Evidence for a Role for AtMYB2 in the Induction of the Arabidopsis Alcohol Dehydrogenase Gene (ADH1) Low Oxygen

Corresponding author: Rudy Dolferus, C.S.I.R.O. Plant Industry, G.P.O. Box 1600, Canberra ACT 2601, Australia, rudy{at}pican.pi.csiro.au (E-mail).

Communicating editor: E. MEYEROWITZ

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The transcription factor AtMYB2 binds to two sequence motifs in the promoter of the Arabidopsis ADH1 gene. The binding to the GT-motif (5'-TGGTTT-3') is essential for induction of ADH1 by low oxygen, while binding to the second motif, MBS-2, is not essential for induction. We show that AtMYB2 is induced by hypoxia with kinetics compatible with a role in the regulation of ADH1. Like ADH1, AtMYB2 has root-limited expression. When driven by a constitutive promoter, AtMYB2 is able to transactivate ADH1 expression in transient assays in both Arabidopsis and Nicotiana plumbaginifolia protoplasts, and in particle bombardment of Pisum sativum leaves. Mutation of the GT-motif abolished binding of AtMYB2 and caused loss of activity of the ADH1 promoter in both transient assays and transgenic Arabidopsis plants. These results are consistent with AtMYB2 being a key regulatory factor in the induction of the ADH1 promoter by low oxygen.

PLANTS respond to conditions of low oxygen by switching carbohydrate metabolism in root cells from an oxidative to a fermentative pathway. In maize, where the molecular events initiated during low oxygen stress have been studied in most detail, transfer to conditions of low oxygen represses aerobic protein synthesis and, at the same time, initiates the synthesis of two transition polypeptides, with molecular weights of approximately 33 kD. After approximately 90 min, a group of about 20 polypeptides, the anaerobic polypeptides (ANPs) are synthesized (SACHS et al. 1980 ; BAILEY-SERRES and FREELING 1990 ). Most of these ANPs are enzymes involved in ethanolic fermentation (alcohol dehydrogenase, ADH; pyruvate decarboxylase, PDC), or in glycolysis (e.g., fructose 1,6-bisphosphate aldolase, sucrose synthase, glucose-6-phosphate isomerase, enolase, glyceraldehyde-3-phosphate dehydrogenase; for review, see SACHS et al. 1996 ). A number of different maize seedling tissues (roots, coleoptile, mesocotyl, endosperm, scutellum, and anther wall) synthesize the ANPs (OKIMOTO et al. 1980 ). Maize leaves, which have emerged from the coleoptile, do not synthesize the ANPs and do not survive even short periods of anaerobiosis (OKIMOTO et al. 1980 ).

Sequence elements in the promoter of the maize ADH1 gene, which are critical for anaerobic induction, have been identified (WALKER et al. 1987 ; OLIVE et al. 1990 , OLIVE et al. 1991A , OLIVE et al. 1991B ). 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 ); both GC-elements are required for expression of Adh1. 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 ; PAUL and FERL 1997 ), suggesting proteins bind to these motifs. No GT-binding protein has been identified in maize.

Arabidopsis has a similar anaerobic response to maize (DOLFERUS et al. 1985 ). Arabidopsis ADH1 is induced by hypoxic conditions and by a number of other environmental stimuli (low temperature, dehydration) and by the phytohormone ABA (DOLFERUS et al. 1994 ; DE BRUXELLES et al. 1996 ). The Arabidopsis ADH1 promoter contains sequences similar to the maize Adh1 ARE between -160 and -140, with the GT-motif in the opposite orientation relative to the maize GT-motifs (GT-motif: 5'-AAACCAA-3'; GC-motif: 5'-GCCCC-3'). The GT- and GC-motifs are both necessary for low oxygen induction (DOLFERUS et al. 1994 ).

The Arabidopsis ADH1 GT-motif contains a potential Myb binding site. Myb transcription factors bind to a consensus sequence with an AAC central motif (5'-T/CAAC[T/G]G-3'; or 5'-CC[T/A]ACC-3'; LUSCHER and EISENMAN 1990 ; GROTEWOLD et al. 1991 ). This prompted us to investigate the involvement of Myb-related transcription factors in the low oxygen induction of the Arabidopsis ADH1 gene. One candidate Myb was AtMYB2, reported by URAO et al. 1993 to be induced by dehydration, salt stress, and exogenous abscisic acid (ABA). AtMYB2 was able to transactivate a promoter containing multimers of the Myb binding site consensus sequence (5'-TAACTG-3'; URAO et al. 1996 ). Recently it was demonstrated that AtMYB2 binds to a Myb recognition site in the Arabidopsis dehydration-responsive gene rd22 (ABE et al. 1997 ).

