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First published online April 25, 2008; 10.1104/pp.108.116897 Plant Physiology 147:528-542 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Functional Identification of Arabidopsis Stress Regulatory Genes Using the Controlled cDNA Overexpression System1,[W],[OA]Institute of Plant Biology, Biological Research Centre, 6726–Szeged, Hungary (C.P., E.A., M.P.J., C.P., C.K., L.S.); and Max-Planck-Institut für Züchtungsforschung, D–50829 Cologne, Germany (C.K.)
Responses to environmental stresses in higher plants are controlled by a complex web of abscisic acid (ABA)-dependent and independent signaling pathways. To perform genetic screens for identification of novel Arabidopsis (Arabidopsis thaliana) loci involved in the control of abiotic stress responses, a complementary DNA (cDNA) expression library was created in a Gateway version of estradiol-inducible XVE binary vector (controlled cDNA overexpression system [COS]). The COS system was tested in three genetic screens by selecting for ABA insensitivity, salt tolerance, and activation of a stress-responsive ADH1-LUC (alcohol dehydrogenase-luciferase) reporter gene. Twenty-seven cDNAs conferring dominant, estradiol-dependent stress tolerance phenotype, were identified by polymerase chain reaction amplification and sequence analysis. Several cDNAs were recloned into the XVE vector and transformed recurrently into Arabidopsis, to confirm that the observed conditional phenotypes were due to their estradiol-dependent expression. Characterization of a cDNA conferring insensitivity to ABA in germination assays has identified the coding region of heat shock protein HSP17.6A suggesting its implication in ABA signal transduction. Screening for enhanced salt tolerance in germination and seedling growth assays revealed that estradiol-controlled overexpression of a 2-alkenal reductase cDNA confers considerable level of salt insensitivity. Screening for transcriptional activation of stress- and ABA-inducible ADH1-LUC reporter gene has identified the ERF/AP2-type transcription factor RAP2.12, which sustained high-level ADH1-LUC bioluminescence, enhanced ADH1 transcription rate, and increased ADH enzyme activity in the presence of estradiol. These data illustrate that application of the COS cDNA expression library provides an efficient strategy for genetic identification and characterization of novel regulators of abiotic stress responses.
Adverse environmental conditions, such as drought, low temperature, and high soil salinity are among the most challenging factors for plant growth and survival. Adaptation to abiotic stress requires coordinate changes in metabolism, cell growth, division, and differentiation, which depend on a large set of genes controlling complex regulatory mechanisms. Cloning of genes whose expression is up-regulated by salt, cold, or drought stress identified several targets and regulators of stress signaling (Serrano and Gaxiola, 1994  ; Ingram and Bartels, 1996; Hasegawa et al., 2000). However, genetic approaches are better suited for the identification of regulatory genes than differential display techniques. In fact, the majority of gene functions controlling responses to high salinity, drought, and cold were discovered using forward genetic screens of mutagenized Arabidopsis (Arabidopsis thaliana) populations. For example, uncovering the SOS (salt overly sensitive) signaling pathway, which controls NaCl homeostasis and salt tolerance (Zhu, 2002), was aided by the isolation of salt hypersensitive mutants assisting the characterization of key determinants of salt tolerance, including the SOS1 Na+/H+ antiporter (Shi et al., 2000), the SnRK3 kinase SOS2 (Guo et al., 2001), and the SOS2-interacting EF-hand Ca2+ binding protein SOS3 (Halfter et al., 2000). Similarly, forward genetic approaches unraveled important components of drought, cold, and abscisic acid (ABA) signaling pathways (for review, see Thomashow, 1999; Finkelstein et al., 2002; Yamaguchi-Shinozaki and Shinozaki, 2006).
