WO1999016878A1 - Method of regulating gene expression - Google Patents

Abstract

The present invention provides genetic constructs and methods for altering the expression of one or more environmental stress-related genes in plant cells. The invention further provides methods of modifying the expression of particular stress-related genes in plants in response to environmental stresses, including anaerobic stress, dehydration stress and cold stress.

Description

METHOD OF REGULATING GENE EXPRESSION

FIELD OF THE INVENTION

The present invention relates generally to reagents and methods for altering gene expression. More particularly, the invention provides genetic constructs and methods for altering the expression of one or more environmental stress-related genes in plant cells. The invention provides the means by which a plant cells response to a wide range of environmental stresses may be modified.

GENERAL

Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.

This specification contains nucleotide and amino acid sequence information prepared using the programme Patentln Version 2.0, presented herein after the bibliography. Each nucleotide or amino acid sequence is identified in the sequence listing by the numeric indicator <210> followed by the sequence identifier (e.g. <210> 1, < 210> 2, etc). The length, type of sequence (DNA, protein (PRT), etc) and source organism for each nucleotide or amino acid sequence are indicated by information provided in the numeric indicator fields <211 > , <212> and <213 > , respectively. Nucleotide and amino acid sequences referred to in the specification are defmed by the information provided in numeric indicator field <400> followed by the sequence identifier (eg. <400 > 1, <400 >2, etc).

The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

The designations for amino acid residues referred to herein are set forth in Table I.

As used herein the term "derived from" shall be taken to indicate that a specified integer may be obtained from a particular source albeit not necessarily directly from that source.

Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated step or element or integer or group of steps or elements or integers but not the exclusion of any other step or element or integer or group of steps or elements or integers.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions or compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps, features, compositions or compounds.

BACKGROUND TO THE INVENTION

Plants are exposed to a wide range of environmental and physiological stresses which lead to reduced productivity and in severe or prolonged situations, cell death may result leading to crop losses. For example, anaerobic stress, flooding, drought, heat, cold and salinity are all common environmental stresses to which plants are exposed and which are capable of reducing plant productivity. The production of plants, in particular agricultural and horticultural crop plants, which have improved tolerance to such environmental stresses is clearly desirable.

Plants may respond to environmental stress by altering cellular metabolism as part of an adaptive stress response, presumably by inducing the expression of specific proteins and/or down-regulating the expression of other proteins. However, for most environmental stresses the nature of the adaptive stress response is, at best, incompletely characterised. In particular, the full complement of proteins induced by any environmental stress is not known.

5 Plants respond to drought stress by inducing the expression of a class of genes known as "dehydrins", at least by increasing transcription of the dehydrin genes (Close et al., 1989). However, the precise function of the dehydrins is unknown.

The response to anaerobic stress, such as occurs during flooding, is one of the best-

10 characterised stress responses of plants, at the physiological and biochemical levels. Plants respond to anaerobic stress by switching from oxidative phosphorylative respiration to fermentative glycolysis, by suppressing normal protein synthesis and inducing the synthesis of so-called "anaerobic polypeptides" (ANPs; Sachs et al., 1980, Sachs et al., 1996). The ANPs are expressed in several different tissues of anaerobically-stressed plants (Okimoto et

15 al., 1980). These ANPs may be classified into at least four functional groups: (i) Glucose- mobilising enzymes, for example sucrose synthase, amongst others; (ii) stem-glycolytic enzymes including phosphoglucomutase, phosphoglucoisomerase, fructose- 1,6-diphosphate aldolase, glyaraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase and enolase, amongst others; (iii) fermentative enzymes including pyruvate decarboxylase and alcohol

20 dehydrogenase, amongst others; and (iv) amino acid biosynthetic enzymes, for example alanine amino transferase, amongst others. The induction of pyruvate decarboxylase and alcohol dehydrogenase is believed to be of particular significance to survival of the plant, because the regeneration of NAD+ facilitated by these enzymes may enable submerged plant roots to continue anaerobic glycolysis for prolonged periods. In the absence of a functional

25 alcohol dehydrogenase- 1 (Adhl) gene, anaerobically-stressed or flooded maize roots reduce pyruvate to lactate, resulting in cytoplasmic acidification, irreversible cell damage and death.

Elements in the promoter of the maize ADH1 gene, which appear to be required for anaerobic induction of gene expression, have been identified (Walker et al., 1987; Olive et al., 1990;

30 1991a, b). The Anaerobic Response Element (ARE) lies between -100 and -140 relative to the transcription start and is a bipartite element with two copies of a GT-element (5'-[T/C]GGTTT-3'), and two GC-elements (5'-GCC[G/C]C-3'). The GC elements bind a GC -Binding Protein (GCBP-1; Olive et al , 1991b) and both appear to be critical for expression, however the nature of the GC-Binding Protein has yet to be elucidated. The GT-motifs are also critical for anaerobic induction and expression (Walker et al., 1987) and are "footprinted" in vivo by dimethyl sulfate (Ferl and Nick, 1987), suggesting proteins bind to the GT-motifs. No GT binding protein has been identified.

Arabidopsis ADHl is a gene induced by anaerobic conditions and a number of other environmental stimuli (low temperature, dehydration) and the phytohormone ABA (Dolferus et al. 1994; de Bruxelles et al., 1996). The Arabidopsis ADHl promoter contains sequences similar to the ARE between -160 and -140, with the GT-motifs in the opposite orientation compared to the maize Adhl promoter (GT-motif: 5'-AAACCAA-3'; GC-motif: 5'-GCCCC-3')- Functional analysis in transgenic Arabidopsis plants showed both motifs to be critical for ADHl induction by low oxygen and by other stress conditions (Dolferus et al., 1994). The Arabidopsis GC-motif, together with two upstream elements between positions -230 to -219, and -197 to -174, were shown, by footprinting, to be contact points for proteins (Ferl and Laughner, 1989). The former region contains the G-box-1 sequence (5'-CCACGTGG-3'; -226 to -219), which was shown to be required for the low temperature, dehydration and ABA responses (Dolferus et al., 1994; de Bruxelles et al, 1996). Mutagenesis of portion of the G-box-2 sequence (5'-CCAAGTGG-3'), in the second footprint region, did not have clearcut effects on ADHl expression (Dolferus et al. , 1994; de Bruxelles et al., 1996).

Notwithstanding that different stress responses may each produce an adaptive stress response, there appears to be limited nexus between responses to different stress situations, making a single solution to improving the tolerance of any plant species to several environmental stress situations a difficult task. Additionally, different plant species appear to mount different responses to the same environmental stress, for example by inducing stress-related genes in different tissues and/or by inducing different stress-related genes. Clearly, a single solution to improving the stress tolerance of several plant species to any one environmental stress is not a straightforward procedure.

Moreover, the absence of knowledge in relation to the precise mechanisms involved in regulating the adaptive stress responses of plants, in particular the transcription factors and second-messengers involved, further complicates the task of producing stress tolerant plants.

SUMMARY OF THE INVENTION

In work leading up to the present invention, the inventors sought to elucidate the nature of transcription factors involved in regulating the expression of different Adhl genes in plants, in response to anaerobic stress and cold stress. The inventors have demonstrated that a family of transcription factors, the MYB2 family of proteins, are capable of inducing the expression of stress-related genes in plant cells, in response to several stress situations. The regulation of stress-related gene expression by the MYB2 family of proteins provides the means for producing plants with improved tolerance to a wide-range of environmental stresses.

Accordingly, one aspect of the present invention provides a method of modifying the expression of a stress-related gene in a cell, said method comprising expressing in said cell a MYB2 protein or a homologue, analogue or derivative thereof for a time and under conditions sufficient for expression of said stress-related gene to be increased, reduced or otherwise altered.

Whilst not limiting the scope of the present invention, the MYB2 protein of the present invention is particularly useful for the purposes of inducing or repressing the expression of plant-expressible genes which are involved in the plant's response to anaerobic stress, flooding stress, cold stress, dehydration stress, drought stress, heat stress or salinity, amongst others, for example those genes selected from the list comprising sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, fructose- 1,6-diphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate mutase, enolase, pyruvate decarboxylase, alcohol dehydrogenase and alanine amino transferase, amongst others. A further aspect of the invention provides a genetic construct when used to express a MYB2 protein in a cell to modify the expression of a stress-related gene in said cell, preferably a gene which encodes an anaerobic polypeptide (ANP).

A further aspect of the invention provides a method of producing a stress-tolerant plant comprising expressing a MYB2 protein or a homologue, analogue or derivative thereof in said plant for a time and under conditions sufficient for expression of a stress-related gene in said plant to be increased, reduced or otherwise altered.

In an alternative embodiment, the invention provides a method of producing a stress-tolerant plant comprising the steps of transforming plant tissue or cells with a genetic construct which at least comprises a nucleotide sequence which encodes a MYB2 protein placed operably under the control of a plant-expressible promoter sequence, regenerating said tissue or cells into a whole plant and expressing said MYB2 protein in said plant for a time and under conditions sufficient for expression of a stress-related gene in said plant to be increased, reduced or otherwise altered.

The present invention provides, in a further aspect, transgenic plants, plant cells and tissues, which comprise a genetic construct as described supra. Preferably, said transgenic plant, cell or tissue has modified expression of a stress-related gene. More preferably, said transgenic plant, cell or tissue is stress-tolerant.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1-1 is a schematic representation of the Arabidopsis thaliana and maize ADHl promoter sequences, comparing their functional organisation. Arrows indicate the orientation of the GT-motifs (GT-1, GT-2) and GC-motifs (GC-1, GC-2) in both promoters. The positions of the G-Box elements (G-Box-1, G-Box-2) in the A. thaliana ADHl promoter are also indicated. The positions of the two Myb-binding sites (MBS-1 and MBS-2) are also indicated. Numbering above the sequences refers to the positions of nucleotides, relative to the start of transcription of each gene. At the bottom of the Figure is a representation of the consensus sequences of MYB-binding sites derived from the genes of both animals (left) and plants (right).

Figure 1-2 is a schematic representation of the nucleotide sequences of the MBS-1 and MBS- 2 ohgonucleotides which comprise the wild-type MBS-1 and MBS-2 binding sites, respectively in the Arabidopsis thaliana ADHl promoter sequence. The nucleotide sequences of ohgonucleotides having mutations in the region of the putative MBS-1 binding site (ΔMBS- 1/1; ΔMBS-1/2) and the putative MBS-2 binding site (ΔMBS-2), in addition to an oligonucleotide comprising multiple repeats of the MBS-1 binding site (4xMBS-l), are also indicated. The nucleotide sequences of ohgonucleotides described by Dolferus et al. (1994) are also indicated (ΔGT, ΔGC, ΔGBOX1, ΔGBOX2). Underlined nucleotides in the MBS-

1, MBS-2 and 4xMBS-l sequences are those which have been mutated in the corresponding mutant sequences. Underlined nucleotides in italics are mutated compared to the wild type sequence. Double stranded equivalents of these ohgonucleotides were used in gel retardation experiments.

Figure 2-1 is- a copy of a photographic representation of a gel retardation autoradiograph showing the binding of purified GST-AtMYB2 fusion protein to radioactively labelled MBS-1 and MBS-2 oligonucleotides (lanes marked 0). Binding is reduced by the addition of unlabelled competitor DNA of the same sequence as the labelled probe (lanes marked 10, 100), but not by the addition of non-specific oligonucleotides (lanes marked NS). Lanes marked FP indicate reaction mixtures containing the labelled probes without added fusion protein. Numbers on the top of each figure indicate fold molar excess of cold competitor.

Figure 2-2 is a copy of a photographic representation of a gel retardation autoradiograph showing the increased binding of purified GST-AtMYB2 fusion protein to radioactively labelled 4xMBS-l oligonucleotides, compared to MBS-1 oligonucleotides as indicated by the higher molar excess of unlabelled competitor DNA that is required to eliminate binding. Binding of fusion protein to MBS-1 and 4XMBS-1 oligonucleotides was carried out in the presence (lanes marked 10, 100, 200, 500 ) of unlabelled competitor DNA of the same sequence as the labelled probe or alternatively, in the absence of unlabelled competitor DNA (lanes marked 0). Numbers on the top of each figure indicate fold molar excess of cold competitor. Lanes marked FP indicate reaction mixtures containing the labelled probes without added fusion protein.

Figure 2-3 is a copy of a photographic representation of three gel retardation autoradiographs showing the increased binding of purified GST-AtMYB2 fusion protein to radioactively labelled MBS-1 oligonucleotide (left and right panels) and radioactively -labelled MBS-2 oligonucleotide (middle panel), in the absence of unlabelled competitor DNA (lanes marked 0 in each panel) or in the presence of varying amounts of unlabelled MBS-1 (left panel, lanes marked 10 and 100), MBS-1/1 (left panel, lanes marked 10, 100 and 500), MBS-1/2 (right panel, lane marked 100) or MBS-2 (middle panel, lane marked 100) competitor oligonucleotide. Numbers on the top of each figure indicate fold molar excess of cold competitor. Lanes marked FP indicate reaction mixtures containing the labelled probes without added fusion protein. Data indicate that the binding of AtMYB2 to MBS-1 and MBS- 2 can not be competed by competitor oligonucleotides with a mutated AAC core sequence

(ΔMBS-1/1 and ΔMBS-2 respectively), however the mutation of the AAC core to GAC

(ΔMBS-1/2 oligonucleotide), did not completely eliminate the capacity of the oligonucleotide to compete with labelled wild type MBS-1 oligonucleotide for binding.

Figure 3-1 is a copy of a photographic representation showing the induction of ADHl gene expression (top panel) and AtMYB2 gene expression (lower panel) by stress treatments. C: control; AN: low oxygen treatment; D: dehydration; CD: low temperature; ABA: ABA treatment. AtMYB2 is induced by all stress treatments, and expression is higher in roots (solid black bars) than in shoots (hatched bars). Error bars represent standard errors for three repeats.

Figure 3-2 is a copy of a photographic representation of a northern hybridisation showing the kinetics of AtMYB2 mRNA accumulation in the leaves (L) and roots (R) of plants, after 0 hours (O H), 2 hours (2 H), 4 hours (4 H), 6 hours (6 H) and 8 hours (8 H) of low oxygen stress treatment. Accumulation of mRNA is preferentially in the roots of the plant.

5 Figure 3-3 is a copy of a graphical representation showing the induction kinetics of AtMYB2 mRNA (D), compared to ADHl mRNA accumulation (O), in Arabidopsis root cultures, under conditions of low oxygen stress (top panel), dehydration stress (second-to-top panel), low temperature stress (second-to bottom panel) and in the presence of abscisic acid (ABA; bottom panel). Results are expressed as fold induction compared with mRNA levels at the 10 start of the treatment.

Figure 4 is a copy of a graphical representation showing the induction kinetics of AtMYB2 (■) mRNA under low oxygen conditions, compared to the kinetics of induction of the ADHl (♦), Arabidopsis thaliana pyruvate decarboxylase (PDC1; A) and Arabidopsis thaliana 15 sucrose synthase (ASUSl; •) genes. Results are expressed as fold induction compared with mRNA levels at the start of the treatment. The expression of the PDC1 and ASUSl genes is root-specific.

Figure 5 is a copy of a photographic representation showing the expression of the genetic 20 constructs CADH-GUS (left panels) and 35S-GUS (right panels) in pea leaves. The expression of the CADH-GUS construct following co-transfection of pea leaves with the construct 35S-AtMYB2 is also indicated (middle panels). The increased spot size and intensity of staining in the samples which were co-transfected with CADH-GUS and 35S-AtMYB2, indicates that AtMYB2 is able to transactivate ADHl -promoter activity. 25

Figure 6-1 is a copy of a graphical representation showing AtMYB2 transactivation of an ADH-GUS construct in Arabidopsis thaliana mesophyll protoplasts and N. plumbaginifolia suspension protoplasts. The ADH-GUS constructs indicated on the x-axis were expressed in protoplasts in the absence (hatched bars) or the presence (solid black bars) of the construct 30 35S-AtMYB2. In control reactions, the construct 35S-GUS was assayed. Transient assays using A. thaliana mesophyll protoplasts indicate that AtMYB2 is able to transactivate ADH1- promoter-driven GUS expression by a factor 2-2.5. This was confirmed also by using protoplasts of N plumbaginifolia suspension cells, where higher transactivation of about 2.5- 4.5-fold were consistently observed.