In this paper we present evidence that AtMYB2 is rapidly induced by low oxygen conditions, and that it binds to the GT-motif in the ADH1 promoter. In transient assays AtMYB2 activates expression of an ADH1-GUS construct, and this transactivation does not occur when the GT-motif is mutated.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant material, growth conditions, and stress treatments:
Arabidopsis thaliana seeds, ecotypes C24 or Columbia (Co-0), used in this study were grown on Murashige and Skoog (MS) medium at 22° (16/8 hr light/dark cycle, 200 µE/sec/cm²). 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% O₂/95% N₂ gas mixture (hypoxic conditions; HOWARD et al. 1987 ), for up to 24 hr at 22° in the dark. Dehydration treatment was carried out by incubating the plantlets in medium containing 0.6 M mannitol, for up to 24 hr at 22°. For cold stress treatment, plantlets were incubated at 4–5° for up to 24 hr. ABA ((±) cis-trans isomers, Sigma, St. Louis) was added to the medium at a final concentration of 0.1 mM for 4 hr. For treatments with the protein synthesis inhibitor cycloheximide, plant material was first preincubated in MS medium containing 10 µM cycloheximide for 1 hr. The solution containing cycloheximide was refreshed before the stress treatment.

Recombinant DNA techniques:
All cloning methods were according to standard procedures (MANIATIS et al. 1982 ; SAMBROOK et al. 1989 ). Plasmid pGEX-RAtmyb 2BE contains the AtMYB2 cDNA fused to the glutathione-S-transferase coding region in plasmid pGEX2T (URAO et al. 1993 ). The plasmid containing the GST-GAMYB fusion protein and the GAMYB probe oligo used in gel retardation were described by GUBLER et al. 1995 . The AtMYB2 fusion protein was purified using the Pharmacia (Piscataway, NJ) GST purification module and used in EMSA (electrophoretic mobility shift assay) experiments. Complementary oligonucleotide probes used in EMSAs (see Figure 1) were annealed, end-labeled using Klenow DNA polymerase, and then gel-purified. Binding reactions (20 µl) contained 1 µl recombinant AtMYB2 (about 50 ng protein), 2 µl 10x 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]dI-dC (1 µg/µl), 15 µl H₂O, and 1 µl labeled 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. Salmon sperm DNA was used as a nonspecific competitor (25 ng per binding reaction; sheared by sonication). Samples were loaded onto a 5% polyacrylamide gel in 0.5x TBE (MANIATIS et al. 1982 ).

View larger version (34K): In this window In a new window Download PPT slide   Figure 1. —(A) Functional organization of the Arabidopsis ADH1 promoter compared to the maize Adh1 promoter. Arrows indicate the orientation of the GT- and GC-motifs in both promoters and the orientation of the arrows shows the orientation of the motifs with respect to the maize ADH1 promoter. The position of the two potential Myb binding sites in Arabidopsis ADH1 (MBS-1 and MBS-2) is indicated. (B) Sequences of the MBS-1 and MBS-2 oligos, as well as substitution mutant oligos for both sequences. Nucleotides which are in underlined italics are mutated compared to the wild-type sequence. Double stranded equivalents of these oligos were used in gel retardation experiments. GT, GC, Gbox-1 and Gbox-2 are the sequences of the substitution mutants used in the original ADH1 promoter mapping (DOLFERUS et al. 1994 ). GBS, GAMYB binding site (GUBLER et al. 1995 ).

A full-length AtMYB2 cDNA, flanked by BamHI sites was obtained using RT-PCR of anaerobically induced root RNA. The resulting cDNA was cloned between the 35S promoter and the 3' NOS terminator sequence of plasmid pART7 (GLEAVE 1992 ). The resulting plasmid p35S-CAtMYB2 was verified by sequencing and used as effector plasmid in all transient assays. Reporter plasmids pADH1-GUS, containing the ADH promoter from position -964 to +53, p35S-GUS, p[GBox-1]ADH-GUS, p[GBox-2]ADH-GUS, p[GT]ADH-GUS, and p[GC]ADH-GUS were described earlier (DOLFERUS et al. 1994 ; Figure 1B). p[MBS-2]ADH-GUS, containing substitution mutations in the MBS-2 site, was constructed by amplifying fragments overlapping the MBS2 site (5'-TAGCAACGCC-3') and transforming this site into a NotI restriction site (5'-GCGGCCGCAT-3'). The full-length ADH1 promoter was reconstructed, and the mutated promoter was cloned into plasmid pADH1-GUS to replace the wild-type promoter. The construct was subcloned in binary vector pBIN19 (BEVAN 1984 ) for transformation to Arabidopsis. Binary vectors were mobilized to Agrobacterium strain AGL1 (LAZO et al. 1991 ) by electroporation (NAGEL et al. 1990 ).