Genetic screens for deregulated expression of stress-responsive reporter gene constructs, such as RD29A-LUC, provide an alternative to isolate mutations in regulatory genes that either activate or repress the activity of the reporter (Xiong et al., 1999
Whereas chemical mutagenesis followed by map-based cloning provides a strategy for identification of weak mutant alleles of essential regulatory genes, wide-scale exploitation of T-DNA and transposon insertion mutagenesis facilitates the isolation of loss-of-function mutations affecting various stress responses (Koiwa et al., 2006
Expression of complementary DNA (cDNA) libraries in plants provides an additional strategy to screen for gain of function phenotypes. This strategy is comparable to the so-called multicopy suppressor screen, which was first invented in yeast to identify functions suppressing salt sensitivity (Bender and Pringle, 1991
The cDNA library transformation approach may also produce dominant loss of function phenotypes, which result from cosuppression of endogenous genes by overexpression of truncated or antisense cDNAs (LeClere and Bartel, 2001
Construction and Testing of the COS
An Arabidopsis cDNA library was constructed in the pDONR201 vector using a SuperSMART cDNA synthesis system (Clontech; Supplemental Fig. S1) in combination with the Gateway cloning technology and RNA templates from different Arabidopsis organs, dark-grown, green, and salt-stressed seedlings, and cultured cells (Supplemental Table S1). The cDNA library was subsequently transferred into pER8GW, a Gateway version of estradiol-inducible expression vector pER8 (Zuo et al., 2000
To test the utility of the COS system, three screening strategies were employed by selecting for transformants showing ABA insensitivity in germination screens, salt tolerance in seedling growth assays, and activation of a stress-inducible ADH1-LUC reporter gene in seedlings (Fig. 2 ). Twenty-thousand to 40,000 transgenic seeds and seedlings were screened in each of these assays using estradiol in the growth medium for transient induction of cDNA expression. Upon selection, the transgenic plants were transferred into estradiol-free medium and then into soil to set seed. The segregation of selected phenotype in the T2 offspring was recurrently assayed by germinating and growing seedlings both in the presence and absence of estradiol, and testing for cosegregation of estradiol-induced conditional phenotype with the hygromycin resistance marker of the pER8GW T-DNA insert. Subsequently, cDNA inserts present in the selected Arabidopsis lines were isolated by PCR amplification from genomic DNA templates using the ER8A and ER8B primers that anneal to the pER8 vector T-DNA sequences flanking the attB recombination sites (Supplemental Fig. S2; Supplemental Table S2). Subsequently, the isolated cDNAs were sequenced and characterized by performing BLAST homology searches with the Arabidopsis sequence database (www.Arabidopsis.org). As each cDNA was flanked by attB1 and attB2 recombination sites, their PCR fragments could easily be recloned in the Gateway entry vector pDONR201 and subsequently into pER8GW for recurrent transformation of Arabidopsis to confirm the phenotype conferred by their estradiol-inducible expression (Supplemental Fig. S2). Using the Gateway technology, the rescue, identification, and subsequent confirmatory recloning of cDNAs required less than 2 weeks providing a high-throughput technology for functional analysis.
Identification of Factors Affecting ABA Sensitivity of Seed Germination To identify cDNAs that confer ABA-insensitive seed germination when expressed conditionally during estradiol induction, one million of the T1 seeds containing about 20,000 transformants were plated on half-strength Murashige and Skoog (MS) agar medium containing 2.5 µM ABA and 4 µM estradiol. Under this selective condition, the germination of wild-type seeds was completely blocked. ABA-insensitive seeds, which germinated within 7 to 10 d producing seedlings with open green cotyledons and emerged radicles, were transferred onto ABA and estradiol free, hygromycin containing medium (Fig. 3A ). Seventy-four ABA-insensitive, hygromycin-resistant plants were identified. T2 progeny of these plants was retested for the ABA insensitive seed germination phenotype in the presence and absence of estradiol along with parallel scoring for single locus segregation of the T-DNA-encoded hygromycin resistance marker. For further analysis, we chose 25 lines, which showed different degrees of estradiol-dependent ABA insensitivity in the germination assay (Fig. 3B). From these, ABA insensitive germination of 19 lines was completely estradiol dependent, whereas six lines displayed some degree of ABA-insensitive germination also in the absence of estradiol. From a subset of selected lines, we have rescued and sequenced 11 cDNAs, and found that they code for proteins that were previously reported to play various roles in different abiotic stress responses, including a glutathione-S-transferase, a SNF1-related kinase regulatory subunit, a lipid transfer protein, a subtilase, and a dehydrin type protein (Table I ; A lines). Although most ABA-insensitive lines carried full-length cDNA inserts, several of them were truncated at the 5' end (Supplemental Fig. S6).