Figure 6-2 is a copy of a graphical representation showing AtMYB2 transactivation of the wild-type ADHl promoter (ADHl -GUS) and several substitution mutant promoter constructs containing mutations in the GT-1 (MBS-l)motif (ΔGT), GC-1 motif (ΔGC), G-Box-1 motif (ΔGboxl), G-Box-2 motif (ΔGbox2) or the MBS-2 motif (ΔMBS2) in Arabidopsis thaliana mesophyll protoplasts and N plumbaginifolia suspension protoplasts. The mutations in each of the mutant constructs are indicated in Figure 1-2. The ADH-GUS constructs were expressed in protoplasts in the absence (hatched bars) or the presence (solid black bars) of the construct 35S-AtMYB2. Data indicate that AtMYB2 is not able to transactivate ADH-GUS expression when the GT-motif (MBS1 motif) is mutated (ΔGT), but activation is not affected when MBS-2 is mutated (ΔMBS2). Transactivation potential is reduced when the GC-motif, the G-box-1 motif or the G-box-2 motif is mutated (ΔGC, ΔGboxl and ΔGbox2, respectively).

Figure 7-1 is a copy of a graphical representation of northern blot hybridisation data showing ADHl mRΝA accumulation under conditions of anaerobic stress (AN), dehydration, (D), cold stress (CD) and abscisic acid treatment (ABA) in four-weeks-old Arabidopsis thaliana plantlets, compared to untreated plants (C), in the absence (filled bars) or presence (stippled bars) of 10 μM cycloheximide (CHX).

Figure 7-2 is a copy of a graphical representation of linear RT-PCR data showing the effect of cycloheximide on AtMYB2 expression after anaerobic stress (AN), dehydration for four hours (D/4H) or ten hours (D/10H), cold stress for four hours (CD/4H) or 24 hours (CD/24H) and abscisic acid treatment (ABA) in four-weeks-old Arabidopsis thaliana plantlets, compared to untreated plants (C), in the absence (filled bars) or presence (stippled bars) of 10 μM cycloheximide (CHX). and ABA treatment. RNA from plants subjected to dehydration (D) or low temperature (CD) was extracted at the 2 time-points showing maximal induction (see Figure 3-3).

Figure 8 is a schematic representation of a model for the functional organisation of the Arabidopsis ADHl promoter, showing regulation of the promoter by AtMYBs and environmental stresses. The cts-acting motifs shown in Figure 1-1 are also indicated, together with proteins which may bind to these regions.

Figure 9 is a schematic representation of plasmid pCMYB/5', containing the 5' RT-PCR fragment of the AtMYB cDNA, from the BamΗI site upstream of the ATG to an internal Xhol site. The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Sαcl, BstX , SacII, Eagl, Notl, Xbάl,Spel, BamHl, Ncol, Xhol, Draϊl,Apal and Kp are also indicated in the Figure.

Figure 10 is a schematic representation of plasmid pCMYB/3', containing the 3' RT-PCR fragment of the AtMYB cDNA, from the BamHl site at the end of the cDNA to an internal Xhol site. The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Sαcl, BstXl, Sacϊl, Eagl, Noil, Xbal,Spel, BamHl, Ncol, Xhol, Draϊl,Apal and Kpήl are also indicated in the Figure.

Figure 11 is a schematic representation of plasmid pCATMYB2/S, containing the spliced 5' RT-PCR and 3' RT-PCR fragments derived from clones pCMYB/5' and pCMYB/3', respectively. The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes EcøRI, Sαcl, Kpήl, Smal, BamHl, Xbal, Sail, Pst/, Sph/ and Hindlll are also indicated in the Figure.

Figure 12 is a schematic representation of the plasmid p35S-cAtmyb2/S, which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the AtMYB2 structural gene sequence (cAtMyb2) in the sense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Not , Xhol, EcøRI, Kpήl, Smal, Clal, Hindϊll, BamHl and Xbal are also indicated in the Figure.

Figure 13: Schematic representation of the plasmid vector pC ADH-GUS, which comprises the Arabidopsis thaliana ADHl promoter sequence (PAdh) operably in connection with the bacterial uidA gene (GUS) in the sense orientation and placed upstream of the nopaline synthase gene terminator (NOS). Restriction enzyme sites for the enzymes EcøRI, Smal, Kpnl, Sad, BamHl, Ncol, Xbal, Sail, Pstl, Sphl and H/raffll are also indicated in the Figure. ATG indicates the translation start site of the uidA gene.

Figure 14 is a schematic representation of plasmid pCATMYB2(251), containing a 3'- truncated AtMYB cDNA. The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes EcøRI, Sαcl, Kpnl, Smal, BamHl, Xbal, Sail, Pst/, Sphl and H dIII are also indicated in the Figure.

Figure 15 is a schematic representation of plasmid pCATMYB2(207), containing a 3'- truncated AtMYB cDNA. The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes EcøRI, Sαcl, Kpήl, Smal, BamHl, Xbal, Sail, Pst/, Sphl and Hindlϊl are also indicated in the Figure.

Figure 16 is a schematic representation of the plasmid p35S-CATMYB2(251), which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the 3'- tnincated AtMYB2 structural gene sequence cAtMyb2(Δ251) in the sense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Nøtl, Xhol, EcøRI, Kpήl, Smal, Clal, Hindlll, BamHl and Xbαl are also indicated in the Figure.

Figure 17 is a schematic representation of the plasmid p35S-CATMYB2(207), which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the 3'- truncated AtMYB2 structural gene sequence cAtMyb2(Δ207) in the sense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Nøtl, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, BamHl and Xbαl are also indicated in the Figure.

Figure 18 is a schematic representation of the binary vector pTi 35S-cAtmyb2/S, which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the AtMYB2 structural gene sequence (cAtMyb2) in the sense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). A plant-expressible chimeric selectable marker gene comprising the nopaline synthase gene promoter operably in connection with the neomycin phosphotransferase structural gene placed upstream of the nopaline synthase terminator (PΝOS-ΝPTII-ΝOS3') is also indicated. The positions of the Ti plasmid left border (TL) and right border (TR) sequences are also indicated flanking the chimeric genes. The position of the Tn7-derived spectinomycin-resistance/ streptomycin-resistance gene (Tn7 SpR/StR) is also indicated. Restriction enzyme sites for the enzymes Not/, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, BamHl and Xbal are also indicated in the Figure.

Figure 19-1 is a schematic representation of the binary genetic construct pTA-CATMYB2, showing the relative arrangement of the Agrobacte um tumefaciens Ti plasmid left-border

(black arrow; LB) and right border (black arrow; RB) sequences; a chimeric gene comprising the CaMV 35S promoter (black arrow; 35S) driving expression of GVG (hatched bar; GVG), placed upstream of the Ε9 terminator (wave-lined bar; E9); a selectable marker gene comprising the NOS promoter (filled arrow; NOS) driving expression of the hygromycin phosphotransferase gene (hatched box; HPT) placed upstream of the NOS terminator

(checkered box; NOS); and a chimeric AtMYB2 gene comprising the glucocorticoid- inducible 6XUAS/-46 chimeric promoter (black arrow) driving expression of a sense-oriented

AtMYB2 structural gene (filled bar; CATMYB2) placed upstream of the rbcS3A terminator

(cross-hatched box; 3A). Restriction enzyme sites for the enzymes BamHl, Xhol, EcøRI, Kp^ήl, Smal, Clal, HindlU, Xbal and Spel are indicated at the top of the Figure. The chimeric gene constructs are contained in the pTA7002 vector backbone, which contains a kanamycin- resistance gene.

Figure 19-2 is a schematic representation of the binary genetic construct pTA-CATMYB2 (Δ251), showing the relative arrangement of the Agrobacteήum tumefaciens Ti plasmid left- border (black arrow; LB) and right border (black arrow; RB) sequences; a chimeric gene comprising the CaMV 35S promoter (black arrow; 35S) driving expression of GVG (hatched bar; GVG), placed upstream of the E9 terminator (wave-lined bar; E9); a selectable marker gene comprising the NOS promoter (filled arrow; NOS) driving expression of the hygromycin phosphotransferase gene (hatched box; HPT) placed upstream of the NOS terminator (checkered box; NOS); and a chimeric AιMYB2 gene comprising the glucocorticoid-inducible 6XUAS/-46 chimeric promoter (black arrow) driving expression of a truncated sense-oriented AtMYB2 structural gene which lacks sequences encoding the carboxy-terminal region downstream of amino acid residue 251 (filled bar; CATMYB2), placed upstream of the rbcS3A terminator (cross-hatched box; 3A). Restriction enzyme sites for the enzymes 5αmHI, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, Xbal and Spel are indicated at the top of the Figure. The chimeric gene constructs are contained in the pTA7002 vector backbone, which contains a kanamycin-resistance gene.

Figure 19-3 is a schematic representation of the binary genetic construct pTA-CATMYB2 (Δ207), showing the relative arrangement of the Agrobacteήum tumefaciens Ti plasmid left- border (black arrow; LB) and right border (black arrow; RB) sequences; a chimeric gene comprising the CaMV 35S promoter (black arrow; 35S) driving expression of GVG (hatched bar; GVG), placed upstream of the Ε9 terminator (wave-lined bar; E9); a selectable marker gene comprising the NOS promoter (filled arrow; NOS) driving expression of the hygromycin phosphotransferase gene (hatched box; HPT) placed upstream of the NOS terminator (checkered box; NOS); and a chimeric AtMYB2 gene comprising the glucocorticoid-inducible 6XUAS/-46 chimeric promoter (black arrow) driving expression of a truncated sense-oriented AtMYB2 structural gene which lacks sequences encoding the carboxy-terminal region downstream of amino acid residue 207 (filled bar; CATMYB2), placed upstream of the rbcS3A terminator (cross-hatched box; 3A). Restriction enzyme sites for the enzymes BamHl, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, Xbal and Spel are indicated at the top of the Figure. The chimeric gene constructs are contained in the pTA7002 vector backbone, which contains a kanamycin-resistance gene.

Figure 20 is a schematic representation of the plasmid p35S-cAtmyb2/A, which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the AtMYB2 structural gene sequence (cAtMyb2) in the antisense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). The position of the ampicillin-resistance gene (Amp) is also indicated. Restriction enzyme sites for the enzymes Not , Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, BamHl and Zbαl are also indicated in the Figure.

Figure 21 is a schematic representation of the binary vector pTi 35S-cAtmyb2/A, which comprises the CaMV 35S promoter sequence (P 35S) operably in connection with the AtMYB2 structural gene sequence (cAtMyb2) in the antisense orientation and placed upstream of the octopine synthase gene terminator (3' OCS). A plant-expressible chimeric selectable marker gene comprising the nopaline synthase gene promoter operably in connection with the neomycin phosphotransferase structural gene placed upstream of the nopaline synthase terminator (PNOS-NPTII-NOS3') is also indicated. The positions of the Ti plasmid left border (TL) and right border (TR) sequences are also indicated flanking the chimeric genes. The position of the Tn7-derived spectinomycin-resistance/ streptomycin- resistance gene (Tn7 SpR/StR) is also indicated. Restriction enzyme sites for the enzymes Not , Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, BamHl and Xbal are also indicated in the Figure.

Figure 22 is a schematic representation of the binary genetic construct pTA-CATMYB2/A, showing the relative arrangement of the Agrobacteήum tumefaciens Ti plasmid left-border (black arrow; LB) and right border (black arrow; RB) sequences; a chimeric gene comprising the CaMV 35S promoter (black arrow; 35S) driving expression of GVG (hatched bar; GVG), placed upstream of the Ε9 terminator (wave-lined bar; E9); a selectable marker gene comprising the NOS promoter (filled arrow; NOS) driving expression of the hygromycin phosphotransferase gene (hatched box; HPT) placed upstream of the NOS terminator (checkered box; NOS); and a chimeric antisense AtMYB2 gene comprising the glucocorticoid-inducible 6XUAS/-46 chimeric promoter (black arrow) driving expression of a truncated antisense-oriented AtMYB2 structural gene (filled bar; CATMYB2), placed upstream of the rbcS3A teπninator (cross-hatched box; 3A). Restriction enzyme sites for the enzymes BamHl, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, Xbal and Spel are indicated at the top of the Figure. The chimeric gene constructs are contained in the pTA7002 vector backbone, which contains a kanamycin-resistance gene.

Figure 23 is a schematic representation of the plasmid pQΕΔ (NH2)AtMYB2, which comprises the 3 '-end of the AtMYB2 cDNA clone in the plasmid pQΕ30, downstream of the poly-Histidine tag (6XHis). The position of the ampicillin-resistance gene (Amp) is also indicated.

Figure 24 is a schematic representation of the plasmid pKBSΔ (NH2)AtMYB2 #29, which comprises the 3 '-end of the AtMYB2 cDNA clone from the plasmid pQE (NH2)AtMYB2, cloned into the BamHl site of plasmid pKBS (Stratagene).The position of the kanamycin- resistance gene (Km) is also indicated.

Figure 25 is a schematic representation of the binary genetic construct pTAΔ(NH₂)ATMYB2, showing the relative arrangement of the Agrobacteήum tumefaciens Ti plasmid left-border (black arrow; LB) and right border (black arrow; RB) sequences; a chimeric gene comprising the CaMV 35S promoter (black arrow; 35S) driving expression of GVG (hatched bar; GVG), placed upstream of the E9 terminator (wave-lined bar; E9); a selectable marker gene comprising the NOS promoter (filled arrow; NOS) driving expression of the hygromycin phosphotransferase gene (hatched box; HPT) placed upstream of the NOS terminator (checkered box; NOS); and a chimeric antisense AtMYB2 gene comprising the glucocorticoid-inducible 6XUAS/-46 chimeric promoter (black arrow) driving expression of a truncated antisense-oriented AtMYB2 structural gene which lacks sequences encoding the amino-terminal region of the protein (filled bar; CATMYB2), placed upstream of the rbcS3A terminator (cross-hatched box; 3A). Restriction enzyme sites for the enzymes BamHl, Xhol, EcøRI, Kpήl, Smal, Clal, HindUl, Xbal and Spel are indicated at the top of the Figure. The chimeric gene constructs are contained in the pTA7002 vector backbone, which contains a kanamycin-resistance gene.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

One aspect of the present invention provides a method of modifying the expression of a stress-related gene in a cell, said method comprising expressing in said cell a MYB2 protein or a homologue, analogue or derivative thereof for a time and under conditions sufficient for expression of said stress-related gene to be increased, reduced or otherwise altered.

In a preferred embodiment of the invention, the cell in which expression of the stress-related gene is modified is a plant cell.

As used herein, the term "expression" in relation to a gene or protein shall be taken to refer to the processes of transcription and/or translation and/or other processes involved in regulating the synthesis of a functional polypeptide. Such "other processes" may include for example (i) processes regulating transcription of a gene such as triple helix formation, DNA:protein complex formation or protei protein interactions, amongst others; (ii) processes regulating translation of a gene such as RNA splicing, polyribosome assembly or disassembly, ribonucleoprotein complex formation, RNA:protein complex formation or protei protein interactions amongst others; (iii) post-translational processes such as glycosylation, peptide splicing, protein folding, compartmentalisation, transport, and turnover amongst others which may regulate activity of a peptide, polypeptide protein or enzyme in a cell.

The invention is not in any way limited by the precise mechanism or steps involved in modifying expression of a stress-related gene, the only requirement being that a MYB2 protein or a homologue, analogue or derivative thereof is involved at some level. Whilst not being bound by any theory or mode of action, the present inventors have demonstrated that MYB2 proteins at least bind to one or more sequence motifs in the promoter of a stress- related gene, thereby modifying transcription of that gene. Additional regulatory mechanisms are clearly possible.

The term "stress-related gene" shall be taken to refer to any gene which is regulated directly or indirectly by an environmental stress in a plant cell, including genes for which expression is increased, reduced or otherwise altered.

Preferred stress-related genes are those genes the expression of which is either induced or repressed by anaerobic stress, flooding stress, cold stress, dehydration stress, drought stress, heat stress or salinity, amongst others. More preferably, the stress-related gene is a gene the expression of which is either induced or repressed by anaerobic stress, flooding stress or cold stress. In an even more preferred embodiment, the stress-related gene is a gene which is capable of being induced by anaerobic stress, flooding stress or cold stress.

In a still more preferred embodiment, the stress-related gene is a gene which encodes an ANP selected from the list comprising sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, fructose- 1 ,6-diphosphate aldolase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate mutase, enolase, pyruvate decarboxylase, alcohol dehydrogenase and alanine amino transf erase, amongst others.

In a particularly preferred embodiment, the stress-related gene is the alcohol dehydrogenase gene or the pyruvate decarboxylase gene.

In an alternative embodiment, the stress-related gene is a gene which is capable of being trans-activated by the MYB2 polypeptide, by virtue of the presence of at least one copy of a cw-acting regulatory sequence, in particular a MBS-1 motif and/or a GC-motif and/or a G- box-1 motif and/or a G-box-2 motif, in the promoter region of said stress-related gene to which the MYB2 polypeptide may bind to facilitate the initiation or enhancement of transcription of said gene.