RNA extractions, Northern and Southern blot analysis:
RNA extraction, gel electrophoresis, Northern blot hybridizations using antisense RNA probes, and filter washing procedures were as described previously (DOLFERUS et al. 1994 ). Filters were placed on phosphor imager screens (Molecular Dynamics, Sunnyvale, CA) and the hybridization signals quantified. 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. AtMYB2 RNA probes were prepared from a clone containing the full-length cDNA. ADH1 probes were transcribed from a clone containing the entire coding region of the gene. Sucrose synthase RNA probes were prepared from a clone containing the coding region of the Arabidopsis ASUS1 gene (MARTIN et al. 1993 ). The PDC1 probe was prepared from a clone containing the entire coding region of the Arabidopsis PDC1 gene (DOLFERUS, PEACOCK and DENNIS, unpublished results). Quantitative RT-PCR was carried out using 1 µg 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 hybridization, and filters were hybridized using the AtMYB2 cDNA. Linearity of signal strength was verified using phosphorimager quantifications. Oligos were used slightly overlapping the 5' end and 3' end of the first and second intron positions of the AtMYB2 gene respectively. These oligos were shown not to amplify genomic DNA as template.

Tissue culture, protoplast transient assays, particle bombardment, and Agrobacterium transformation:
Arabidopsis root cultures were established by placing 1-month-old leaf cuttings (ecotype C24) on callus-induction medium (VALVEKENS et al. 1988 ) for 3 days, prior to infection with Agrobacterium rhizogenes (strain A4RS; VILAINE and CASSE-DELBART 1987 ). The leaf disks were cocultivated for 3 days on callus-induction medium, washed in a 200 mg/ml timentin solution (Smithkline Beecham, Dandenong, Australia), and placed on solid MS medium including 100 mg/ml timentin. After 3–4 wk the hairy root explants were transferred to liquid MS medium, and refreshed monthly. Arabidopsis mesophyll protoplasts were prepared from ecotype Co-0, 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 x 10^-6 protoplasts and 15–20 µg reporter plasmid DNA, plus or minus the same amount of effector plasmid (p35S-CAtMYB2). Nicotiana plumbaginifolia suspension cells were maintained and protoplasts were prepared according to NEGRUTIU et al. (1981), using the media described by KAO and MICHAYLUK 1975 . Plasmid DNA was introduced using the PEG method for both Arabidopsis and N. plumbaginifolia protoplasts (ABEL and THEOLOGIS 1994 ).

Particle bombardment of pea leaves was carried out using a homemade helium gun. A total of 25 µl of particles (100 mg/ml in 50% glycerol; 50:50 mixture of tungsten and gold particles) was mixed with 2–6 µg plasmid DNA (1 µg/µl), 25 µl 2.5 M CaCl₂, 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 one leaf. Reporter and effector plasmids were used in a 1:1 ratio. Leaves were incubated on MS plates for 16 hr before GUS staining.

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 (Richmond, CA) protein assay kit (BRADFORD 1976 ). Fluorometric and protein assays were carried out in microwell plates, and analyzed using Labsystems (Marlboro, MA) Fluoroskan II and Multiskan Plus readers, in conjunction with Delta Soft II software (Biometallics Inc., Princeton, NJ; BREYNE et al. 1993 ). For in vivo GUS assays, the staining solution was vacuum infiltrated into the plant tissue. Plant material was fixed in 70% ethanol after GUS staining.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The Arabidopsis ADH1 promoter has binding sites for AtMYB2:
The Arabidopsis ADH1 promoter contains two potential Myb Binding Sites (MBS) in the 200 bp immediately upstream of the start of transcription (Figure 1A). The more distal MBS-2 at -189 to -187 lies in the footprinted region containing G-box-2 (FERL and LAUGHNER 1989 ; DOLFERUS et al. 1994 ). The second motif, MBS-1 at -150 to -148, coincides with the GT-motif (Figure 1A) necessary for mediating the ADH1 low oxygen response (DOLFERUS et al. 1994 ; DE BRUXELLES et al. 1996 ). The MBS-2 motif resembles the classical vertebrate Myb binding sequence (LUSCHER and EISENMAN 1990 ; WESTON 1992 ), whereas MBS-1 (within the GT-motif) shows more homology to the maize P-Myb binding site (GROTEWOLD et al. 1994 ; Figure 1A). The possibility arises that AtMYB2 binds to one or both of the two putative Myb binding sequences of the ADH1 promoter (Figure 1A).