Line A26 carried a full-length cDNA of class II small heat shock protein 17.6A (HSP17.6A, At5g12030) gene, including a 5'-untranslated leader of 53 bp and 3'-UTR sequences of 127 bp (Supplemental Fig. S3). Because HSP17.6A has not been implicated so far in the control of ABA response of seed germination, we have performed further characterization of line A26. In the T2 generation, the conditional ABA insensitivity of line A26 was dominant, and 3:1 segregation of hygromycin-resistant and sensitive offspring indicated that this trait is linked to a single T-DNA insertion. A26 seeds germinated in the presence of 3 µM ABA and 4 µM estradiol, while their ABA sensitivity was similar to wild-type seeds in the absence of estradiol (Fig. 3, C and D). Semiquantitative reverse transcription (RT)-PCR analysis confirmed that HSP17.6A expression in line A26 was indeed induced only by estradiol, but not by ABA (Fig. 3E). As expected, HSP17.6A transcription was activated by heat shock and salt stress in wild-type plants (Fig. 3F). HSP17.6A showed very low expression in most organs except for roots and wilted leaves (Fig. 3G).
ABA sensitivity of seed germination is controlled by the transcription factors ABI3, ABI4, and AB5 (Finkelstein et al., 2002
Identification of a cDNA Conferring Increased Salt Tolerance To screen for enhanced salt tolerance of seed germination and seedling growth, a population of 40,000 transgenic T1 seed was germinated on plates containing hygromycin, and resistant plantlets were transferred to selective half-strength MS agar plates supplemented with 225 mM NaCl and 4 µM estradiol. Alternatively, 20,000 seeds were germinated on half-strength MS agar plates containing 225 mM NaCl and 4 µM estradiol. Under this condition, wild-type seeds either did not germinate or the seedlings died after germination. Lines displaying salt-tolerant germination and subsequent development of green seedlings within 15 to 20 d on the selective medium (Fig. 5A ) were transferred to soil to set seed. Salt tolerance of T2 offspring of selected lines was recurrently tested in germination and growth assays using salt selection in the presence or absence of 4 µM estradiol. Estradiol-dependent conditional salt tolerance characterized by at least 2-fold higher germination rate compared to wild-type seeds was confirmed for 14 lines (Fig. 5B), from which the cDNA inserts were subsequently PCR amplified. Nine from the sequenced cDNAs carried full-length coding regions of a CDK2-related Ser/Thr kinase, a seed storage/lipid transfer protein (LTP), AtSTE24 CAAX protease, AtISU1 (iron-sulfur cluster assembly complex protein), 60S ribosomal protein L27 (RPL27B), and several unknown proteins (Table I; N lines). Five lines carried inserts with 5' truncated cDNAs (Supplemental Fig. S6). From this population, line N180 showing high salt tolerance and 3:1 segregation of hygromycin resistance marker of a single copy pER8GW T-DNA insert was characterized. In the presence of estradiol and 225 mM NaCl the T2 offspring of line N180 germinated at least 2 d earlier than wild-type and estradiol-untreated N180 seeds, and the germination efficiency reached nearly 100% already 10 d after sawing. By contrast, line N180 displayed only the emergence of radicles and similar germination efficiency as control wild-type seed on estradiol-free selective medium containing 225 mM NaCl (Fig. 5, C and E). When 5-d-old seedlings were germinated on half-strength MS medium and were transferred to medium containing 175 mM NaCl, N180 plantlets continued to grow only in the presence of estradiol (Fig. 5D). From line N180 a single cDNA insert was PCR amplified and proved to carry the full-length coding region of the At5g16970 2-alkenal reductase enzyme (2AER, EC 1.3.1.74; Supplemental Fig. S4). Compared to wild-type and estradiol-untreated N180 seedlings, semiquantitative RT-PCR analysis indicated that the transcription of 2AER is estradiol induced and correlates with conditional salt tolerance of line N180 (Fig. 6A ). However, upon increasing the sensitivity of RT-PCR detection (i.e. higher cycle number) transcription of the 2AER gene could also be detected in all tested organs with highest abundance in cauline and rosette leaves, wilted leaves, and developed siliques (Fig. 6B).