As used herein, the terms "MBS-1 motif", "GC-motif", "G-box-1 motif" and "G-box-2 motif" are defmed with reference to the nucleotide sequences set forth in Figure 1-1, wherein "GC- motif" extends to include both "GC-1 " and"GC-2" motifs set forth in Figure 1-1. However, it is to be understood that the nucleotide sequences of such motifs as set forth in Figure 1-1 may comprise additional nucleotides at their 5 '-end and/or 3 '-end without affecting their function, provided that the "core" nucleotide sequence is present. It is also to be understood that the terms "MBS-1 motif", "GC-motif", "G-box-1 motif" and "G-box-2 motif" extend further to encompass the complementary nucleotide sequences of the nucleotide sequence set forth in Figure 1-1.

Genes possessing such elements include those stress-related genes encoding the enzymes alcohol dehydrogenase, aldolase, glyceraldehyde phosphate dehydrogenase, pyruvate decarboxylase, sucrose synthase and lactate dehydrogenase, amongst others. The present invention therefore extends to the use of MYB transcription factors, in particular AtMYB2, to regulate the expression of these genes.

Those skilled in the art will be aware that by "induction" of expression of a stress-related gene is meant that expression of said gene is enhanced or increased, independent of the actual level of expression either before or after induction has occurred. Similarly, "repression" of expression means that expression is reduced, inhibited or delayed, independent of the level of expression either before or after repression has occurred. Thus, it is not essential for gene expression to be increased from an undetectable or zero level to qualify as induction or alternatively, to be reduced to an undetectable level to qualify as repression, the only requirement being that the level of expression is detectably altered.

The term "stress" as used herein refers to any alteration in cellular metabolism, reduced cell growth or cell death which is produced by an environmental stressor or by a hormone, second messenger or other molecule which is related to or induced by said stressor.

The term "anaerobic stress" means any reduction in oxygen levels sufficient to produce a stress as hereinbefore defined, including hypoxia and anoxia.

The term "flooding stress" refers to any stress which is associated with or induced by prolonged or transient immersion of a plant, plant part, tissue or isolated cell in a liquid medium such as occurs during monsoon, wet season, flash flooding or excessive irrigation of plants, etc.

"Cold stress" and "heat stress" are stresses induced by temperatures which are respectively, below or above, the optimum range of growth temperatures for a particular plant species. Such optimum growth temperature ranges are readily determined or known to those skilled in the art.

"Dehydration stress" is any stress which is associated with or induced by the loss of water, reduced turgor or reduced water content of a cell, tissued, organ or whole plant.

"Drought stress" refers to any stress which is induced by or associated with the deprivation of water or reduced supply of water to a cell, tissue, organ or organism.

The terms "salinity-induced stress", "salt-stress" or similar term refers to any stress which is associated with or induced by a perturbation in the osmotic potential of the intracellular or extracellular environment of a cell.

The term "MYB2 protein" or "MYB2" or similar term shall be taken to refer to any peptide, polypeptide, monomeric or multimeric protein, fusion polypeptide, fusion protein or enzyme which comprises a sequence of amino acids which is at least about 30% identical to the amino acid sequence of Arabidopsis thaliana MYB2 [hereinafter referred to as "AtMYB2" (Urao et al. , 1993)] wherein said peptide, polypeptide, monomeric or multimeric protein, fusion polypeptide, fusion protein or enzyme further includes an amino acid sequence which is at least about 85% , preferably at least about 90% and more preferably at least about 95% identical to the R2 domain and/or R3 domain of AtMYB2.

In determining whether or not two amino acid sequences fall within these percentage limits, those skilled in the art will be aware that it is necessary to conduct a side-by-side comparison or multiple alignment of sequences. In such comparisons or alignments, differences will arise in the positioning of non-identical residues, depending upon the algorithm used to perform the alignment. In the present context, reference to a percentage identity or similarity between two or more amino acid sequences shall be taken to refer to the number of identical and similar residues respectively, between said sequences as determined using any standard algorithm known to those skilled in the art. For example, amino acid sequence identities or similarities may be calculated using the GAP programme of the Computer Genetics Group, Inc., University Research Park, Madison, Wisconsin, United States of America (Devereaux et al, 1984). The GAP programme utilizes the algorithm of Needleman and Wunsch (1970) to maximise the number of identical/similar residues and to minimise the number and/or length of sequence gaps in the alignment. Alternatively or in addition, wherein more than two amino acid sequences are being compared, the ClustalW programme of Thompson et al (1990) is used.

For the purposes of clarification, the nucleotide sequence and derived amino acid sequence of AtMYB2 derived from Arabidopsis thaliana ecotype Columbia are set forth in < 400 > 1 and <400> 2, respectively. The nucleotide sequence and derived amino acid sequence of AtMYB2 derived from Arabidopsis thaliana ecotype C24 are set forth in < 400 > 3 and < 400 > 4 , respectively .

The R2 and R3 domains of a MYB-like polypeptide are known by those skilled in the art to comprise amino acid repeat regions having 2 to 3 conserved tryptophan residues spaced approximately 18-20 residues apart in each repeat region, believed to play a critical role in stabilising the DNA-binding domain of such proteins. The particular AtMYB polypeptide sequence set forth in <400 > 2 or <400>4 comprises only two R2 and R3 domains, each of 51 to 53 amino acid residues in length, located at amino acid residues 20 to 72 and residues 73 to 123 thereof. The present inventors have found that whilst there is a high degree of conservation between different MYB2 proteins in these R2R3 domains, there is very little amino acid sequence identity between MYB2 proteins outside the R2/R3 domains.

The present invention clearly extends to the use of homologue, analogue or derivatives of a MYB2 protein, such as the AtMYB2 protein set forth in <400> 2 or <400>4, the only requirement being that said homologue, analogue or derivative is capable of modulating the expression of a stress-related protein in a cell, in particular a plant cell.

In the present context, "homologues" of an amino acid sequence refer to those polypeptides, enzymes or proteins which have a similar activity in modulating expression of a stress-related gene as the amino acid sequence set forth in <400 >2 or <400>4, notwithstanding any amino acid substitutions, additions or deletions thereto. A homologue may be isolated or derived from any plant, animal or yeast cell, tissue, organ or organism, including the plant species from which AtMYB2 is derived.

Furthermore, the amino acids of a homologous polypeptide may be replaced by other amino acids having similar properties, for example hydrophobicity, hydrophilicity, hydrophobic moment or antigenicity, and so on.

"Analogues" encompass polypeptides having the amino acid sequence of MYB2 notwithstanding the occurrence of any non-naturally occurring amino acid analogues therein.

The term "derivative" shall be taken to refer hereinafter to mutants, parts or fragments of a complete MYB2 polypeptide as defined herein which are useful at least for the purposes of utilising the DNA-binding domain and/or transcriptional activation domain in the regulation of expression of a stress-related gene, for example in the production of fusion polypeptides. Derivatives include modified peptides in which ligands are attached to one or more of the amino acid residues contained therein, such as carbohydrates, enzymes, proteins, polypeptides or reporter molecules such as radionuclides or fluorescent compounds. Glycosylated, fluorescent, acylated or alkylated forms of the subject peptides are particularly contemplated by the present invention. Additionally, derivatives of MYB2 which comprise fragments parts of the subject amino acid sequences are within the scope of the invention, as are homopolymers or heteropolymers comprising two or more copies of the subject polypeptides. Particularly contemplated derivatives are fusion polypeptides between MYB2 and a second regulatory polypeptide which are capable of binding to the 5 '-upstream region or promoter sequence of a stress-related gene, but which are capable of modulating activity of said gene is a different manner or to a different degree compared to MYB2. For example, the extent of induction or repression of expression of the stress-related gene may be greater using such derivatives, compared to using the un-derivatised MYB2. Procedures for derivatizing proteins are well-known in the art.

Substitutions encompass amino acid alterations in which an amino acid is replaced with a different naturally-occurring or a non-conventional amino acid residue. Such substitutions may be classified as "conservative", in which an amino acid residue contained in MYB2 is replaced with another naturally-occurring amino acid of similar character, for example Gly÷ Ala, Val<→Ile-<-»Leu, Asp÷ Glu, Lys-^Arg, Asn<->-Gln or Phe<->Trp«-»Tyr.

Substitutions encompassed by the present invention may also be "non-conservative", in which an amino acid residue which is present in MYB2 is substituted with an amino acid with different properties, such as a naturally-occurring amino acid from a different group (eg. substituted a charged or hydrophobic amino acid with alanine), or alternatively, in which a naturally-occurring amino acid is substituted with a non-conventional amino acid.

Amino acid substitutions are typically of single residues, but may be of multiple residues, either clustered or dispersed.

Amino acid deletions will usually be of the order of about 1-50 amino acid residues, while insertions may be of any length. Those skilled in the art will readily be able to determine the length of a MYB2 protein which may deleted without significantly reducing the ability of the protein to modulate expression of a stress-related protein in a cell. Deletions and insertions may be made to the N-terminus, the C-terminus or be internal deletions or insertions. Generally, insertions within the amino acid sequence will be smaller than amino-or carboxyl- terminal fusions and of the order of 1-4 amino acid residues.

Preferred homologues, analogues and derivatives of the MYB2 polypeptide described herein will comprise an amino acid sequence that is at least about 70% identical to the amino acid sequence set forth in <400 > 2 or <400 >4, and preferably at least about 80% identical, more preferably at least about 90% identical and even more preferably at least about 95% identical to said amino acid sequences. It is also particularly preferred that such homologues, analogues and derivatives are functionally-equivalent to the MYB2 polypeptides exemplified herein in so far as their ability to trans-activate the expression of a stress-related gene derived from a plant species is concerned and preferably, in so far as their ability to modify the stress responses of plants, at least under conditions of anoxia, hypoxia or other anaerobic stress and/or cold stress and/or dehydration stress is concerned.

In an alternative embodiment of the invention, the subject method comprises the additional first step of introducing into the cell an isolated nucleic acid molecule which encodes a MYB2 protein or a homologue, analogue or derivative thereof operably connected in the sense orientation to a promoter sequence.

Optionally, the isolated nucleic acid molecule may further be operably connected to a terminator sequence.

The isolated nucleic acid molecule may comprise any sequence of nucleotides capable of encoding a MYB2 polypeptide or a homologue, analogue or derivative thereof as hereinbefore defined. Preferably, the isolated nucleic acid molecule is at least about 20-30% identical to <400 > 1, more preferably at least about 30-50% identical to <400> 1 and even more preferably at least about 50-75% identical to <400> 1 and still more preferably at least about 75-95 % identical to < 400 > 1 or a part or fragment thereof.

The isolated nucleic acid molecule may be one or more of the following molecules: (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences);

(ii) mRNA or cDNA corresponding to the coding region or a part thereof or one or more exon sequences, and 5 '-untranslated sequences and/or 3'- untranslated sequences of the gene; (iii) a structural region corresponding to the coding region or a part thereof or one or more exon sequences; and/or

(iv) a synthetic or fusion molecule encoding a MYB2 functional product.

Preferably, the isolated nucleic acid molecule, promoter and terminator sequences are contained within a genetic construct suitable for introduction into a plant cell for transient expression therein or alternatively, which is suitable for the stable transformation of the cell by any means known to those skilled in the art. Such genetic constructs preferably include one or more selectable marker genes or reporter genes for efficient selection of cells carrying the subject nucleic acid molecule.

Optionally, the genetic construct may further comprise sequences for integration into cellular DNA, in particular the left border (LB) and/or right border (RB) sequences of T-DNA required for Agrobacteήum tumefaciens mediated transformation of plant cells.

The term "selectable marker gene" shall be taken to refer to any gene as hereinbefore defined, the expression of which in a cell may be utilised to detect and/or select for the presence of a transgene to which said selectable marker gene is linked or which said selectable marker gene has been co-transformed.

Preferred selectable marker genes include genes which when expressed are capable of conferring resistance on a cell to a compound which would, absent expression of said selectable marker gene, prevent or slow cell proliferation or result in cell death. Preferred selectable marker genes contemplated herein include, but are not limited to antibiotic- resistance genes such as those conferring resistance to ampicillin, Claforan, gentamycin, G- 418, hygromycin, kanamycin, neomycin, spectinomycin, tetracycline or a derivative or related compound thereof or alternatively, herbicide-resistance genes such as those conferring resistance to the compounds atrazine, Basta, bialaphos, bromoxinol, Buctril, 2,4-D, glyphosate, phosphinothricin, suphonylurea or a derivative or related compound thereof, amongst others. The compound names "Basta", "Buctril", "claforan" and "G-418" are trademarks.

In a particularly preferred embodiment, the selectable marker gene is the neomycin phosphotransferase gene npt II, which when expressed confers resistance on a cell to neomycin and kanamycin and related compounds thereof. More preferably, the nptll selectable marker gene is placed operably under the control of a promoter suitable for expression in a plant cell.

The term "reporter gene" shall be taken to refer to any gene which, when expressed, produces a polypeptide or enzyme capable of being assayed, for example the bacterial chloramphenicol acetyltransferase gene, β-glucuronidase gene and firefly lucif erase gene, amongst others. Those skilled in the art will be aware that the coding region of a reporter gene may be placed in operable connection with a promoter sequence such that expression of said reporter gene may be monitored to determine the pattern of expression regulated by said promoter sequence.

Preferred reporter genes are those genes for which their expression is capable of being assayed, for example the bacterial chloramphenicol acetyl transferase (CAT) gene, bacterial β-glucuronidase (uidA, GUS or gusA) gene, firefly luciferase (luc) gene, green fluorescent protein (gfp) gene or other gene which is at least useful as an indicator of expression.

The term "terminator" refers to a DNA sequence at the end of a transcriptional unit which signals termination of transcription. Terminators are 3 '-non-translated DNA sequences containing a polyadenylation signal, which facilitates the addition of polyadenylate sequences to the 3 '-end of a primary transcript. Terminators active in plant cells are known and described in the literature. They may be isolated from bacteria, fungi, viruses, animals and/or plants. Examples of terminators particularly suitable for use in the genetic constructs of the present invention include the nopaline synthase (NOS) gene terminator of Agrobacteήum tumefaciens, the terminator of the Cauliflower mosaic virus (CaMV) 35S gene, the zein gene terminator from Zea mays, the ribulose -1, 5-bisphosphate carboxylase small subunit gene (rbcS la or rbcS 3a) terminator, and the isopentenyladenine transferase (ipt) terminator, amongst others.

Reference herein to a "promoter" is to be taken in its broadest context and includes the transcriptional regulatory sequences of a classical 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. A promoter is usually, but not necessarily, positioned upstream or 5', of a structural gene, the expression of which it regulates. Furthermore, the regulatory elements comprising a promoter are usually positioned within 2 kb of the start site of transcription of the gene.

In the present context, the term "promoter" is also used to describe a synthetic or fusion molecule, or derivative which confers, activates or enhances expression of the subject MYB2 protein in a cell, in particular a plant cell. Preferred promoters may contain additional copies of one or more specific regulatory elements, to further enhance expression of the gene and/or to alter the spatial expression and/or temporal expression. For example, regulatory elements which confer copper inducibility may be placed adjacent to a heterologous promoter sequence driving expression of the MYB2 protein, thereby conferring copper inducibility on the expression of said gene. Placing a gene operably under the control of a promoter sequence means positioning the said gene such that its expression is controlled by the promoter sequence. Promoters are generally positioned 5' (upstream) to the genes that they control. In the construction of heterologous promoter/structural gene combinations it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function. Similarly, the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the genes from which it is derived. Again, as is known in the art, some variation in this distance can also occur.

Examples of promoters suitable for use in expressing a MYB2 protein in a cell include viral, fungal, animal and plant derived promoters. Preferred promoters are capable of conferring expression in a eukaryotic cell, especially a plant cell. The promoter may regulate the expression of a gene constitutively, or differentially with respect to the tissue in which expression occurs or, with respect to the developmental stage at which expression occurs, or in response to external stimuli such as environmental stress, or hormones amongst others. Examples of preferred promoters according to the present invention include, but are not limited to the CaMV 35S promoter, NOS promoter, octopine synthase (OCS) promoter, Sc4 promoter from subterranean clover stunt virus, seed-specific promoter such as the vicilin promoter or a derivative thereof, floral-specific promoter such as apetala-3, anther-specific promoter, tapetum-specific promoter, root-specific promoter, leaf-specific promoter such as the Arabidopsis thaliana rbcS la promoter or other rbcS promoter sequence, stem-specific promoter, light-inducible promoter such as the Arabidopsis thaliana rbcS la promoter or other rbcS promoter sequence, metal-inducible promoter such as the copper-inducible promoter, a glucocorticoid-inducible promoter, heat-shock promoter or other environmentally-inducible promoter such as those induced by anaerobiosis or hypoxia, flooding stress, cold stress, dehydration stress or salt stress, or wound-inducible promoter, amongst others. Preferred promoters may also be synthetic sequences comprising one or more upstream activating sequences derived from any of the promoters referred to herein. Those skilled in the art will recognise that the choice of promoter will depend upon the nature of the cell being transformed and when expression of the recombinase, structural gene or other gene contained in the genetic construct of the invention is required.