AtMYB2 was expressed in Escherichia coli as a glutathione-S-transferase fusion protein (AtMYB2-GST) and affinity purified. The fusion protein was used in EMSAs with oligonucleotides corresponding to the MBS-1 and MBS-2 motifs (Figure 1B). The recombinant AtMYB2 protein retarded both classes of oligonucleotides (Figure 2A). Binding was competed by unlabeled homologues, but not by a nonspecific competitor (salmon sperm DNA; Figure 2A). Binding to the monomers was competed out by 100–200-fold molar excess of either of the MBS-1 or MBS-2 oligos. Multimerization of the MBS-1 oligo gave significantly stronger binding than the monomer (Figure 2A), a 500-fold molar excess excluding all binding to the tetramer; at this level, some degree of competition was also observed with the nonspecific competitor (salmon sperm DNA; Figure 2A). AtMYB2-GST did not bind to other motifs of the ADH1 promoter (G-box-1 or GC-motif sequences; data not shown). Another plant Myb transcription factor, GAMYB (GUBLER et al. 1995 ), which binds to a GARE sequence (GA response element; 5'-TAACAAA-3') of the gibberellic acid inducible -amylase promoter, did not interact with either MBS-1 or MBS-2 when expressed as a GAMyb-GST fusion protein (Figure 2A).

View larger version (71K): In this window In a new window Download PPT slide   Figure 2. —Gel retardation results, showing binding of purified GST-AtMYB2 fusion protein to 32P-labeled MBS-1 and MBS-2 oligos (see Figure 1B). Numbers on the top of each figure indicate fold molar excess of cold competitor. FP indicates lanes with free probes (no protein added), and NS indicates lanes where nonspecific salmon sperm competitor DNA was used. (A) The left panel shows binding of AtMYB2 to both MBS-1 and MBS-2 sequences. Binding is reduced by cold competitor DNA of the same sequence as the labeled probe, but not by the nonspecific competitor. Middle panel: Binding of AtMYB2 to multimerized (4 x MBS-1) oligos. Higher molar excess of cold competitor is required to eliminate binding. Right panel: GAMYB does not bind to MBS-1 or MBS-2, but shows strong binding to the GAMYB Binding Site (GBS). (B) Binding of AtMYB2 to MBS-1 and MBS-2 can not be competed by competitor oligos with a mutated AAC core sequence (MBS-1/1 and MBS-2 respectively). Mutation of the AAC core to GAC (MBS-1/2 oligo) showed weak competition for binding to labeled wild-type MBS-1 oligo.

AtMYB2 binding to MBS-1 and MBS-2 requires the AAC-core:
The AAC-core sequence of MBS-1 and MBS-2 was mutated to CCC (MBS-1/1 and MBS-2) or GAC (MBS-1/2; Figure 1B). Similar mutations in vertebrate and plant Myb factors abolished binding (WESTON 1992 ; GUBLER et al. 1995 ). The CCC core sequence (MBS-1/1 and MBS-2) did not compete for binding to wild-type MBS-1 and MBS-2 probe sequence (Figure 2B); the GAC core (MBS-1/2) had much reduced ability to compete for binding to wild-type MBS-1 (Figure 2B). The EMSA results indicate that AtMYB2 binding requires the AAC-core sequence of both MBS-1 and MBS-2. The fact that GAMYB did not interact with MBS-1 and MBS-2 further suggests that both motifs are specific interaction sites for AtMYB2.

AtMYB2 expression is induced by low oxygen stress in roots:
We found that AtMYB2 mRNA levels were increased significantly by low oxygen treatment, with higher induction in roots than leaves (Figure 3A). There was an average of 5.6-fold induction, with root expression levels about seven times higher than in shoots (Figure 3B). Expression peaks within 4 hr, declines by 6–8 hr, and increases again (Figure 3A). The timing of AtMYB2 induction by hypoxia was compared to that of ADH1, using RNA extracted from Arabidopsis root cultures (Figure 3C). Induction of ADH1 mRNA is tightly coupled to the first rise (2–4 hr) in AtMYB2 mRNA (Figure 3C). Peak ADH1 levels were obtained between 4 and 6 hr, followed by a decline and a second increase reaching maximal expression after 24 hr, mirroring a second rise in AtMYB2 mRNA levels.

