X-Gluc (Europa Bioproducts ML 113A) in 500 μl DMSO)]. Vacuum infiltration was applied for 15 to 30 minutes. The plants or plant parts were incubated for up to 16 hours at 37° C. until development of blue colour was visible. The samples were washed 3 times for 5 minutes with Tris buffer. Chlorophyll was extracted in ethanol series of 50%, 70% and 90% (each for 30 minutes).

## Claims

1. A method for increasing above-ground area of a monocotyledonous plant, comprising the steps of:
(a) transforming plant cells from a monocotyledonous plant with a genetic construct which comprises a nucleic acid sequence that encodes a polypeptide comprising an amino acid sequence having at least 95% identity to SEQ ID NO: 2, and wherein the nucleic acid sequence is operably linked to a shoot-specific promoter that is at least 5 times stronger in shoot than in other plant organs;
(b) expressing said polypeptide in the transformed plant cells;
(c) regenerating transgenic plants from said transformed plant cells; and
(d) identifying a transgenic plant from said transgenic plants, which exhibits an increase in above-ground area of 8% compared to an untransformed plant of the same species.

2. The method of claim 1, wherein said polypeptide has the amino acid sequence as set forth in SEQ ID NO: 2.

3. The method of claim 1, wherein said monocotyledonous plant is selected from the group consisting of rice, wheat, barley, maize, millet, rye, oat, sugar cane and sorghum.