In a particularly preferred embodiment, the promoter is the glucocorticoid-inducible promoter or the CaMV 35S promoter sequence and/or the terminator is the A. thaliana rbcS 3a terminator or the octopine synthase terminator and/or the selectable marker gene is selected from the list comprising hygromycin transferase and a gene encoding resistance to Spectinomycin, Streptomycin, ampicillin or kanamycin.

In a further preferred embodiment of the invention, the isolated nucleic acid molecule encoding MYB2 is contained within a genetic construct diagrammatically represented in the accompanying Figures.

Although intended for the transformation of a eukaryotic organism and/or the expression of genes contained therein, the genetic constructs of the present invention may need to be propagated in a prokaryotic organism such as the bacteria Escheήchia coli or Agrobacteήum tumefaciens. Accordingly, the genetic constructs described herein may further comprise genetic sequences corresponding to a bacterial origin of replication and/or a selectable marker gene such as an antibiotic-resistance gene, suitable for the maintenance and replication of said genetic construct in a prokaryotic organism. Such sequences are well-known in the art. Usually, an origin of replication or a selectable marker gene suitable for use in bacteria is physically-separated from those genetic sequences contained in the genetic construct which are intended to be expressed or transferred to a eukaryotic cell, or integrated into the genome of a eukaryotic cell.

Those skilled in the art will be aware of how to produce a genetic construct expression of the MYB2 protein and of the requirements for obtaining the expression thereof, when so desired, in a specific cell or cell-type under the conditions desired. The present invention extends to all genetic constructs essentially as described herein.

The genetic constructs described herein are particularly useful for the production of stress- tolerant transgenic cells and transgenic organisms, in particular transgenic plants.

Accordingly, a further aspect of the invention provides a stress-tolerant cell or organism and methods of producing same.

The term "transgenic organism" refers to any organism that has a nucleic acid molecule, including, but not limited to DNA, cDNA, mRNA, tRNA, rRNA, synthetic oligonucleotide molecule, ribozyme, antisense molecule, co-suppression molecule, structural gene, wherein said nucleic acid molecule is introduced into the genome of a cell as an addition to the complement of genetic material present in said cell in the absence of said nucleic acid molecule.

By "stress-tolerant transgenic" is meant that the transgenic cell or organism is able to withstand a particular stress for a longer period of time than an otherwise isogenic non- transgenic organism without resulting in cell damage or death.

The term "stress-tolerant transgenic" also encompasses transgenic cells and organisms in which a stress response occurs more rapidly or to a greater degree, for example by inducing expression of an ANP compared to an otherwise isogenic non-transgenic cell or organism. The earlier or more intense expression of a stress-related protein, in particular an ANP, enables a cell to modify its metabolism as part of an adaptive response earlier than would otherwise be the case. However, the enhanced survival of the cell is probably also dependent upon the repression of expression of stress-related proteins which are not ANPs, because the amino acids normally required for the synthesis of such proteins in the absence of stress, must be diverted into synthesis of ANPs. Accordingly, the present invention also encompasses stress-tolerant plants wherein one or more genes encoding a non-ANP stress-related protein is repressed by a MYB2 protein. Preferably, the stress-tolerant cell or organism is at least tolerant to anaerobic stress and/or flooding stress and/or cold stress. Compared to otherwise isogenic non-transformed cells and organisms, the stress-tolerant cell or organism preferably has increased expression levels of alcohol dehydrogenase and/or pyruvate decarboxylase enzymes in the absence of stress and/or immediately following stress induction and/or is capable of inducing expression of these enzymes earlier.

Methods of producing a transgenic plant are well-known to those skilled in the art. The technique used for a given plant species or specific type of plant tissue depends on the known successful techniques.

Means for introducing recombinant DNA into plant tissue include, but are not limited to, direct DNA uptake into protoplasts (Krens et al, 1982; Paszkowski et al, 1984), PEG- mediated uptake to protoplasts (Armstrong et al, 1990) microparticle bombardment electroporation (Fromm et al, 1985), microinjection of DNA (Crossway et al , 1986), microparticle bombardment of tissue explants or cells (Christou et al, 1988; Sanford, 1988), vacuum-infiltration of plant tissue with nucleic acid, or T-DNA-mediated transfer from Agrobacteήum to the plant tissue. Representative T-DNA vector systems are described in the following references: An et α/.(1985); Herrera-Estrella et al. (1983a,b); Herrera-Estrella et al (1985).

For microparticle bombardment of cells, a microparticle is propelled into a plant cell, in particular a plant cell not amenable to Agrobacteήum mediated transformation, to produce a transformed cell. Wherein the cell is a plant cell, a whole plant may be regenerated from the transformed plant cell. Alternatively, other non-animal cells derived from multicellular species may be regenerated into whole organisms by means known to those skilled in the art. Any suitable ballistic cell transformation methodology and apparatus can be used in practising the present invention. Exemplary apparatus and procedures are disclosed by Stomp et al (U.S. Patent No. 5,122,466) and Sanford and Wolf (U.S. Patent No. 4,945,050). When using ballistic transformation procedures, the genetic construct may incorporate a plasmid capable of replicating in the cell to be transformed.

Examples of microparticles suitable for use in such systems include 1 to 5 μm gold spheres. The DNA construct may be deposited on the microparticle by any suitable technique, such as by precipitation.

Plant species may be transformed with the genetic construct of the present invention by the DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a vector of the present invention. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, 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 term "organogenesis", as used herein, means a process by which shoots and roots are developed sequentially from meristematic centers.

The term "embryogenesis", as used herein, means a process by which shoots and roots develop together in a concerted fashion (not sequentially), whether from somatic cells or gametes.

Plants of the present invention may take a variety of forms. The plants may be chimeras of transformed cells and non-transformed cells; the plants may be clonal transformants (e.g., all cells transformed to contain the MYB2 expression cassette); the plants may comprise grafts of transformed and untransformed tissues (e.g., a transformed root stock grafted to an untransformed scion in citrus species). The 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 Tl) transformed plants may be selfed to give homozygous second generation (or T2) transformed plants, and the T2 plants further propagated through classical breeding techniques.

Plants which may be employed in practising the present invention include both horticultural plant species and agricultural plant species, tree species and ornamental plant species.

Preferred recipient plants include Arabidopsis thaliana, wheat, barley, rice, rye, maize, or sorghum, oil seed rape (Canola), Linola, cotton and sugar cane. Additional species are not excluded.

Once introduced into the plant tissue, the expression of the introduced gene may be assayed in a transient expression system, or it may be determined after selection for stable integration within the plant genome. Techniques are known for the in vitro culture of plant tissue, and in a number of cases, for regeneration into whole plants. Procedures for transferring the introduced genetic construct from the originally transformed plant into commercially useful cultivars are known to those skilled in the art.

The present invention further extends to the progeny of said transgenic plant.

The present invention is further described with reference to the accompanying non-limiting Figures and Examples.

EXAMPLE 1

Plant material, growth conditions and stress treatments.

Arabidopsis thaliana seeds, ecotypes C24 or Columbia (Co-O), used in this study were grown on Murashige and Skoog (MS) medium at 220 C (16/8 hours light/dark cycle, 200 E.s-l.cm-2). Stress and ABA treatments were carried out hydroponically, in dishes containing 15 ml liquid MS medium as previously described (Dolferus et al., 1994; de Bruxelles et al, 1996). Low oxygen treatments were carried out by incubating plantlets in a 5% 02/95% N2 gas mixture (hypoxic conditions; Howard et al, 1987), for 24 h at 220 C in the dark. Dehydration treatment was carried out by incubating the plantlets in medium containing 0.6 M mannitol, for 24 h at 220 C. For cold stress treatment, plantlets were 5 incubated at 4-50 C for 24 h. Abscisic acid (ABA, (+) cis-trans isomers, Sigma) was added to the medium at a final concentration of 0.1 mM for 4 h. For treatments with the protein synthesis inhibitor cycloheximide, plant material was first pre-incubated in MS medium containing 10 μM cycloheximide for 1 h. The solution containing cycloheximide was refreshed for incubation during the stress treatment.

10

EXAMPLE 2

Isolation of a full-length AtMYB2 cDNA

The nucleotide sequence of the Arabidopsis thaliana ecotype Columbia AtMYB gene is presented in < 400 > 1 , with the corresponding amino acid sequence encoded therefor

15 presented in <400> 2.

A full-length AtMYB2 cDNA, flanked by BamHl sites was obtained using RT-PCR of anaerobically induced root RNA derived from Arabidopsis thaliana ecotype C24. The 5 '-end and the 3'- end of the cDNA were each amplified and subcloned separately and their identities

20 confirmed by nucleotide sequence analysis. A BamHl site was created at both the 5 '-end and 3'- end of the cDNA and an Xhol site within the cDNA was used to amplify both ends as BamHl/ Xhol fragments. The resultant plasmids were called pCMYB/5'(5'-end; Figure 9) and pCMYB/3' (3 '-end; Figure 10). These clones were used to reconstruct the complete cDNA as a BamHl fragment in plasmid pGEM3Zf(-), producing the plasmid pCATMYB2/S; Figure

25 11).

The nucleotide sequence of the full-length cDNA present in pCATMYB2/S is set forth in <400>3. The nucleotide sequence of the cDNA differs in a few positions from the genomic sequence derived from ecotype Columbia (GenBank Accession No. D 14712). 30 EXAMPLE 3 Gel Retardation assays

All cloning methods were according to standard procedures (Maniatis et al, 1982; Sambrook et al , 1989). Plasmid pGEX-RAtmyb 2BE, containing the AtMYB2 cDNA fused to the glutathione-S-transferase coding region in plasmid pGEX2T was kindly supplied by Dr. Shinozaki (Urao et al. , 1993). The AtMYB2 fusion protein was purified using the Pharmacia GST purification module and used in GMSA experiments. Complementary oligonucleotide probes used in gel retardation assays (see Figure 1-2 ) were annealed, end-labeled using Klenow DNA polymerase, and then gel-purified. Binding reactions contained 1 μl recombinant AtMYB2 (1:100 diluted; about 50 ng protein), 2 μl lOx EMSA buffer (100 mM Tris-HCl, pH 8.0, 500 mM NaCl, 10 mM EDTA, 0.5 % skimmed milk powder, 50% glycerol, and 10 mM DTT), 1 μl DTT (10 mM), 1 μl poly dl-dC (1 μg/μl), 15 μl H20, and 1 μl labelled target oligonucleotide (0.1 ng; 10,000 cpm). Reactions were incubated at room temperature for 10-15 min. Competition experiments were performed by adding unlabeled competitor oligonucleotide to the reaction prior to the addition of radiolabeled oligonucleotide. Samples were loaded on a 5% polyacrylamide gel in 0.5x TBE (Maniatis et al , 1982).

EXAMPLE 4

RNA extractions, northern and southern blot analysis

RNA extraction, gel electrophoresis, northern blot hybridisations using antisense RNA probes, and filter washing procedures were completely as described previously (Dolferus et al, 1994). Filters were placed on phosphor imager screens (Molecular Dynamics, Sunnyvale, CA) and the hybridization signals quantified. Uniformity of gel-loading was assessed visually using ethidium bromide staining. The Arabidopsis ubiquitin gene (Burke et al, 1988) was used as a probe to correct for variation in sample loading, by dividing all signal strengths by their respective ubiquitin signal. Quantitative RT-PCR was carried out using lμg total RNA and the Promega Access RT-PCR system (Madison, WI). Samples were taken during the PCR reaction after 5, 10, 15 and 25 cycles and loaded on agarose gels. Gels were treated for Southern blot hybridisation, and filters were hybridised using the AtMYB2 cDNA. Linearity of signal strength was verified using phosphorimager quantifications. Oligonucleotides were used slightly overlapping the 5' end and 3' end of the first and second intron positions of the AtMYB2 gene respectively. These oligonucleotides were shown not to amplify using genomic DNA as template.

EXAMPLE 5 Tissue culture, protoplast transient assays, particle bombardment and Agrobacterium-mediated transformation.

Binary vectors were mobilised to Agrobacteήum strain AGL1 (Lazo et al , 1991) by electroporation (Nagel et al, 1990).

Arabidopsis root cultures were established by placing one-month-old leaf cuttings (ecotype C24) on callus-induction medium (CIM; Valvekens et al , 1 988) for three days, prior to infection with Agrobacterium rhizogenes (strain A4RS; Vilaine and Casse-Delbart, 1987). The leaf disks were co-cultivated for three days on CIM-medium, washed in a 200 mg/ml timentin solution (Beecham), and placed on solid MS-medium including 100 mg/ml timentin. After 3-4 weeks the explants were transferred to liquid MS medium, and refreshed monthly. Transgenic Arabidopsis plants were obtained using a slightly modified root explant transformation method (Valvekens et al, 1988; Dolferus et al, 1994). Arabidopsis mesophyll protoplasts were prepared from ecotype Cø-O, using a modification of previously published procedures (Damm and Willmitzer, 1988; Damm et al, 1989; Abel and Theologis, 1994). Typically, transient assays were carried out using 2.10-6 protoplasts and 15-20 μg reporter plasmid DNA, plus or minus the same amount of effector plasmid (p35S-cAtMYB2/S; Figure 12). Suspension cell protoplasts from N. plumbaginifolia were prepared according to Negrutiu et al. (1981), and plasmid DNA was introduced using the same methods as for Arabidopsis protoplasts.

Particle bombardment of pea leafs was carried out using a home made helium gun. A mixture of tungsten and gold particles (50:50 ratio) was used as coating material: 25 μl of the particles (lOOmg/ml in 50% glycerol) were mixed with 2-6 μg plasmid DNA (1 μg/ μl), 25 μl 2.5 M CaC12, and 10 μl spermidine (0.1 M). The total volume was then adjusted to 40 μl, and 4 μl was used for the bombardment of 1 leaf. Reporter and effector plasmid were used in a 1: 1 ratio. Leafs were incubated on MS plates for 16 h before GUS staining.

EXAMPLE 6

GUS histochemical staining and fluorometric assays

GUS fluorometric assays and in vivo histochemical stainings were carried out as previously described (Jefferson, 1987; De Block and De Brouwer, 1992). The total protein concentration in the extracts was determined using a Bio-Rad protein assay kit (Bradford, 1976). Fluorometric and protein assays were carried out in micro well plates, and analysed using Labsystems Fluoroskan II and Multiskan Plus readers, in conjunction with Delta Soft II software (Biometallics Inc., Princeton, USA; Breyne et al, 1993). For in vivo GUS assays, the staiiiing solution was vacuum infiltrated into the plant tissue. Plant material was fixed in 70% ethanol after GUS staining.

EXAMPLE 7 The Arabidopsis ADHl promoter has binding sites for AtMYB2.

Nucleotide sequences of oligonucleotides containing the wild-type Adhl promoter sequence and several mutant sequences were derived from the sequences of known genetic constructs (Dolferus et al, 1994). The nucleotide sequences of the wild-type sequence and these mutations are set forth in Figures 1-1 and 1-2 herein.

The MBS-2 site was mutated by amplifying fragments overlapping the MBS2 site (5'-TAGCAΛCGCC-3') and transforming this site into a Nøtl restriction site (5'-GCGGCCGCAT-3'). The nucleotide sequences of the wild-type sequence and these mutations contained therein are set forth in Figures 1-1 and 1-2 herein. The AtMYB2 gene was expressed in E. coli as a glutathione-S-transferase fusion protein and affinity purified. This fusion protein was used in electrophoretic mobility shift assays (EMS A), to assay binding to oligonucleotides corresponding to the MBS-1 (GT-motif) and MBS-2 regions of the ADHl promoter (Figure 1-2). The recombinant AtMYB2 protein retarded oligonucleotides containing both MBS-1 (GT-motif) and MBS-2 binding sites (Figure 2-1). Binding was marginally stronger for the oligonucleotides containing the GT-motif. No binding was observed for oligonucleotides containing either the G-box-1 or GC-motif sequences, nor did the recombinant Arabidopsis AtMYB2 protein (Urao et al , 1993) bind to the MBS-1 and MBS-2 oligonucleotides (data not shown). Multimerisation of the MBS-1 (GT-motif) oligonucleotide gave significantly stronger binding than the monomers (Figure 2- 2). Binding to the monomers was competed out by hundred to two hundred-fold molar excess for the MBS-1 and MBS-2 oligonucleotides. Higher amounts of cold multimeric oligonucleotide were required to compete out binding to the tetramers, with 500-fold molar excess excluding all binding (Figure 2-2). Addition of non-specific competitors such as the G-box-1 and GC-motif sequences did not compete for binding (Figure 2-1).