View larger version (32K): In this window In a new window Download PPT slide   Figure 3. —Northern blot hybridization results showing the AtMYB2 expression pattern under different imposed stresses as described in MATERIALS AND METHODS. Ubiquitin was used as a control to standardize expression levels. (A) Northern blot showing kinetics of AtMYB2 mRNA accumulation during low oxygen stress treatment. Accumulation of mRNA is preferentially in the roots of the plant (L, leaves; R, roots) over the 8 hours of treatment. (B) Induction of AtMYB2 and ADH1 mRNA in shoots and roots by stress treatments. C, control; AN, low oxygen treatment (24 hr); D, dehydration; CD, low temperature; ABA, ABA treatment. AtMYB2 is induced by all stress treatments, and expression is higher in roots () than in shoots (). Error bars represent standard errors for three repeats. (C) Induction kinetics of AtMYB2 mRNA (), compared to ADH1 mRNA accumulation (), in Arabidopsis root cultures. Results are expressed as a percentage of maximum mRNA induction obtained for each treatment (24 hr).

The induction of AtMYB2 is also coordinated with the induction of other anaerobically induced Arabidopsis genes, such as the pyruvate decarboxylase (PDC1; DOLFERUS, PEACOCK and DENNIS, unpublished results) and sucrose synthase genes (ASUS1; MARTIN et al. 1993 ). All these genes contain GT-motifs which are potential binding sites for AtMYB2 (Table 1), and they display similar induction kinetics following low oxygen treatment, with peak expression levels found immediately after AtMYB2 mRNA levels have reached a maximum (Figure 4). These experiments indicate that the timing of AtMYB2 mRNA accumulation is tightly coupled to expression of anaerobically induced genes, supporting a role for AtMYB2 in the induction of anaerobic proteins.

View larger version (21K): In this window In a new window Download PPT slide   Figure 4. —Induction kinetics of AtMYB2 mRNA under low oxygen conditions over 24 hr, compared to the kinetics of induction of ADH1 and two other anaerobically induced genes: PDC1, Arabidopsis pyruvate decarboxylase; ASUS1, Arabidopsis sucrose synthase. The expression of these genes is root-specific (data not shown). Results are expressed as a percentage of the maximum induction of mRNA obtained for each treatment over 24 hr.

Induction of AtMYB2 by other stresses correlates with ADH1 induction:
Maximal induction of ADH1 occurs after 8–10 hr of dehydration stress, 20–24 hr of low temperature, and 4 hr of ABA treatment (DOLFERUS et al. 1994 ; DE BRUXELLES et al. 1996 ). AtMYB2 is induced by all these treatments (URAO et al. 1993 ; Figure 3B). Low temperature stress, like hypoxic stress, shows root-limited AtMYB2 induction. Dehydration and ABA treatment induce AtMYB2 in both leaves and roots, even though ADH1 is induced predominantly in roots by these treatments (Figure 3B).

AtMYB2 mRNA accumulates following dehydration with kinetics similar to those of ADH1 mRNA, with two peaks (4 and 10 hr) in both (Figure 3C). We also found two peaks of ABA induction of AtMYB2 (2 and 10 hr; 24- and 15-fold induction respectively), and of ADH1 (peaks at 4 and 24 hr; Figure 3C). Low temperature treatment resulted in transient AtMYB2 mRNA accumulation between 2 and 6 hr (5-fold induction), with induction of ADH1 mRNA reaching a peak level between 12 and 24 hr. These data suggest that AtMYB2 expression is correlated both temporally and spatially with ADH1 expression. The Arabidopsis rab18 gene is strongly induced by dehydration and ABA in both leaves and roots (LANG and PALVA 1992 ), but not by low oxygen stress (data not shown). rab18 has a G-box-like element but no GT-motif, and induction kinetics following dehydration and especially ABA treatment are considerably slower than AtMYB2 and ADH1 (data not shown), suggesting this gene is regulated by a different set of factors (data not shown).