Oxidative stress imposed by 4 µM paraquat treatment for 3 and 8 h on 14-d-old, in vitro germinated wild-type seedlings (plantlets with four leaves), as well as treatment of similar seedlings with 200 mM NaCl for 3 and 8 h, resulted in moderate increase of 2AER transcript levels (Fig. 6C). Similarly to our results, inspection of public transcript profiling data (http://www.genevestigator.ethz.ch) indicated that transcription of the 2AER At5g16970 gene is up-regulated by hydrogen peroxide, senescence, and wounding.
Screening for ABA insensitivity and salt tolerance has also resulted in the identification of several lines carrying truncated cDNA inserts (Table I). As in all other cases examined, the estradiol-dependent ABA insensitivity and salt tolerance phenotypes of four of these lines were repeatedly tested in three independent experiments. Sequence analysis showed that the identified cDNA inserts contained in frame ATG codons for potential translation of N-terminally truncated proteins carrying some functionally important regulatory domains (Supplemental Fig. S6). However, in the absence of suitable antibodies against these proteins, it remained an open question whether the observed estradiol-dependent dominant stress tolerance phenotypes resulted from overproduction of truncated proteins, or from dominant cosuppression mediated by the corresponding truncated transcripts as suggested by LeClere and Bartel (2001)
To test the applicability of COS technology in screening for factors that confer trans-activation of a stress-regulated promoter, we have constructed an ADH1-LUC luciferase reporter gene, the activation of which can be monitored using nondestructive low-light imaging (Alvarado et al., 2004
Upon transformation of ADH1-LUC reporter line with the COS cDNA library, we screened 20,000 hygromycin-resistant T1 seedlings and identified 11 plants displaying enhanced LUC activity in the presence of 4 µM estradiol (Fig. 8A ). Recurrent assays confirmed estradiol-dependent LUC activation in the T2 progeny of two lines (Table I; ADH lines), whereas all other candidates found in the primary screen showed constitutive estradiol-independent LUC activity. One of the two estradiol-inducible lines, ADH121, showed 3:1 segregation of hygromycin-resistant versus sensitive progeny, and inducible expression of the ADH1-LUC reporter in all hygromycin-resistant T2 seedlings.
Whereas estradiol did not induce the ADH1-LUC reporter in the parental control plants, the ADH121 line showed gradually increasing bioluminescence after estradiol treatment (Fig. 8, B and C). In the absence of estradiol, transfer of the parental ADH1-LUC and ADH121 seedlings to medium containing 200 mM NaCl led to transient increase of bioluminescence within 3 to 4 h followed by gradual decrease of ADH1-LUC expression (Fig. 8, B and C). Whereas the parental ADH1-LUC seedlings showed a similar pattern of luciferase expression also in response to combined treatment with both estradiol and 200 mM NaCl, the COS cDNA transformed ADH121 line displayed persistent maintenance of high level luciferase expression for at least 16 h (Fig. 8, B and C). Similarly to salt treatment, spraying of ADH121 plantlets with 50 µM ABA in the absence of estradiol yielded a transient luminescence peak within 4 h in leaves and roots followed by a gradual decline of LUC activity. This reflected normal ABA-mediated activation of the ADH1-LUC reporter. By contrast, the parental ADH-LUC line showed no response to estradiol induction, whereas treatment of ADH121 seedlings with 4 µM estradiol triggered gradual increase and long-term maintenance of LUC activity in roots but not in leaves. Combined ABA and estradiol treatment of line ADH121 resulted in sustained LUC activation without apparent decline of light emission for at least 24 h (Fig. 8D). These results indicated that activation of a cDNA encoded function conferred root-specific activation of the ADH1-LUC reporter in the ADH121 line, which was superimposed onto the ABA- and salt-induced activation pattern of ADH-LUC (i.e. leaves and roots) in plants subjected to combined treatment with estradiol and ABA or salt. To compare the induction of the endogenous ADH1 gene with activation of the ADH1-LUC reporter, we have monitored the ABA- and estradiol-induced changes in ADH1 mRNA levels by quantitative RT-PCR in the ADH121 line. In seedlings harvested 6 h after treatment with 50 µM ABA a 3-fold increase of ADH1 transcript levels was observed, but following 24 h the ADH1 mRNA levels declined and were comparable to those of untreated control plants. Upon estradiol treatment, the endogenous ADH1 transcript level was increased 1.8- to 2-fold and also remained at the same level 24 h following the treatment. This indicated that estradiol-induced expression of a cDNA in line ADH121 conferred limited activation of the endogenous ADH1 gene. Combination of estradiol with ABA treatment resulted in a 3-fold increase of ADH1 mRNA levels as seen upon ABA induction, but the transcript levels failed to decline even 24 h after the induction (i.e. due to synergistic effect of estradiol-induced cDNA overexpression; Fig. 8E).