EXAMPLE 8 AtMYB2 binding to MBS-1 and MBS-2 requires the AAC-core.

We tested the specificity of binding of AtMYB2 by determining whether the central AAC-core sequence in the MBS-1 and MBS-2 sequences was essential (Figure 2-3).

Mutations were introduced within the core sequence (Figure 1-2), and the effect on binding to AtMYB2 was determined using EMSA (Figure 2-3). The first mutation tested was a substitution, C for A, in the first A of the AAC-core in the MBS-1 and MBS-2 motifs. A similar mutation in a vertebrate system (Weston et al, 1992) abolished Myb-binding, and the same result was seen in the case of the plant GA-Myb (Gubler et al. , 1995). This substitution did not abolish binding of AtMYB2 to either motif, but greatly reduced binding to each of the substituted boxes relative to the wild type sequence (data not shown). Replacing the AAC core with CCC (MBS1/1) completely abolished binding to both sites, and the mutated oligonucleotides were unable to compete out binding by the wild type sequence (Figure 2-3). When the AAC core sequence was mutated to GAC (MBS- 1/2), some degree of competition with wild type MBS-1 binding remained (Figure 2-3). The MBS-1 (5^*-AAACCAAA-3') and MBS-2 (5'-CAACGCCA-3') sequences competed with each other despite differences in sequence (data not shown). In conclusion, the EMSA results indicate that the potential Myb binding sites in the ADHl promoter, MBS-1 (GT-motif) and MBS-2 (upstream of the G-box-2 sequence), are each able to interact with AtMYB2, and that the AAC sequence is an important core element for both these interactions.

EXAMPLE 9 AtMYB2 expression is induced by low oxygen stress in roots. We investigated the effect of low oxygen stress on AtMYB2 expression in roots and leaves of one-month-old Arabidopsis plantlets (Figure 3-1). Maximal induction levels for ADHl are obtained after 4-6 hours of low oxygen stress treatment (Dolferus et al., 1994; de Bruxelles et al. , 1996). Low oxygen inductions were carried out for 4 hours. We found that AtMYB2 is induced strongly by low oxygen (average: 5.6-fold induction), and expression is significantly higher in roots than in shoots (about 7 times on average; Figure 3-1 and Figure 3-2).

The timing of AtMYB2 induction by low oxygen stress was compared to that of ADHl, using RNA extracted from Arabidopsis root cultures (Figure 3-3). AtMYB2 levels peak after 2-4 hours, preceding maximal levels of ADHl mRNA (4-6 hours). The induction of AtMYB2 is also coordinated with the induction of other anaerobically-induced Arabidopsis genes, such as the pyruvate decarboxylase (PDC1; Dolferus et al., in preparation), and sucrose synthase genes (ASUSl; Martin et al , 1993; Figure 4). All these genes contain GT-motifs which are potential binding sites for AtMYB2 (Table I). These genes show similar induction kinetics following low oxygen treatment, with peak expression levels found immediately after AtMYB2 mRNA levels have reached a maximum, suggesting that AtMYB2 is required for their activation following low oxygen stress. EXAMPLE 10 AtMYB2 is able to transactivate ADHL

1. Base Plasmids

A. The effector plasmid p35S-CAtMYB2/S The full-length AtMYB2 cDNA was sub-cloned from plasmid ρCATMYB2/S (Figure 11) into plasmid pART7 (Gleave, 1992), between the 35S promoter and the 3' NOS terminator sequence. The resulting plasmid, designated p35S-CAtMYB2/S (Figure 12) was verified by sequencing and used as effector plasmid in all transient assays.

B. The reporter plasmid pC ADH-GUS

The reporter plasmid pC ADH-GUS is described previously (Dolferus et al., 1994). The map of this pasmid is set frith in Figure 13.

2. Experimental To investigate whether AtMYB2 could transactivate hypoxically induced genes in the absence of hypoxia, we set up two different transient assay systems. As reporter construct we used an DHZ-promoter-GUS fusion containing all cis elements of the ADHl promoter necessary for anaerobic induction (CADΗ-GUS; Figure 13; Dolferus et al, 1994). The effector plasmid was a 35S Cauliflower mosaic virus promoter, driving a full-length AtMYB2 cDNA (p35S-CAtMYB2/S; Figure 12). Pea leaves were used as target tissue and DNA was introduced by biolistics. The AtMYB2 gene product did activate the ADΗ-GUS reporter plasmid as shown by the overall increased intensity of GUS staining and the size of the blue spots on the leaves co-bombarded with pCADΗ-GUS and p35S-AtMYB2/S. Data are presented in Figure 5. This experiment was repeated four times in duplicate and gave essentially similar results, with a 2-3 fold enhancement of DH/-promoter-driven GUS expression in the presence of the 35S-AtMYB2 construct.

In order to obtain more quantitative data about AtMYB2 transactivation of the ADHl promoter, we carried out transient assays using protoplasts with the same constructs. AtMYB2 expression was able to transactivate ADHl promoter activity by a factor of 2-2.5 (Figure 6-1). Higher stimulations of ADHl-promoter-driven GUS expression were found when Nicotiana plumbaginifolia suspension cell protoplasts were used (Figure 6-1; about 2.5-4.5-fold stimulation on average). Levels of transactivation were not increased by higher amounts of effector plasmid compared to the reporter plasmid (data not shown).

EXAMPLE 11 AtMYB2 transactivates the ADHl promoter via the GT-motif (MBS-1).

1. Base Plasmids

A. The effector plasmid p35S-CAtMYB2/S Plasmid p35S-CAtMYB2/S (Figure 12) is described in the preceding Example.

B. The reporter plasmid pC ADH-GUS and derivatives thereof

The reporter plasmid pCADH-GUS is in the preceding Example. Plasmids p35S-GUS, p[GBox-l]ADH-GUS, p[GBox-2] ADH-GUS p[GT]ADH-GUS p[GC]ADH-GUS were described earlier (Dolferus et al. , 1994).

Plasmid p[MBS-2] ADH-GUS, contaiiiing a deletion in the MBS-2 site (Figures 1-1 and 1-2), was constructed by amplifying fragments overlapping the MBS2 site (5 ' -TAGC AACGCC-3 ' ) and transforming this site into a Nøtl restriction site (5'-GCGGCCGCAT-3').

2. Experimental

The nucleotide sequences of the wild-type Adhl sequence and the mutations contained therein are set forth in Figures 1-1 and 1-2 herein.

Mutations in the GT-motif decreased ADHl expression drastically (Dolferus et al , 1994). The presence of a second potential Myb binding site MBS-2 in the ADHl promoter which is able to bind AtMYB2 suggested the Arabidopsis ADHl promoter may resemble the maize Adhl promoter more closely, where two GT-motifs were shown to be functionally important (Figure 1-1). MBS-2 is located in a region which is part of a larger in vivo footprinted area. The area previously mutagenised within the G-box-2, did not affect ADHl expression (Dolferus et al, 1994).

We mutagenised the MBS-2 region (5'-TAGCAACGCC-3') to a NotI restriction site

(5'-GCGGCCGCAT-3'; p(ΔAMBS-2)C ADH-GUS) and checked the effect of these changes on GUS expression and transactivation by AtMYB2. Using Arabidopsis mesophyll protoplasts, we found that AtMYB2 was unable to transactivate constructs with a mutated MBS-1 (GT-motif; Dolferus et al , 1994) sequence, but the transactivation potential of AtMYB2 was not affected by mutations in the MBS-2 sequence (Figure 6-2). Essentially the same results were obtained using N. plumbaginifolia protoplasts. These data indicate that, although AtMYB2 is able to bind to the MBS-2 site, binding is not functional and MBS-2 is, in the presence of an intact MBS-1 site, not of prime importance for the ADHl promoter.

Mutation of the GC-motif (Figure 1-2; Dolferus et al, 1994) strongly reduced AtMYB2 transactivation of ADH-GUS expression in both Arabidopsis mesophyll protoplasts and in N. plumbaginifolia suspension cell protoplasts (Figure 6-2). These results indicate that anaerobic induction of the ADHl promoter requires AtMYB2 and the GT-motif, but also a factor binding to an intact GC-motif plays an important role in promoter activation. Transactivation levels of the G-Box-1 mutants (Figure 1-2) were also reduced compared to the wild type promoter in both Arabidopsis mesophyll and N. plumbaginifolia suspension protoplasts, with transactivation levels similar to the GC-mutants (Figure 6-2), indicating that the G-box binding factor plays a role in stabilising binding of AtMYB2 to the promoter. Close to normal transactivation levels were found for the G-Box-2 mutants (Figure 1-2) in both Arabidopsis and N. plumbaginifolia protoplasts, suggesting that this mutation is not interfering dramatically with the assembly of a functional promoter complex. This result is consistent with the results obtained in transgenic plants, where the effect of G-Box-2 mutants was less obvious than G-Box-1 mutants (Dolferus et al, 1994; de Bruxelles et al., 1996).

EXAMPLE 12 Induction of ADHl by stress requires protein synthesis but AtMYB2 is hyper-induced. Protein synthesis is required before ADHl mRNA is induced by low oxygen, dehydration, ABA, and low temperature conditions (Figure 7-1). The addition of cycloheximide (10 μM) 1 h prior to and during stress treatment prevents accumulation of ADHl mRNA. Because AtMYB2 accumulation is induced by the same stress treatments, we tested the effect of cycloheximide on AtMYB2 expression using a combination of linear RT-PCR and Southern blot hybridisation (Figure 7-2). The results indicate that cycloheximide treatment caused a massive increase in AtMYB2 mRNA levels for all treatments (100-800%). It is not clear at this stage whether this effect of cycloheximide is at the transcriptional or post-transcriptional level, or due to increased mRNA stability. Experiments with AtMYB2-promoter-GUS constructs have indicated that stress-induced expression of AtMYB2 is at least partially controlled at the transcriptional level (Urao et al. , 1993). This indicates that AtMYB2 can be induced without protein synthesis occurring, while ADHl induction does depend on protein synthesis.

EXAMPLE 13 Induction of AtMYB2 by other stresses correlates with ADHl induction.

Maximal induction levels for ADHl are obtained after 8-10 hours of dehydration stress, 20-24 hours of low temperature, and 4 hours of ABA treatment (Dolferus et al, 1994; de Bruxelles et al, 1996). AtMYB2 is induced by all these treatments, including low temperature which was not previously shown to induce AtMYB2; (Figure 3-1). Expression is significantly higher in roots than in shoots for low temperature treatment, but expression levels are similar in leaves and roots for dehydration and ABA treatment, even though ADHl is expressed predominantly in roots in response to these treatments (Figure 3-1). Equal induction levels of AtMYB2 in leaves and roots for both dehydration and ABA treatments agrees well with the fact that dehydration treatment is associated with increased ABA levels in Arabidopsis leaves and roots (de Bruxelles et al , 1996). The root-specificity of ADHl expression under these conditions stresses the involvement of other factors, eg. the factor binding to the G-box-1 sequence.

The kinetics of AtMYB2 mRNA accumulation correspond well with the induction kinetics of ADHl mRNA. Two peaks (4 and 10 hours) were obtained for dehydration, which is in agreement with the two phases in the time course of ADHl mRNA induction (Figure 3-3). Low temperature treatment resulted in transient AtMYB2 mRNA accumulation between 2 and 6 hours (5 -fold induction), followed by induction of ADHl mRNA reaching peak levels between 12 and 24 hours. We also found two peaks for ABA induction (2 and 10 hours; 24- and 15 -fold induction respectively), which was mimicked by the ADHl time course (peaks at 4 and 10 hours; Figure 3-3). AtMYB2 expression is correlated well both temporally and spatially with expression of the ADHl gene: both genes are induced in a coordinated way by the same stresses and both show root-specific expression.

EXAMPLE 14

Effects of over-expressing AtMYB and derivatives thereof in transgenic plants

Truncated versions of the AtMYBl cDNA which lacked the region of the gene encoding the C-terminal protein of the ATMYB protein were obtained by PCR, using the following primers: 5' Primer: 5'-GGATCCGAAATGGAAGATTACGAGCG-3'

3' Primer (Δ251): 5'-GGATCCTTACCACATGTTGTCCCCACCAACACAGC-3' 3' Primer (Δ207): 5'-GGATCCTTAAGAATTCGAAGACGTTGCTGAC-3'

In all cases, the 5' Primer contained the translation start site of the gene.

One truncated gene fragment was produced using the 5' Primer in combination with the 3' Primer (Δ251) and is translated to yield a polypeptide product that is truncated at amino acid position 251 of AtMYB. The fragment was sub-cloned as a BamHl fragment into pGEM3Zf(-) to produce the clone designated pCATMYB2 (Δ251) (Figure 14).

Another truncated gene fragment was produced using the 5' Primer in combination with the 3' Primer (Δ207) and is translated to yield a polypeptide product that is truncated at amino acid position 207 of AtMYB. The fragment was sub-cloned as a BamHl fragment into pGEM3Zf(-) to produce the clone designated pCATMYB2 (Δ207) (Figure 15). The truncated cDNAs in plasmids pCATMYB2 (251) and pCATMYB2 (207) were used to make constructs for expression studies in transgenic Arabidopsis thaliana plants. In a first attempt, we used the strong constitutive CaMV 35S promoter to drive expression of the truncated AtMYB2 cDNAs. The cDNAs were cloned as ZtømHI fragments into plasmid pART7 (Gleave, 1992), producing plasmids p35S-CATMYB2(251) (Figure 16) and p35S- CATMYB2(207) (Figure 17), respectively.

The chimeric genes containing the full-length AtMYB coding region (p35S-cATMYB2/S; Figure 12) and the truncated versions p35S-CATMYB2(251) (Figure 16) and p35S- CATMYB2(207) (Figure 17) were transferred as Nøtl fragments into the binary vector pART27 (Gleave, 1992) for transformation using Agrobacteήum tumefaciens. The resultant plasmids, pTi35S-cAtMYB2/S, pTi35S-cAtMYB2/S(251) and pTi35S-cAtMYB2/S(207) are represented in Figure 18.

Transgenic Arabidopsis thaliana plants have been obtained for all these constructs. Plants containing the plasmid pTi35S-cAtMYB2/S were analysed by northern blot hybridisation. None of the 14 independent transformants showed over expression of AtMYB2 mRNA. Whilst not being bound by any theory or mode of action, these data suggest that constitutive over-expression of AtMYB2 in Arabidopsis thaliana may be lethal, and possibly leads to selection of transformants where the gene is deleted or inactivated. This possibility is investigated using Southern blot hybridisations.

To avoid a possible lethal effect of AtMYB2 expression in transgenic plants, we prepared a set of new constructs with AtMYB2 under the control of a glucocorticoid-inducible promoter (Figures 19-1, 19-2, 19-3, 19-4 and 19-5). The full-length and truncated versions of the AtMYB2 cDNAs were subcloned into plasmid pTA7002 (Aoyama and Chua, 1997) in the sense orientation. Plasmid pTA7002 allows glucocorticoid-mediated transcriptional induction in transgenic plants. The cDNAs were isolated as .X7zøI(partial)/.XbαI fragments from plasmids p35S-cAtMYB2/S (Figure 12), p35S-CATMYB2(251) (Figure 16) and p35S-cAtMYB2(207) (Figure 17), and cloned into the XhollSpel sites of plasmid pTA7002, to yield plasmids pTA- CATMYB2 (Figure 19-1), pTA-CATMYB2(251) (Figure 19-2) and pTA-CATMYB2(207) (Figure 19-3). These constructs were transformed to Arabidopsis thaliana.

Transgenic plants are analysed for improved stress tolerance.

EXAMPLE 15 Effects of under-expressing AtMYB and derivatives thereof in transgenic plants

In order to study the effect of decreased levels of AtMYBl gene expression on ADHl expression, we created antisense constructs. The full-length AtMYB2 cDNA was subcloned as a jBαmHI fragment into plasmid pART7 to yield plasmid p35S-cAtmyb2/A (Figure 20). The presence of the AtMYBl gene in the antisense orientation in this plasmid was confirmed by sequencing.

The chimeric construct was then subcloned as a Nøtl fragment in plasmid pART27, to give pTi35S-cAtmyb2/A (Figure 21). This construct was transformed to Arabidopsis thaliana and the resulting transgenic plants were analysed by northern blot hybridisations. A total of 12 independent antisense transformants were analysed and preliminary data show that there is no significant effect of CaMV 35S promoter-driven expression of the antisense AtMYBl gene on the expression of the endogenous AtMYBl gene, nor is there any apparent effect on ADHl gene expression.