AtMYB2 does transactivate ADH1:
To investigate whether AtMYB2 could transactivate ADH1 in the absence of hypoxia, an ADH1-promoter-GUS reporter construct (ADH1-GUS; DOLFERUS et al. 1994 ) was cointroduced with a 35S-promoter-AtMYB2 construct as effector plasmid (p35S-CAtMYB2). We first used biolistics, with pea leaves as target tissue, because of the availability of this system. The ADH1-GUS reporter plasmid showed increased intensity and size of the blue spots only when the effector construct was present (Figure 5).

View larger version (73K): In this window In a new window Download PPT slide   Figure 5. —Particle bombardment using 35S-GUS, ADH1-GUS, and 35S-AtMYB2 plasmids and pea leaves. Samples which were cobombarded on three separate occasions by ADH1-GUS and 35S-AtMYB2 show larger spot size than samples which were bombarded with the ADH1-GUS construct only. Representative leaves are shown for each experiment and construct used.

For quantitative data, we carried out transient assays in Arabidopsis mesophyll protoplasts. AtMYB2 transactivated ADH1 promoter activity, increasing expression by a factor of 2–2.5-fold (Figure 6A). A greater stimulation (2.5–4.5-fold) was observed in N. plumbaginifolia suspension cell protoplasts (Figure 6A). Transactivation was low when lower amounts of effector plasmid compared to the reporter plasmid were used (data not shown).

View larger version (35K): In this window In a new window Download PPT slide   Figure 6. —Transient assays showing AtMYB2 transactivation of an ADH1-GUS construct in Arabidopsis mesophyll and N. plumbaginifolia suspension protoplasts. , -35S-ATMYB2; , +35S-AtMYB2. All transient assays were repeated at least three times, each assay containing a repetition of each transformation. Although expression levels varied between different protoplast isolations, fold-induction values were very reproducible. Data shown are average fold-inductions over six repeats (standard errors shown as error bars). (A) Transient assays using Arabidopsis 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 transactivations of about 2.5–4.5-fold were consistently observed. (B) Transient assays using the substitution mutant constructs used to map the ADH1 promoter elements (DOLFERUS et al. 1994 ; Figure 1B). AtMYB2 is not able to transactivate ADH1-GUS expression when the GT-motif (MBS-1) is mutated, but transactivation is unaffected when MBS-2 (p[MBS-2]ADH-GUS) is mutated and is even increased in N. plumbaginifolia. Transactivation potential is reduced for the GC-motif, the G-box-1, and G-box-2 mutants.

AtMYB2 transactivates the ADH1 promoter via the GT-motif (MBS-1):
The presence of a second potential AtMYB2 binding site, MBS-2, in the ADH1 promoter suggested the promoter may resemble the maize Adh1 promoter in having two functionally important GT-motifs (Figure 1). MBS-2 is in an in vivo footprinted segment. The area previously mutagenized (G-box-2) did not affect ADH1 expression (DOLFERUS et al. 1994 ). We mutagenized the MBS-2 region (5'-TAGCAACGCC-3'), replacing the core AAC with CCG (5'-GCGGCCGCAT-3'; p(MBS-2)ADH1-GUS). Mutation of all the bases of the AAC core eliminates binding to AtMYB2 in EMSA assays (Figure 2B). Mutations of MBS-1 did abolish transactivation in Arabidopsis mesophyll protoplasts (Figure 6B). In contrast, we found that MBS-2 mutations increased transactivation by about 1.5-fold over wild-type levels in N. plumbaginifolia protoplasts. This could indicate that different factors interact with the ADH1 promoter in suspension cells compared to the mesophyll protoplast system, or that different factors interact with the ADH1 promoter in N. plumbaginifolia. Alternatively, mutation of MBS-2 could make more AtMYB2 factor available for binding to MBS-1.

Mutation of the GC-motif (Figure 1B; DOLFERUS et al. 1994 ) also reduced AtMYB2 transactivation of ADH1-GUS expression in both Arabidopsis mesophyll protoplasts and in N. plumbaginifolia suspension cell protoplasts (Figure 6B). These results indicate that anaerobic induction of the ADH1 promoter requires not only AtMYB2 and the GT-motif, but also a factor binding to the GC-motif. Transactivation levels of G-Box-1 mutants (Figure 1B) were reduced in both Arabidopsis mesophyll and N. plumbaginifolia suspension protoplasts (Figure 6B), suggesting that the G-box binding factor may play a role in effective binding of AtMYB2. In contrast, G-Box-2 mutants (Figure 1B) in either Arabidopsis and N. plumbaginifolia protoplasts did not affect transactivation potential.