To confirm that estradiol-mediated induction of a cDNA construct in ADH121 also leads to root-specific activation of endogenous ADH1 gene (i.e. as seen for estradiol-induced activation of the ADH-LUC reported in the ADH121 line in Fig. 8D), we have compared the ADH enzyme activities in roots of parental ADH1-LUC and cDNA transformed ADH121 lines using a histochemical assay (Baud and Graham, 2006
PCR amplification and sequence analysis revealed that a single pER8GW T-DNA insert in the ADH121 line carried a full-length cDNA of the At1g53910 gene encoding RAP2.12, a yet uncharacterized member of the AP2/ERF (ethylene responsive element binding factor) transcription factor family (Supplemental Fig. S5). AP2/ERF-like transcription factors carry one or two AP2-type DNA binding domains and are represented by 122 genes in Arabidopsis (Nakano et al., 2006
To determine whether RAP2.12 expression correlates with the induction of the ADH1-LUC reporter, we monitored the RAP2.12 mRNA levels in the parental ADH1-LUC and pER8-cDNA transformed ADH121 lines by RT-PCR. High levels of RAP2.12 transcript was exclusively detected in estradiol-treated ADH121 seedlings, whereas in the absence of estradiol or in the presence of ABA only very low levels of RAP2.12 RNA could be detected in ADH121 seedlings and the parental ADH1-LUC line (Fig. 9B). This data thus showed that RAP2.12 transcription was not induced by ABA and could only be activated by estradiol treatment in ADH121. At higher sensitivity, the RAP2.12 transcript was detected in all organs of wild-type plants, showing the highest levels in roots, flower buds, and stems. The fact that the RAP2.12 mRNA level was somewhat higher in leaves of wilted than well-watered plants suggested possible drought regulation of RAP2.12 (Fig. 9C). These results well agreed with the available transcript profiling data (http://www.genevestigator.ethz.ch) indicating that transcription of the RAP2.12 (At1g53910) gene is not affected by ABA, ethylene, and other plant hormones, and is only slightly up-regulated by senescence and osmotic stress. To confirm that estradiol-inducible activation of RAP2.12 transcription was indeed responsible for activation of the ADH1-LUC reporter in the ADH121 line, we have recloned the isolated cDNA into the pDONR201 plasmid, and upon moving it into the pER8GW vector (Supplemental Fig. S2) we introduced it by transformation into the parental ADH1-LUC line again. Most of the ADH1-LUC transformants (10 out of 13) carrying the pER8GW-RAP2.12 construct showed estradiol-inducible luciferase activity, which was similar to the activity of the ADH121 line described above (Fig. 8G). In conclusion, this reconstruction experiment confirmed that RAP2.12 is a positive regulator of the ADH1 gene, which is known to be induced in Arabidopsis by anoxia, high salinity, cold, and drought stress.
The COS offers a simple and powerful technology to screen for gene functions implicated in the regulation of specific stress responses. The COS cDNA library was prepared in a chemically inducible expression vector using the Gateway technology, which offers precise transcriptional control and easy recloning of the cDNA inserts. The rationale of opting for an inducible system was that constitutive overexpression of cDNAs encoding regulatory factors in stress signaling was observed to result frequently in severe developmental deficiencies. For example, constitutive overexpression of DREB1-type transcription factors resulted in growth retardation, abnormal development, late flowering, and reduced fertility (Liu et al., 1998 ; Kasuga et al., 1999; Gilmour et al., 2000). The estradiol-inducible XVE/pER8 expression vector (Zuo et al., 2000) was therefore chosen for construction of the COS library to secure conditional and controlled expression of cDNAs to avoid potentially deleterious effects of their overexpression.