In order to anticipate any problems with lethality of constitutive AtMYBl under expression in transgenic Arabidopsis thaliana plants, we constructed an antisense construct using the glucocorticoid-inducible promoter. To construct plasmid pTA-CATMYB2/A (Figure 22), we isolated the AtMYB2 cDΝA as a SaWSmal fragment from pCATMYB2/S (Figure 11) and cloned this fragment into the Xhol/ Spel(fύ\ed-m) sites of pTA7002.

Different Myb proteins show a higher degree of sequence similarity in the DΝA binding domain, located at the amino terminal end of the protein. In contrast, the carboxyl terminal end of the protein contains the transactivation domain of the protein, and shows a high degree of variability. As a consequence, we made a further antisense construct with only the —COOH end of the AtMYB2 protein, in order to avoid lethal effects caused by non-specific suppression of other Myb genes due to their homology with AtMYB2 in the DNA binding domain. The — COOH-encoding part of the AtMYB2 cDN A was obtained as a JSαmHI PCR fragment using the following primers.

5' Primer(ΔNH₂): 5'-GGATCCGACTATGAGAAATGTTTGGATGCCGAG-3' 3' Primer: 5'-GGATCCTTAATTATACGAATACGATGTCGTATCG-3'

The amplified DNA was cloned into the BamHl site of plasmid pQE30, to produce the plasmid designated pQEΔ (NH₂)AtMYB2 (Figure 23). The sequence of the amplified fragment was confirmed and the 5 '-truncated gene was subsequently subcloned as a βαmHI fragment into the kanamycin-resistant version of pBluescript (pKBS) (Stratagene), to give plasmid pKBSΔ(NH2)AtMYB2 #29 (Figure 24). The Δ(NH2)AtMYB2 gene fragment from this plasmid was then cloned as an Xhol/Spel fragment into the corresponding sites of pTA7002 to give plasmid pTAΔ(NH₂)ATMYB2/A (Figure 25).

The antisense plasmids pTAΔC ATMYB2/A (Figure 22) and pTAΔ(NH₂)ATMYB2/A (Figure 25) have been confirmed by sequence analysis. The plasmids are used to transform Arabidopsis thaliana to modify the stress responses of plants.

EXAMPLE 16 DISCUSSION

Several lines of evidence support the idea that the transcription factor AtMYB2 is a key regulatory factor in environmentally-induced ADHl gene expression. First, AtMYB2 binds to two potential Myb binding sites in the Arabidopsis ADHl promoter, the GT- (MBS- 1 ) and (MBS-2) motifs. The presence of two Myb binding sites in the ADHl promoter, both resembling the GT-motifs found in the maize Adhl promoter, suggested that Arabidopsis ADHl may have a double GT-element like the maize ARE (Figure 1-1). The spacing between the two GT elements is different in Arabidopsis and maize, but variations in spacing between GT-motifs do not affect performance of the maize ARE (Olive et al., 1991a, b). Mutations in the GT-motif (MBS-I) which eliminated binding of AtMYB2, also eliminated ADHl expression, both uninduced and following treatment with low oxygen tension, dehydration, low temperature or ABA (Dolferus et al, 1994; de Bruxelles et al., 1996). MBS-2 is located just upstream of the previously mutated G-Box-2 sequence, which is not critical for expression of ADHl (Dolferus et al., 1994). Because the previous analysis concentrated on residues resembling a G-Box motif, the mutations introduced did not remove key residues from the MBS-2 site (Dolferus et al., 1994; see Figure 1-1). Transient expression assays using constructs mutated in the MBS-2 site showed that this AtMYB2 binding site is not important for ADHl promoter activity (Figure 6-2), and that AtMYB2 binding to MBS-2 on its own is not functionally important. The GT-motif (MBS-1) is crucial for activation of gene expression by AtMYB2.

Secondly, AtMYB2 has an expression pattern that is similar to ADHl, and AtMYB2 mRNA accumulation is tightly coupled to ADHl expression. Highest levels of expression of AtMYB2 mRNA precede peak levels of ADHl mRNA by 2-4 hours. Induction of other anaerobically-induced Arabidopsis genes such as pyruvate decarboxylase (Pdcl) and sucrose synthase (ASusl; Figure 4) shows a similar temporal relationship to AtMYB2 expression.

The observed root specificity of ADHl expression could be a consequence of the tissue-specificity of AtMYB2 expression. If AtMYB2-availability is the limiting factor in ADHl expression, and AtMYB2 is only expressed in roots, then ADHl will also be expressed only in roots. If AtMYB2 is not expressed in leaves then neither will ADHl be. The transactivation results suggest that ATMYB2 is the limiting factor for ADHl expression.

We have shown using cycloheximide, that induction of ADHl by stress conditions requires protein synthesis, as cycloheximide blocks ADHl mRNA accumulation. The fact that

AtMYB2 is induced before ADHl suggests that synthesis of AtMYB2 could be the protein synthesis required for ADHl expression. AtMYB2 mRNA accumulation is not inhibited, but rather super-induced by cycloheximide treatment. Cycloheximide similarly induces other transcription factors and signal transduction components, such as the maize cold-inducible leucine zipper transcription factor mliplS, the calcium-dependent protein kinase ZmCDPKl (Berberich and Kusano, 1997), and HVA11 (Shen et al, 1993). Nuclear run-on experiments will be required to show whether AtMYB2 accumulation under stress conditions is controlled at the transcriptional or post-transcriptional level and whether the effect of cycloheximide is due to changes in mRNA stability, or to the absence of a repressor. Induction of the maize anaerobic proteins was shown to be preceded by the transient accumulation of two proteins with MW approximately 30 KD (Sachs et al. , 1980; MWAtMYB2 =27.5 KD). It is therefore conceivable that AtMYB2 could belong to the maize transition proteins, and that these proteins are required for induction of all the ANPs.

Finally, the third and most important experimental evidence is that the AtMYB2 protein coding region expressed under the control of a strong promoter is able to activate expression of a reporter gene driven by the ADHl promoter. Levels of transactivation seen when AtMYB2 is over-expressed in protoplasts (2-3-fold for biolistics and protoplasts) are similar to those obtained after low oxygen treatment of protoplasts (Figure 6-1 and Figure 6-2). These induction levels were not increased by higher ratios of 35S-AtMYB2: CADH-GUS (data not shown), and were lower than induction levels following low oxygen tension treatment of roots (5-1 0-fold at protein level; 20-50-fold at mRNA level). It is possible that the endogenous ADHl gene is already somewhat induced after protoplast isolation, and that this leads to higher background activity of the CADH-GUS reporter gene and apparently lower fold induction. ADHl gene expression in Arabidopsis callus and suspension cultures is generally very high and constitutive (Dolferus et al, 1985). Transient assays using N. plumbaginifolia suspension cell protoplasts, gave consistently higher transactivation levels.

Mutation analysis has shown that the GC-motif is equally as crucial as the GT-motif for

ADHl expression, and AtMYB2 is unable to transactivate ADHl -promoter function in the absence of a functional GC-motif (Figure 6-2). In maize, the transcription factor GCBP-1 was shown to interact with the GC-motifs of Adhl, and the human general transcription factor Spl was able to bind to the maize Adhl GC-motifs (Olive et al. , 1991b). It is therefore possible that ADHl expression in Arabidopsis requires a general transcription factor in addition to AtMYB2, to obtain high levels of expression. The degree of transactivation by AtMYB2 could be limited by other components or processes, such as the requirement for 5 phosphorylation and activation of a signal transduction pathway. It was recently demonstrated that the C terminus of AtMYB2 protein contains a phosphorylation site (Urao et al., 1996), and that truncation of the protein at the C terminus to amino acid 251 leads to higher transactivation levels, indicating that an interaction site with a repressor could also be present in this region (Urao et al , 1996).

10

Vertebrate and yeast Myb transcription factors commonly activate transcription in close association with other factors and to work in a synergistic manner with these factors (Burk et al, 1993; Tice-Baldwin et al., 1989). It has been demonstrated that the maize Cl myb and B protein (basic helix-loop-helix factor) interact directly on the maize Bronze-1 promoter and

15 activate transcription in a cooperative way (Goff et al., 1992). Our data from mutation analysis (Figure 6-2) indicate there may be a similar close association between AtMYB2 and the proteins binding to the GC-motif, and possibly the factor binding to the G-box- 1 sequence. In the maize Adhl promoter both GT-motifs are closely linked to a GC-motif, while in Arabidopsis ADHl the absence of a GC-motif close to the MBS-2 site could explain

20 why this site is not critical for promoter function.

While only one and three Myb genes are known in Drosophila and humans respectively (Katzen et al, 1985; Luscher and Eisenmann, 1990), plant Mybs are encoded by large multigene families. An estimated 20-30 Myb genes are present in Petunia (Avila et al,

25 1993). Plant Myb-proteins are involved in the regulation of a variety of processes. Myb transcription factors regulate pigmentation of floral organs, via the regulation of the phenylpropanoid (Sablowski et al. , 1994) and flavanoid (Grotewold et al, 1994) biosynthetic pathways. They are also involved in hormone-regulated gene expression and developmental processes, such as the induction of a-amylase biosynthesis in barley aleurone cells by

30 gibberellic acid (Gubler et al., 1995), trichome formation (Oppenheimer et al., 1991), the proliferation of meristematic tissues (Wissenbach et al., 1993), and the regulation of cell shape in petal epidermis cells (Noda et al., 1994). Myb transcription factors are also involved in the regulation of gene expression by environmental stimuli such as induction of anthocyanin biosynthesis by UV-light (Paz-Ares et al., 1987), and in the light-regulation of an Arabidopsis Lhcb gene by phytochrome (Wang et al., 1997). Another desiccation and ABA-induced Myb unrelated to AtMYB2 was found in the resurrection plant Craterostigma plantagineum (Iturriaga et al, 1996).

The homology between Myb proteins is restricted to the DNA binding region at the N terminus. Although plant Mybs can be isolated successfully from cDNA libraries using the animal v-Myb consensus sequence as a probe (Quaedvlieg et al, 1996), the target gene and recognition sequence of many plant Mybs remains unknown. Different plant Mybs show small differences in the recognition sequence (Table II). Maize P Myb, Antirrhinum Myb305, potato Myb Stl, and especially the Arabidopsis ACC 1 factor differ from the vertebrate Myb consensus sequence (Table II). AtMYB2 can bind to the animal Myb consensus sequence, and activate transcription via this sequence (Urao et al, 1993). Our work has shown that AtMYB2 binds specifically to the GT-motif (MBS-1) and MBS-2 of the ADHl promoter. It is able to transactivate the ADHl promoter only via the GT-motif, a motif functionally important in a number of anaerobically induced genes (Table I), and which is functional in either orientation in monocots and dicots (Olive et al., 1991a,b). The GT-motif consensus sequence is 5'-AAACCA-3' , and is usually located between positions -300 and -100 relative to the start of transcription (Table I). Conservation of the sequence is generally higher in the 5' end of the central AAC core sequence. This is partly due to the fact that in many cases, such as the maize Adhl GT- 1 sequence (Figure 1-1), the GT-motif is flanked immediately at the 3'-end by a GC-motif (consensus: 5'-GC[G/C]CC-3'; Olive et al, 1991b).

In conclusion, we have identified the transcription factor AtMYB2 as a key factor in the regulation of ADHl expression under a variety of environmental stress situations. AtMYB2, together with a factor binding to the GC-motif, likely to be a general enhancer element, are crucial for the attainment of high levels of root-specific expression under low oxygen, and also low temperature and dehydration conditions (Figure 8). The GT- and GC-motifs together are sufficient for low oxygen induction of the ADHl promoter, but low temperature, dehydration, and induction by the phytohormone ABA also require the G-box-1 sequence for high expression levels (Dolferus et al, 1994; de Bruxelles et al., 1996; Figure 8). It has recently been shown that the abscisic acid response complex has a modular nature, and consists of several sequences necessary for obtaining a specific ABA-induced response (Shen et al, 1993; 1996; Shen and Ho, 1995). The combination of the GT-motif and the G-box-1 sequence could be specific for ABA-induced metabolic genes such as the Arabidopsis ADHl gene. Further experiments, using transgenic plants over- and under-expressing AtMYB2 are in progress, with the aim of investigating which genes are induced by AtMYB2, and to verify the similarity between different stress-induced protein patterns and the pattern induced by AtMYB2.

TABLE 1

TABLE 1 (conf)

TABLE 1 (conf)

REFERENCES

1. Abel, S., and Theologis, A. (1994) Transient transformation of Arabidopsis leaf protoplasts: a versatile experimental system to study gene expression. Plant J 5, 421-

427.

2. An et al. (1985) EMBO J. 4:277-284.

3. Aoyama and Chua, N-H. (1997) Plant Journal 11, 605-612.

4. Armstrong, C.L., Peterson, W.L., Buchholz, W.G., Bowen, B.A. Sulc, S.L. (1990). Plant Cell Reports 9, 335-339.

5. Ausubel, F. M., Brent, R., Kingston, RE, Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (1987). In: Current Protocols in Molecular Biology. Wiley Interscience (ISBN 047150338).

6. Avila, J. , Niet, C, Canas, L., Benito, M.J. and Paz-Ares, J. (1983). Petunia hybrida genes related to the maize regulatory Cl gene and to animal myb proto- oncogenes. Plant J 3, 553-562.

7. Bailey-Serres, J. and Freeling, M. (1990). Hypoxic stress-induced changes in ribosomes of maize seedling roots. Plant Physiol 94, 1237-1243.

8. Baranowskij, N., Frohberg, C, Prat, S. and Willmitzer, L. (1994) A novel DNA binding protein with homology to Myb oncoproteins containing only one repeat can function as a transcriptional activator. EMBO J 13, 5383-5392.

9. Berberich, T. and Kusano, T. (1997). Cycloheximide induces a subset of low temperature-inducible genes in maize. Mol Gen Genet 254, 257-283. 10. Bevan, M. (1984). Binary Agrobacterium vectors for plant transformation. Nucl. Ac. Res. 12, 8711-8721.

11. Bradford, M.M. (1976). A rapid and sensitive method for quantification of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-254.

12. Breyne, P., De Loose, M., Dedonder, A., Van Montagu, M. and Depicker, A. (1993) Quantitative kinetic analysis of β-Glucuronidase activities using a computer- directed microtiter plate reader. Plant Mol. Biol. Rep. 11, 21-31.

13. Burk, O., Mink, S., Ringwald, M. and Klempnauer, K.H. (1993). Synergistic activation of the chicken mt^'m-1 gene by v-myb and C/EBP transcription factors. EMBO J 12, 2027-2038.

14. Burke, T.J., Callis, J. and Vierstra, R.D. (1988). Characterisation of a polyubiquitin gene from Arabidopsis thaliana. Mol Gen Genet 213, 435-443.

15. Christou, P., McCabe, D.E., Swain, W.F. (1988). Plant Physiol 87, 671-674.

16. Crossway et al. (1986) Mol. Gen. Genet. 202,179-185.

17. Damm, B. and WiUmitzer, L. (1988.) Regeneration of fertile plants from protoplasts of different Arabidopsis thaliana genotypes. Mol Gen Genet 213, 15-20.

18. Damm, D., Schmidt, R. and WiUmitzer, L. (1989). Efficient transformation of Arabidopsis thaliana using direct gene transfer to protoplasts. Mol Gen Genet 217, 6-12.

19. De Block, M. and De Brouwer, D. (1992). ln-situ enzyme histochemistry on plastic- embedded plant material. The development of an artefact-free β-glucuronidase assay. Plant J 2, 261-266.

20. De Bruxelles, G.L., Peacock, W.J., Dennis, E.S. and Dolferus, R. (1996). Abscisic acid induces the alcohol dehydrogenase gene in Arabidopsis. Plant Physiol. Ill, 381- 391.

21. De Lisle, A. J. and Ferl, R. J. (1990). Characterisation of the Arabidopsis ADH G-box binding factor. Plant Cell 2, 547-557.

22. Dennis, E.S., Sachs, M.M., Gerlach, W.L. , Finnegan, E.J. and Peacock, W.J. (1985). Molecular analysis of the alcohol dehydrogenase 2 (Adhl) gene of maize. Nucl. Ac. Res. 13, 121-1 '43.

23. Dennis, E.S., Gerlach, W.L., Walker, J.C. , Lavin, M. and Peacock, W.J. (1988) Anaerobically regulated aldolase gene of maize. A chimeric origin ? J. Mol. Biol. 202, 759-767.

24. Dolferus, R. , Marbaix, G. and Jacobs, M. (1985). Alcohol dehydrogenase in Arabidopsis: analysis of the induction phenomenon in plants and tissue cultures. Mol. Gen. Genet. 199, 256-264.

25. Dolferus, R., Peacock, W.J. and Dennis, E.S. (1994). Differential interactions of promoter elements in stress responses of the Arabidopsis ADH gene. Plant Physiol 105, 1075-1078.