In transgenic plants, ADH1-GUS expression was decreased dramatically when mutations were introduced into the GT+GC motifs (DOLFERUS et al. 1994 ). Gbox1 and Gbox2 mutations had expression levels similar to the wild type construct.

Cycloheximide inhibits ADH1 induction but increases AtMYB2 expression:
If AtMYB2 accumulation is necessary for ADH1 expression then induction of ADH1 mRNA would require protein synthesis. Figure 7A shows cycloheximide prevents accumulation of ADH1 mRNA following inductive conditions, indicating that protein synthesis is required. In contrast, cycloheximide caused a 2–9-fold increase in AtMYB2 mRNA levels for all treatments (Figure 7B). It is not clear whether this effect is at the transcriptional or post-transcriptional level, but the results do show that AtMYB2 mRNA can be induced without prior de novo protein synthesis.

View larger version (34K): In this window In a new window Download PPT slide   Figure 7. —Effect of cycloheximide on AtMYB2 and ADH1 mRNA expression levels. , control treatments without cycloheximide; , treatments in the presence of 10 µmol cycloheximide. The scale of the Y-axis is empirical and shows relative mRNA expression levels as measured using the phosphorimager (signal strength divided by signal strength of ubiquitin mRNA expression levels). Ubiquitin mRNA expression levels were not significantly affected by the treatments (data not shown). (A) Northern blot hybridization results showing the inhibition of ADH1 mRNA accumulation by cycloheximide in four-week-old Arabidopsis plants treated with different stresses. Abbreviations as in Figure 3. (B) RT-PCR combined with Southern blot hybridization was used to study the effect of cycloheximide on AtMYB2 expression during stress and ABA treatment. RNA from dehydration (D) and low temperature (CD) treated roots was extracted at the two time-points showing maximal induction (see Figure 3C).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Our results suggest that AtMYB2 is a key transcription factor in stress-induced ADH1 gene expression. AtMYB2 binds to two sites in the Arabidopsis ADH1 promoter, the MBS-1 and MBS-2 motifs. The binding is specific to AtMYB2; neither of two other plant Myb factors, GAMYB (GUBLER et al. 1995 ) or AtMYB1 (URAO et al. 1993 ) binds to the motifs. Mutations in MBS-1 eliminate both binding of AtMYB2 and ADH1 expression, indicating that the binding is critical for ADH1 expression. Mutation of MBS-2 reduces AtMYB2 binding but does not alter ADH1 expression (Figure 6), showing that binding of AtMYB2 to MBS-2 does not make any functional contribution to ADH1 expression.

AtMYB2 has tissue and temporal expression patterns compatible with the proposed role as key regulator of ADH1 transcription. The tissue-specificity of AtMYB2-GUS expression (URAO et al. 1993 ) is similar to the pattern observed for ADH1-GUS constructs (DOLFERUS et al. 1994 ). AtMYB2 mRNA begins to accumulate soon after the initiation of low oxygen treatment, preceding ADH1 mRNA accumulation. Other anaerobically induced genes such as PDC1 and sucrose synthase (ASus1) show a similar temporal relationship to AtMYB2 induction (Figure 4). AtMYB2 may well be a key transcription factor in the regulation of ADH1 during other environmental stresses, since a similar relationship exists between the induction of AtMYB2 and ADH1 mRNA for low temperature stress, dehydration and exogenous application of ABA. This suggestion is consistent with our previous findings that the GT-motif is necessary for all these responses (DOLFERUS et al. 1994 ; DE BRUXELLES et al. 1996 ).

The low temperature, dehydration and ABA responses also require the G-box-1 sequence (DOLFERUS et al. 1994 ; DE BRUXELLES et al. 1996 ), suggesting that these responses involve a transcription factor which binds to the G-Box-1. AtMYB2 appears to be a transcription factor needed in all stress responses, but interacts with other factors which may differ with the different stress conditions.