26. Ferl, R.J. and Nick, H.S. (1987). In vivo detection of regulatory factor binding sites in the 5' flanking region of maize ADHl. J. Biol. Chem. 262, 7947-7950.

27. Ferl, R.J. and Laughner, B.H. (1989). In vivo detection of regulatory factor binding sites of Arabidopsis thaliana ADH. Plant Mol. Biol. 12, 357-366.

28. Fromm et al (1985) Proc. Natl. Acad. Sci. (USA) 82,5824-5828.

29. Gleave, A. P. (1992). A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol. Biol. 20, 1203-1207.

30. Goff, S.A., Cone, K.C. and Chandler, V.C. (1992). Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes Dev. 6, 864-875.

31. Herrera-Estrella et al. (1983a) Nature 303, 209-213.

32. Herrera-Estrella et al (1983b) EMBO J. 2, 987-995.

33. Herrera-Estrella et al. (1985) In: Plant Genetic Engineering, Cambridge University Press, NY, pp 63-93.

34. Jefferson, R.A. (1987). Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5, 387-405.

35. Katzen, A.L., Kornberg, T.B. and Bishop, J.M. (1985) Isolation of the proto- oncogene c-myb from D.melanogaster. Cell 41, 449-456.

36. Kohler, U., Liaud, M.F., Mendel, R.R., Cerff, R. and Hehl, R. (1995). The maize GapC4 promoter confers anaerobic reporter gene expression and shows homology to the maize anthocyanin regulatory locus Cl. Plant Mol. Biol. 29, 1293-1298.

37. Krens, F.A., Molendijk, L., Wullems, G.J. and Schilperoort, R.A. (1982). Nature 296, 72-74.

38. Lazo, G.R., Stein, P. A. and Ludwig, R.A. (1991). Bio/technology 9, 963-967.

39. Lazo, G.R., Stein, P. A. and Ludwig, R.A. (1991). A DNA transformation competent Arabidopsis genomic library in Agrobacterium. Bio/Techniques 9, 963- 967.

40. Llewellyn, D.J., Finnegan, E.J., Ellis, J.G., Dennis, E.S. and Peacock, W.J. (1987). Structure and expression of an alcohol dehydrogenase 1 gene from Pisum sativum (cv. "Greenfasf ). J. Mol. Biol. 195, 115-123.

41. Luscher, B. and Eisemann, R. (1990). New light on Myc and Myb. Part II. Myb. Genes Dev. 4, 7092-7096.

42. Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982). Molecular cloning, a laboratory manual. Cold Spring Harbor, New York.

43. Martin, T., Frommer, W.B., Salanoubat, M. and Willmitzer, L. (1983). Expression of an Arabidopsis sucrose synthase gene indicates a role in metabolization of sucrose both during phloem loading and in sink organs. Plant J. 4, 367-377.

44. McKendree, W.L., Paul, A-L., DeLisle, A.J. and Ferl, R.J. (1990) In vivo and in vitro characterisation of protein interactions with the dyad G-box of the Arabidopsis ADH gene. Plant Cell 2, 207-214.

45. Millar, A. A. and Dennis, E.S. (1966). The alcohol dehydrogenase genes of cotton. Plant Mol. Biol. 31, 897-904.

46. Mitchell, L.E., Dennis, E.S. and Peacock, W.J. (1989) Molecular analysis of an alcohol dehydrogenase (Adh) gene from chromosome 1 of wheat. Genome 32, 349- 358.

47. Nagel, R. , Elliot, A., Masel, A. , Birch, R.G. and Manners, J.M. (1990). Electroporation of binary Ti plasmid vector into Agrobacterium tumefaciens and Agrobacteήum rhizogenes. FEMS Microbiol Lett 67, 325-328.

48. Negrutiu, I. (1981). Improved conditions for large-scale culture, mutagenesis and selection of haploid protplasts of Nicotiana plumbaginifolia Viviani. Z. Pflanzenphysiol 104, 431-442.

49. Noda, K., Glover, B.J., Linstead, P. and Martin, C. (1994). Flower colour intensity depends on specialized cell shape controlled by a Myb-related transcription factor. Nature 369, 661-664.

50. Okimoto, R., Sachs, M.M. , Porter, E. and Freeling, M. (1980). Patterns of polypeptide synthesis in various maize organs under hypoxia. Planta 150, 89-94.

51. Olive, M.R., Walker, J.C., Singh, K., Dennis, E.S. and Peacock, W.J. (1990). Functional properties of the anaerobic responsive element of the maize ADHl gene. Plant Mol. Biol. 15, 593-604.

52. Olive, M.R., Walker, J.C., Singh, K., Ellis, J.G., Llewellyn, D., Peacock, W.J. and Dennis, E.S. (1991a). The anaerobic responsive element. Plant Mol. Biol. 2, 673- 684.

53. Olive, M.R., Peacock, W.J. and Dennis, E.S. (1991b). The anaerobic responsive element contains two GC-rich sequences essential for binding a nuclear protein and hypoxic activation of the maize ADHl promoter. Nucl. Ac. Res. 19, 7053-7060. 54. Oppenheimer, D.G., Herman, P.L. , Sivakumaran, S., Esch, J. and Marks, M.D. (1991). A Myb gene required for leaf trichome differentiation in Arabidopsis is expressed in stipules. Cell 67, 483-493.

55. Paul, A-L. and Ferl, R. (1997). The hypoxic response of three alcohol dehydrogenase genes: in vivo and in vitro footprinting of DN A/protein interactions describes multiple signalling connections. Annals Bot. 79, (Sup. A), 33-37.

56. Paz-Ares, J., Ghosal, D., Wienand, U., Peterson, P. A. and Saedler, H. (1987). The regulatory cl locus of Zea mays encodes a protein with homology to myb proto- oncogene products and with structural similarities to transcriptional activators. EMBO J. 6, 3553-3558.

57. Pazkowski et al. (1984) EMBO J. 3, 2717-2722.

58. Pietrzak, M., Shillito, R.D., Hohn, T. and Potrykus, I. (1986). Nucl. Acids Res. 14, 5857-5868.

59. Quaedvlieg, N., Dockx, J., Keultjens, G., Kock, P., Wilmering, J., Weisbeek, P. and Smeekens, S. (1996). Identification of a light-regulated MYB gene from an Arabidopsis transcription factor gene collection. Plant Mol. Biol. 32, 987-993.

60. Roth, B.A., Goff, S.A., Klein, T.M. and Fromm, M.E. (1991). Cl- and R- dependent expression of the maize Bzl gene requires sequences with homology to mammalian myb and myc binding sites. Plant Cell 3, 317-325.

61. Sablowski, R.W.M., Moyano, E., Culianez-Macia, F.A. , Schuch, W., Martin, C. and Bevan, M. (1994). A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. EMBO J. 13, 128-137. 62. Sachs, M.M., Freeling, M. and Okimoto, R. (1980). The anaerobic proteins of maize. Cell 20, 761-767.

63. Sachs, M.M., Subbaiah, CC. and Saab, I.M. (1996). Anaerobic gene expression and flooding tolerance in maize. J. Exp. Bot. 47, 1-15.

64. Sambrook, J., Fritsch, E. and Maniatis, T. (1989). Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.

65. Shen, Q. , Uknes, S.J. and Ho, T-H.D. (1993). Hormone response complex of a novel abscisic acid and cycloheximide inducible barley gene. J. Biol. Chem. 268, 23652-23660.

66. Shen, Q. and Ho, T-H.D. (1995). Functional dissection of an abscisic acid (ABA)- inducible gene reveals two independent ABA-responsive complexes each containing a G-box and a novel cts-acting element. Plant Cell 7, 295-307.

67. Shen, Q., Zhang, P. and Ho, T-H.D. (1996). Modular nature of abscisic acid (ABA) response complexes: composite promoter units that are necessary and sufficient for ABA induction of gene expression in Barley. Plant Cell 8, 1107-1119.

68. Solano, R., Concepcion, N., Avila, J., Canas, L., Diaz, I. and Paz-Ares, J. (1995) Dual DNA binding specificity of a petal epidermis-specific MYB transcription factor (MYB.Ph3) from Petunia hybrida. EMBO J. 14, 1773-1784.

69. Tice-Baldwin, K., Fink, G.R. and Arndt, K.T. (1989). BAS1 has a Myb motif and activates HIS4 transcription only in combination with BAS2. Science 246, 931-935.

70. Trick, M., Dennis, E.S., Edwards, K.J. and Peacock, W.J. (1988). Molecular analysis of the alcohol dehydrogenase gene family of barley. Plant Mol. Biol. 11, 147-160.

71. Urao, T. , Yamaguchi-Shinozaki, K., Urao, S. and Shinozaki, K. (1993). An Arabidopsis Myb homolog is induced be dehydration stress and its gene product binds to the conserved Myb recognition sequence. Plant Cell 5, 1529-1539.

72. Urao, T. , Noji, M. , Yamaguchi-Shinozaki, K. and Shinozaki, K. (1996). A transcriptional activation domain of ATMYB2, a drought-inducible Arabidopsis Myb- related protein. Plant J. 10, 1145-1148.

73. Valvekens, D., Van Montagu, M. and Van Lijsebettens, M. (1988). Agrobacterium tumefaciens- mediated transformation Arabidopsis thaliana root explants by using kanamycin selection. Proc. Natl. Acad. Sci. USA 85, 5536-5540.

74. Vilaine, F. and Casse-Delbart, F. (1987). Independent induction of transformed roots by the TL and TR regions of the Ri plasmid of agropine type Agrobacterium rhozogenes. Mol. Gen. Genet. 206, 17-23.

75. Walker, J.C. , Howard, E.A., Dennis, E.S. and Peacock, W.J. (1987) DNA sequences required for anaerobic expression of the maize ADHl gene. Proc. Natl. Acad. Sci. USA, 84, 6624-6629.

76. Wang, Z-Y, Kenigsbuch, D., Sun, L., Harel, E. , Ong, M.S. and Tobin, E.M. (1997). A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene. Plant Cell 9, 491-507.

77. Werr, W. , Frommer, W-B, Maas, C. and Starlinger, P. (1985). Structure of the sucrose synthase gene on chromosome 9 of Zea mays L. EMBO J 4, 1373-1380.

78. Weston, K. (1992). Extension of the DNA binding domain consensus of the chicken c-Myb and v-Myb proteins. Nucl. Ac. Res. 20, 3043-3049.

79. Wissenbach, M., Uberlacker, B., Vogt, F., Becker, D., Salamini, F. and Rohde, W. (1993) myb genes from Hordeum vulgare: tissue-specific expression of chimeric Myb promoter/Gus genes in transgenic tobacco. Plant J. 4, 411-422.

80. Wolyn, D.J. and Jelenkovic, G. (1990). Nucleotide sequence of an alcohol dehydrogenase gene in octaploid strawberry (Fragaria XAnanassa Duch.). Plant Mol. Biol. 14, 855-857.

81. Xie, Y. and Wu, R. (1990). Molecular analysis of an alcohol dehydrogenase- encoding genomic clone (adhl) from rice. Gene 87, 185-191.

82. Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453.

SEQUENCE LISTING

<110> COMMONWEALTH SCIENTIFIC AND INDUSTRIAL RESEARCH ORGANISATION

<120> METHOD OF REGULATING GENE EXPRESSION

<130> 194353 -PCT/MRO

<140> PCT/AU98/00000

<141> 1998-09-25

<150> P09479

<151> 1997-09-26

<160> 4

<170> Patentln Ver. 2.0

<210> 1

<211> 2521

<212> DNA

<213> Arabidopsis thaliana

<220>

<221> exon <222> (983) .. (1139)

<220>

<221> exon

<222> (1231) (1360)

<220>

<221> exon

<222> (1462) .. (1996)

<400> 1

tcccaacgta cgataaaatt ttgtttgata aaactcacaa aagtttagta tatttctgta 60

aatatatgca tagaagtcat agtgattgac agaggaaaga cgtctctttg actactcttc 120

aatcgaaaac aaaatgtgct aaaatttcaa aagcaaagtc cctaaactcg cctaactcca 180

tttctcattt gactttgcct taaatgtcaa tagtcctatg ttatttagct acattcaaaa 240

ttgggaatta agaattattt tagactccat atacttagaa acgaatccgt attggaaact 300

aatttccatt tagtaatagt atttaagtat tattatgctt accgagtagc atgtagtagt 360

ttcttaatgt tagctaattt caaagcaaac gcgaatatta aatcgtgatt gcacacaaca 420

tgaagtgatt aataaatcat taatttcgcc catagtaatc actaaccaga gtaattttga 480

acaagactaa ggatgtacaa taacttacgt ctgcgatact gtgtgggaat aaatatagaa 540 actttatgaa atagaactct cttaccaaag aaagaaaaag caagagtgcg tgttactata 600

catctgaaca ggtcaaaggg tcaaacctta gtattttaaa attacacaaa caattgacca 660

aatggagagc taattatgtt tagcataatt tactatatag ttgcaaaatt ccactttaga 720

aacaggataa aaaaataaaa attgaacata acacttatgc cgtgttccag tcactcttca 780

acattctact cattttcaaa tatccttttt ataaaatact acttttaaaa ttatttgatt 840

cttatgttcc ttcactatat atatataaat atcgcacatt gctttaatca cttcaaatct 900

aatccacaaa accattcaca ccatctcatc ttctttctct ctatctcttt tcctttcctc 960

atttaaagtt tctttataag aa atg gaa gat tac gag cga ata aac tea aac 1012

tct cca aca cat gaa gaa gat tct gat gta egg aaa ggt cca tgg ace 1060

gag gaa gaa gat gca ate eta gtc aac ttc gtc tct att cat ggc gat 1108

get cgt tgg aac cac ate get cgt tec tct ggt ateatcaaaa cateaatcte 1161

taaatataca tatacacaaa ggctttatat atagataaac ttatatatat tttactcata 1221

tatgtgcag ggc taa age gaa ctg gta aga gtt gta gat taa gat ggc tta 1272

att act tac gtc cag atg tta gaa gag gca aca tea etc teg aag aac 1320

aat tta tga tec tea aac tec att etc ttt ggg gca ata ggt 1362

acacactata tatctcaaat gttaatattt tgctattcta tatgttacta gttaagtatg 1422

ttattaatat atgtcttttt tttcaattga atatataag gtg gtc gaa gat tgc 1476

gca ata tct ace ggg aag aac aga taa tga aat aaa gaa tta ttg gag 1524

aac teg agt cca aaa gca age caa aca cct aag atg cga tgt taa cag 1572

taa tct ttt caa gga gac tat gag aaa tgt ttg gat gee gag att agt 1620 gga acg aat caa cgc cca ate att ace cac cac gtg tga aca agt gga 1668

gtc aat gat cac cga ccc aag tea ace agt taa cga ace gag tec ggt 1716

cga gec ggg ttt cgt tea att cag cca gaa tea tea tea gca att cgt 1764

ace ggc tac gga att gtc age aac gtc ttc gaa ttc tec ggc tga gac 1812

gtt ttc gga cgt teg agg tgg ggt ggt gaa egg gtc agg tta tga tec 1860

gtc ggg tea aac ggg ttt egg aga gtt caa cga ttg ggg ctg tgt tgg 1908

tgg gga caa cat gtg gac tga cga gga gag ttt ttg gtt ctt gca gga 1956

cca gtt ctg ccc cga tac gac ate gta ttc gta taa tta agg 1998

aaatatacga ttactatacg taacgaggaa ttcaattgcg tcacgtttgg tgtaatattc 2058

attegtgegt gatgecaatt ttagatacgg cettggtata cgaatctttg acttaattat 2118

tatcttttct tttcctctct tgttttaaac ccctgattaa attaagattt gatcatcaga 2178

cgaggatatt tgtgattcac tgatttgtga tattgatata tgtgaattat ttgatataac 2238

gttttaaaaa ceaacaaaaa aaaaaaatca ttecaaggaa aagttcttaa ttttgatact 2298

cgaaaagagc gtagactgac tcgaatcagt tcatattttc tttggttcgt tttatttacg 2358

acaaaattca ctaacaaaaa ttaaaaaacg acaaaacgaa aatatgacta aatttatttt 2418

tttgtcagtt aaccactgat tataggttga aattgtcaca acacatgatt tatcttgata 2478

gaaatttagt agtccagaat gctgcatggt tgatcctaag aaa 2521

<210> 2 <211> 273

<212 > PRT

<213 > Arabidopsis thaliana

<400> 2

Met Glu Asp Tyr Glu Arg lie Asn Ser Asn Ser Pro Thr His Glu Glu 1 5 10 15

Asp Ser Asp Val Arg Lys Gly Pro Trp Thr Glu Glu Glu Asp Ala lie 20 25 30

Leu Val Asn Phe Val Ser lie His Gly Asp Ala Arg Trp Asn His lie 35 40 45

Ala Arg Ser Ser Gly Leu Lys Arg Thr Gly Lys Ser Cys Arg Leu Arg 50 55 60

Trp Leu Asn Tyr Leu Arg Pro Asp Val Arg Arg Gly Asn lie Thr Leu 65 70 75 80

Glu Glu Gin Phe Met He Leu Lys Leu His Ser Leu Trp Gly Asn Arg 85 90 95

Trp Ser Lys He Ala Gin Tyr Leu Pro Gly Arg Thr Asp Asn Glu He 100 105 110

Lys Asn Tyr Trp Arg Thr Arg Val Gin Lys Gin Ala Lys His Leu Arg 115 120 125 Cys Asp Val Asn Ser Asn Leu Phe Lys Glu Thr Met Arg Asn Val Trp 130 135 140

Met Pro Arg Leu Val Glu Arg He Asn Ala Gin Ser Leu Pro Thr Thr 145 150 155 160

Cys Glu Gin Val Glu Ser Met He Thr Asp Pro Ser Gin Pro Val Asn 165 170 175

Glu Pro Ser Pro Val Glu Pro Gly Phe Val Gin Phe Ser Gin Asn Hiε 180 185 190

His Gin Gin Phe Val Pro Ala Thr Glu Leu Ser Ala Thr Ser Ser Asn 195 200 205

Ser Pro Ala Glu Thr Phe Ser Asp Val Arg Gly Gly Val Val Asn Gly 210 215 220

Ser Gly Tyr Asp Pro Ser Gly Gin Thr Gly Phe Gly Glu Phe Asn Asp 225 230 235 240

Trp Gly Cys Val Gly Gly Asp Asn Met Trp Thr Asp Glu Glu Ser Phe 245 250 255

Trp Phe Leu Gin Asp Gin Phe Cys Pro Asp Thr Thr Ser Tyr Ser Tyr 260 265 270

Asn

<210> 3

<211> 837

<212> DNA

<213> Arabidopsis thaliana

<220>

<221> CDS

<222> (10) .. (828)

<400> 3

ggateegaa atg gaa gat tac gag cga ata aac tea aac tct cca aca cat 51 Met Glu Asp Tyr Glu Arg He Asn Ser Asn Ser Pro Thr His

1 5 10

gaa gaa gat tct gat gta egg aaa ggt cca tgg ace gag gaa gaa gat 99 Glu Glu Asp Ser Asp Val Arg Lys Gly Pro Trp Thr Glu Glu Glu Asp 15 20 25 30

gca ate eta gtc aac ttc gtc tct att cat ggc gat get cgt tgg aac 147 Ala He Leu Val Asn Phe Val Ser He His Gly Asp Ala Arg Trp Asn 35 40 45 cac ate get cgt tec tct ggg eta aag cga act ggt aag agt tgt aga 195 His He Ala Arg Ser Ser Gly Leu Lys Arg Thr Gly Lys Ser Cys Arg 50 55 60

tta aga tgg ctt aat tac tta cgt cca gat gtt aga aga ggc aac ate 243 Leu Arg Trp Leu Asn Tyr Leu Arg Pro Asp Val Arg Arg Gly Asn He 65 70 75

act etc gaa gaa caa ttt atg ate etc aaa etc cat tct ctt tgg ggc 291 Thr Leu Glu Glu Gin Phe Met He Leu Lys Leu His Ser Leu Trp Gly 80 85 90

aat agg tgg teg aag att gcg caa tat eta ccg gga aga aca gat aat 339 Asn Arg Trp Ser Lys He Ala Gin Tyr Leu Pro Gly Arg Thr Asp Asn 95 100 105 110

gaa ata aag aat tat tgg aga act cga gtc caa aag caa gec aaa cac 387 Glu He Lys Asn Tyr Trp Arg Thr Arg Val Gin Lys Gin Ala Lys His 115 120 125

eta aga tgc gat gtt aac agt aat ctt ttc aag gag act atg aga aat 435 Leu Arg Cys Asp Val Asn Ser Asn Leu Phe Lys Glu Thr Met Arg Asn 130 135 140

gtt tgg atg ccg aga tta gtg gaa cga ate aac gee caa tea tta ccc 483 Val Trp Met Pro Arg Leu Val Glu Arg He Asn Ala Gin Ser Leu Pro 145 150 155

ace acg tgt gaa caa gtg gag tea atg ate ace gac cca agt caa cca 531 Thr Thr Cys Glu Gin Val Glu Ser Met He Thr Asp Pro Ser Gin Pro 160 165 170 gtt aac gaa ccg agt ccg gtc gag ccg ggt ttc gtt caa ttc age cag 579

Val Asn Glu Pro Ser Pro Val Glu Pro Gly Phe Val Gin Phe Ser Gin 175 180 185 190

aat cat cat cag caa ttc gta ccg get acg gaa ttg tea gca acg tct 627 Asn His His Gin Gin Phe Val Pro Ala Thr Glu Leu Ser Ala Thr Ser 195 200 205

teg aat tct ccg get gag acg ttt teg gac gtt cga ggt ggg gtg gtg 675 Ser Asn Ser Pro Ala Glu Thr Phe Ser Asp Val Arg Gly Gly Val Val 210 215 220

aac ggg tea ggt tat gat ccg teg ggt caa acg ggt ttc gga gag ttc 723 Asn Gly Ser Gly Tyr Asp Pro Ser Gly Gin Thr Gly Phe Gly Glu Phe 225 230 235

aac gat tgg ggc tgt gtt ggt ggg gac aac atg tgg act gac gag gag 771 Asn Asp Trp Gly Cys Val Gly Gly Asp Asn Met Trp Thr Asp Glu Glu 240 245 250

agt ttt tgg ttc ttg cag gac cag ttc tgc ccc gat acg aca teg tat 819 Ser Phe Trp Phe Leu Gin Asp Gin Phe Cys Pro Asp Thr Thr Ser Tyr 255 260 265 270

teg tat aat taaggatcc 837 Ser Tyr Asn

<210> 4 <211> 273 <212> PRT <213> Arabidopsis thaliana

<400> 4

Met Glu Asp Tyr Glu Arg He Asn Ser Asn Ser Pro Thr His Glu Glu 1 5 10 15

Asp Ser Asp Val Arg Lys Gly Pro Trp Thr Glu Glu Glu Asp Ala He 20 25 30

Leu Val Asn Phe Val Ser He His Gly Asp Ala Arg Trp Asn His He 35 40 45

Ala Arg Ser Ser Gly Leu Lys Arg Thr Gly Lys Ser Cys Arg Leu Arg 50 55 60

Trp Leu Asn Tyr Leu Arg Pro Asp Val Arg Arg Gly Asn He Thr Leu 65 70 75 80

Glu Glu Gin Phe Met He Leu Lys Leu His Ser Leu Trp Gly Asn Arg

85 90 95

Trp Ser Lys He Ala Gin Tyr Leu Pro Gly Arg Thr Asp Asn Glu He 100 105 110

Lys Asn Tyr Trp Arg Thr Arg Val Gin Lys Gin Ala Lys His Leu Arg 115 120 125

Cys Asp Val Asn Ser Asn Leu Phe Lys Glu Thr Met Arg Asn Val Trp 130 135 140

Met Pro Arg Leu Val Glu Arg He Asn Ala Gin Ser Leu Pro Thr Thr 5 145 150 155 160

Cys Glu Gin Val Glu Ser Met He Thr Asp Pro Ser Gin Pro Val Asn 165 170 175

10

Glu Pro Ser Pro Val Glu Pro Gly Phe Val Gin Phe Ser Gin Asn His 180 185 190

15

His Gin Gin Phe Val Pro Ala Thr Glu Leu Ser Ala Thr Ser Ser Asn 195 200 205

20 Ser Pro Ala Glu Thr Phe Ser Asp Val Arg Gly Gly Val Val Asn Gly 210 215 220

Ser Gly Tyr Asp Pro Ser Gly Gin Thr Gly Phe Gly Glu Phe Asn Asp 25 225 230 235 240

Trp Gly Cys Val Gly Gly Asp Asn Met Trp Thr Asp Glu Glu Ser Phe 30 245 250 255

Trp Phe Leu Gin Asp Gin Phe Cys Pro Asp Thr Thr Ser Tyr Ser Tyr 260 265 270

35

Asn

Claims

CLAIMS:

1. A method of modifying the expression of a stress-related gene in a plant cell, tissue or organ or a whole plant, said method at least comprising the step of expressing therein a MYB2 protein or a functionally-equivalent homologue, analogue or derivative thereof for a time and under conditions sufficient for expression of said stress-related gene to be increased, reduced or otherwise altered.

2. The method according to claim 1, wherein the expression of the stress-related gene is increased by expression of the MYB2 protein.

3. The method according to claims 1 or 2, wherein the MYB2 protein comprises an amino acid sequence set forth in <400>2 or <400>4 or a functionally-equivalent homologue, analogue or derivative thereof which is at least about 70% identical thereto.

4. The method according to any one of claims 1 to 3, wherein the stress-related gene comprises a promoter having at least one copy of a cw-acting regulatory nucleotide sequence designated "MBS-1 motif" as hereinbefore defined or a complementary nucleotide sequence thereto.

5. The method according to any one of claims 1 to 4, wherein the stress-related gene comprises a promoter having at least one copy of a cw-acting regulatory nucleotide sequence designated "GC-motif" as hereinbefore defined or a complementary nucleotide sequence thereto.

6. The method according to any one of claims 1 to 5, wherein the stress-related gene comprises a promoter having at least one copy of a m-acting regulatory nucleotide sequence designated "G-box-1 " as hereinbefore defined or a complementary nucleotide sequence thereto.

7. The method according to any one of claims 1 to 6, wherein the stress-related gene comprises a promoter having at least one copy of a czs-acting regulatory nucleotide sequence designated "G-box-2" as hereinbefore defined or a complementary nucleotide sequence thereto.

8. The method according to any one of claims 4 to 7, wherein the stress-related gene encodes an enzyme selected from the list comprising alcohol dehydrogenase, aldolase, glyceraldehyde phosphate dehydrogenase, pyruvate decarboxylase, sucrose synthase and lactate dehydrogenase.

9. The method according to claim 8, wherein the stress-related gene encodes alcohol dehydrogenase.

10. The method according to any one of claims 1 to 3, wherein the stress-related gene is induced in response to anaerobic stress.

11. The method according to claim 10, wherein the stress-related gene encodes alcohol dehydrogenase.

12. The method according to claim 10, wherein the stress-related gene encodes pyruvate decarboxylase.

13. The method according to claim 10, wherein the stress-related gene encodes an enzyme selected from the list comprising sucrose synthase, phosphoglucomutase, phosphoglucose isomerase, fructose- 1,6-diphosphate aldolase, glyceraldehyde-3 -phosphate dehydrogenase, phosphogly cerate mutase, enolase and alanine amino transferase.

14. The method according to any one of claims 1 to 3, wherein the stress-related gene is induced in response to dehydration or water loss or reduced turgor in the plant.

15. The method according to claim 14, wherein the stress-related gene encodes alcohol dehydrogenase.

16. The method according to any one of claims 1 to 3, wherein the stress-related gene is induced in response to cold stress.

17. The method according to claim 16, wherein the stress-related gene encodes alcohol dehydrogenase.

18. The method according to any one of claims 1 to 3, wherein the stress-related gene is induced by an environmental stress selected from the list comprising flooding stress, heat stress, drought stress and salt stress.

19. The method according to any one of claims 1 to 3, wherein the stress-related gene is induced by the application of abscisic acid.

20. The method according to claim 19, wherein the stress-related gene encodes alcohol dehydrogenase.

21. The method according to any one of claims 1 to 20 comprising the additional first step of introducing into the plant cell, tissue, organ or whole plant an isolated nucleic acid molecule which encodes said MYB2 protein or functionally-equivalent homologue, analogue or derivative thereof.

22. The method according to claim 21 wherein the isolated nucleic acid molecule comprises the nucleotide sequence set forth in <400 > 1 or <400 > 3 or a homologue, analogue or derivative that is at least about 75% identical thereto or a complementary nucleotide sequence thereof.

23. The method according to claims 21 or 22, wherein the isolated nucleotide sequence is operably linked to a promoter sequence that is operable in the plant cell, tissue, organ or whole plant.

24. The method according to claim 23 wherein the promoter is the CaMV 35S promoter sequence.

25. The method according to claim 23 wherein the promoter is a glucocorticoid-inducible promoter sequence.

26. A genetic construct which comprises a nucleotide sequence set forth in <400> 1 or <400> 3 or a homologue, analogue or derivative thereof which encodes a functional MYB2 polypeptide and is at least about 75% identical to said nucleotide sequence, wherein said genetic construct expresses a MYB2 protein in a plant cell, tissue, organ or whole plant to modify the expression of a stress-related gene therein.

27. The genetic construct according to claim 26, wherein the nucleotide sequence is placed operably under the control of a promoter sequence that is operable in the plant cell, tissue, organ or whole plant.

28. The genetic construct according to claim 27 wherein the promoter is the CaMV 35S promoter sequence.

29. The genetic construct according to claim 27 wherein the promoter is a glucocorticoid- inducible promoter sequence.

30. A method of producing a stress-tolerant plant comprising the steps of transforming plant tissue or cells with the genetic construct according to any one of claims 26 to 29, regenerating said tissue or cells into a whole plant and expressing the MYB2 protein encoded by said genetic construct therein for a time and under conditions sufficient for expression of a stress-related gene in said plant to be increased, reduced or otherwise altered.

31. A method of producing a stress-tolerant plant comprising at least the step of expressing a MYB2 protein or a homologue, analogue or derivative thereof in said plant or a cell, tissue or organ thereof for a time and under conditions sufficient for expression of a stress-related gene in said plant to be increased, reduced or otherwise altered.

32. The method according to claims 30 or 31 wherein the stress-related gene in said plant is increased.

33. The method according to any one of claims 30 to 32, wherein the MYB2 protein comprises an amino acid sequence set forth in <400 >2 or <400 >4 or a functionally- equivalent homologue, analogue or derivative thereof which is at least about 70% identical thereto.

34. The method according to any one of claims 30 to 33, wherein the stress-related gene comprises a promoter having at least one copy of a cw-acting regulatory nucleotide sequence designated "MBS-1 motif" as hereinbefore defined or a complementary nucleotide sequence thereto.

35. The method according to any one of claims 30 to 34, wherein the stress-related gene comprises a promoter having at least one copy of a s-acting regulatory nucleotide sequence designated "GC-motif" as hereinbefore defined or a complementary nucleotide sequence thereto.

36. The method according to any one of claims 30 to 35, wherein the stress-related gene comprises a promoter having at least one copy of a cw-acting regulatory nucleotide sequence designated "G-box-1 " as hereinbefore defined or a complementary nucleotide sequence thereto.

37. The method according to any one of claims 30 to 36, wherein the stress-related gene comprises a promoter having at least one copy of a cw-acting regulatory nucleotide sequence designated "G-box-2" as hereinbefore defmed or a complementary nucleotide sequence thereto.

38. The method according to any one of claims 30 to 33, wherein the stress-related gene encodes an enzyme selected from the list comprising alcohol dehydrogenase, aldolase, pyruvate decarboxylase, sucrose synthase, lactate dehydrogenase, phosphoglucose isomerase, fructose- 1,6-diphosphate aldolase, glyceraldehyde-3 -phosphate dehydrogenase, phosphoglycerate mutase, enolase and alanine amino transferase.

39. The method according to claim 38, wherein the stress-related gene encodes alcohol dehydrogenase.

40. The method according to claim 38, wherein the stress-related gene encodes pyruvate decarboxylase.

41. The method according to any one of claims 30 to 40, wherein the stress to which the plant is made tolerant comprises anaerobic stress.

42. The method according to any one of claims 30 to 40, wherein the stress to which the plant is made tolerant comprises dehydration or water loss or reduced turgor in the plant.

43. The method according to any one of claims 30 to 40, wherein the stress to which the plant is made tolerant comprises cold stress.

44. The method according to any one of claims 30 to 40, wherein the stress to which the plant is made tolerant comprises an environmental stress selected from the list comprising flooding stress, heat stress, drought stress and salt stress.

45. A transgenic plant produced by the method according to any one of claims 30 to 44.

46. A plant having modified expression of a stress-related gene, wherein said plant is produced by the method according to any one of claims 1 to 25.

47. A cell, tissue or organ derived from the plant according to claims 45 or 46, wherein said cell, tissue or organ has modified expression of a stress-related gene.

48. A plant cell, tissue or organ other than a whole plant which exhibits modified expression of a stress-related gene by virtue of the expression therein of a MYB2 protein.