Our finding that the induction of transcription and subsequent translation of ADH1 by low oxygen is sensitive to cycloheximide implies that protein synthesis is required for the operation of this response. On the other hand, cycloheximide does not inhibit AtMYB2 induction, but actually increases it. We conclude that induction of AtMYB2 may be the initial response, and that its synthesis is required for the induction of ADH1, and probably for the other anaerobic polypeptides. Transition proteins are synthesized before the induction of the anaerobic proteins (SACHS et al. 1980 ), and their molecular weight (33 kD) is similar to the mass of AtMYB2 (27.5 kD). It is possible that AtMYB2 is one of the transition proteins. Independence to cycloheximide may be a feature of transcription factors involved in switching on a coordinate response, and other transcription factors and signal transduction components, such as the maize cold-inducible leucine-zipper transcription factor mLip15, the calcium-dependent protein kinase ZmCDPK1 (BERBERICH and KUSANO 1997 ), and HVA22 (SHEN et al. 1993 ) show cycloheximide insensitivity. Neither the GT- nor the GC-motifs are found in the promoter of the AtMYB2 gene, suggesting that AtMYB2 expression is not subject to autoregulation, again suggesting that AtMYB2 is the initial step in the response.

The transient expression experiment with cotransfection of 35S-AtMYB2 and ADH1-GUS also showed that AtMYB2 is a key transcription factor for the ADH1 promoter. While transactivation levels in protoplasts (2–3-fold) were lower than induction levels observed in roots following low oxygen treatment (5–10-fold at protein level; 20–50-fold at mRNA level), they were of the same magnitude as those observed in N. plumbaginifolia suspension protoplasts following low oxygen treatment (LLEWELLYN et al. 1987 ).

In plants, AtMYB2 expression under low oxygen conditions is confined to the roots. Following dehydration stress or ABA treatment AtMYB2 mRNA is induced both in leaves and roots, as is ADH1 (Figure 3B), paralleling the increase in ABA levels in these two tissues (DE BRUXELLES et al. 1996 ). ADH1 expression remains root-specific, which suggests that another transcription factor needed for ADH1 expression is not present in the leaves. In the ABA response of ADH1 the factor binding to the G-Box-1 may interact with AtMYB2. In the barley HVA22 promoter, the ABA response is dependent on the G-Box and other motifs (SHEN et al. 1993 , SHEN et al. 1996 ; SHEN and HO 1995 ). Recently, AtMYB2 was proposed to act in conjunction with a Myc-related transcription factor in the drought- and ABA-regulated rd22 gene (ABE et al. 1997 ). ADH1 does not have an obvious Myc recognition site, suggesting that in this promoter AtMYB2 must interact with other classes of transcription factors.

Our mutation analysis has shown that AtMYB2 probably also requires association with the protein binding to the GC-motif (Figure 6; possibly the Arabidopsis homologue of GCBP-1; OLIVE et al. 1991B ). In the maize Adh1 promoter both GT-motifs are closely linked to the GC-motifs. In Arabidopsis the lack of a GC-motif close to the MBS-2 site could explain why this site is not critical for ADH1 induction. Vertebrate and yeast Myb transcription factors commonly activate transcription in close association with other factors and work in a synergistic manner with these factors (TICE-BALDWIN et al. 1989 ; BURK et al. 1993 ). In plants, the maize C1 Myb interacts directly with the B protein (basic helix-loop-helix factor) on the maize Bronze-1 promoter and activates transcription in a cooperative way (GOFF et al. 1992 ).

The GT-motif is present in all anaerobically induced genes (Table 1), and is usually located between positions -300 and -100 relative to the start of transcription. The consensus sequence is 5'-AAACCA-3'. Depending on whether or not a GC-motif is next to the GT-motif, the consensus sequence can be extended to 5'-AAACCAAA-3' or 5'-AAACCG[G/C][G/C]-3' respectively (Table 1). The core AtMYB2 recognition sequence in the rd22 promoter (5'-TAACCA-3') is similar to the GT-motif (ABE et al. 1997 ; Table 1). The preference for a 5'-[A]AACC[A]-3' core binding site differentiates AtMYB2 from other known plant Mybs (Table 2). The second AtMYB2 binding site in the ADH1 promoter (MBS-2; 5'-CAACGCC-3') is quite different from the GT-motif (MBS-1), and could explain why binding of AtMYB2 to MBS-2 is not functional.

	ACKNOWLEDGMENTS

The authors wish to thank S. STOPS for excellent technical assistance, K. SHINOZAKI for providing us with the AtMYB2-GST clone, L. WILLMITZER for providing us with the Arabidopsis ASUS1 probe, P. LARKIN for providing us with the N. plumbaginifolia suspension cell protoplasts, and F. GUBLER for many helpful discussions throughout the work. F. HOEREN was supported by grants from the Australian Research Council (ARC), Alexander von Humboldt, and the Deutsche Forschungs Gemeinschaft (DFG grant HO 1824).

Manuscript received January 6, 1998; Accepted for publication March 23, 1998.

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