The COS system was tested in several screening strategies, each of them aiming at a particular aspect of a stress response. Screening for salt tolerance at seedling level permitted the identification of genes which, upon overexpression, could enhance the germination rate or increase the survival of seedlings in saline environment. As an example, line N180 was characterized to show that overexpression of 2AER cDNA confers salt tolerance to transgenic plants. Previous studies document that the 2AER enzyme has a NADPH-dependent oxidoreductase activity, which probably plays a role in the detoxification of reactive carbonyls, and hence in the protection of cells against oxidative stress (Mano et al., 2005
Screening for ABA insensitive germination aimed at the identification of novel negative regulators of ABA signaling. Isolation of numerous lines displaying estradiol-dependent ABA insensitivity indicates that the COS technology could also effectively support this screening strategy. As an example, we showed that regulated overexpression of small heat shock protein gene AtHSP17.6A in line A26 conferred conditional ABA insensitivity, pointing to a novel function of this gene. It is well documented that expression of small heat shock proteins is induced by high temperature but some of them, including AtHSP17.6A, are also produced in developing seeds and in response to water stress (Vierling, 1991
The application of luciferase reporter gene constructs driven by different stress-induced promoters facilitates nondestructive detection of gene activation in mutant screens, as well as the identification of transcription factors controlling the expression of a particular target gene. Expression of the alcohol dehydrogenase gene ADH1 is controlled by multiple regulatory pathways, including ABA and ethylene signaling (Jarillo et al., 1993 The above-described examples illustrate the COS library transformation method facilitates high-throughput screening for phenotypes conferred by inducible overexpression of Arabidopsis transcripts in an Arabidopsis genetic background. Although the COS approach utilizes artificial overexpression of mRNAs in analogy to multicopy suppressor screens, in the example of the ADH1-luc reporter activation experiment we illustrated that RAP2.12 can also control the expression of the native endogeneous ADH1 gene. In any case, further analysis of functions identified by the COS approach should be supported logically by a series of confirmatory studies using e.g. insertion mutations, inducible artificial RNAi, microarray transcript profiling, and chromatin immunoprecipitation to thoroughly characterize and validate the newly identified regulatory functions. It is also evident that application of the COS technology is not restricted to intraspecies studies using Arabidopsis as the model but can also be extended to interspecies library screens, in which cDNAs from natural variants of drought-, salt-, or cold-tolerant plant species are tested in Arabidopsis or other model species. This extended COS approach provides the possibility for identification of natural sequence variations in known regulatory genes (i.e. based on cross-species sequence comparisons) that confer either increase or decrease in stress tolerance, or are associated with characteristically altered regulatory functions of signaling factors (i.e. transcription factors, protein kinases, protein phosphatases, etc.) controlling a set of target genes in response to well-defined stress or hormonal stimuli.
Construction of the COS Library
Flow chart of the cDNA library construction is shown in Supplemental Figure S1. The library was constructed from Arabidopsis (Arabidsopsis thaliana; Col-0) RNA samples, which were collected from 10 different tissue sources (Supplemental Table S1). RNA was isolated according to Chomczynski and Sacchi (1987)
Promoter region of the Arabidopsis ADH1 gene (At1g77120) was amplified by PCR using the gene-specific primers ADH-1 and ADH-2 (Supplemental Table S2). The amplified fragment contains the 5' region of the ADH1 gene extending from position –2,385 to –20 upstream of the ATG codon (position +39 downstream of the transcription start; Fig. 7A). Following sequence verification, the amplified promoter fragment was inserted into the HindIII site of the promoter test vector pBinLuc+ (Mullineaux et al., 1990
The pER8GW COS cDNA library was introduced into Arabidopsis (Col-0) by large-scale in planta transformation (Clough and Bent, 1998
cDNAs carried by the pER8GW T-DNA inserts were rescued by PCR amplification using genomic DNA templates prepared from transgenic plants according to Dellaporta et al. (1983)
To monitor estradiol-induced production of cDNA encoded transcripts, either real-time or semiquantitative RT-PCR was performed. Hormone and stress treatments were carried out with 3-week-old plants grown in sterile culture in vitro under short day photoperiod (8-h light/16-h dark) by transferring them into liquid culture medium supplemented by different additives. If not stated otherwise, the following treatments were employed: 20 µM ABA, 200 mM NaCl, 400 mM Suc, 10 mM H2O2, 4 µM paraquat in liquid half-strength MS medium for 3 to 24 h. Control plants were incubated for the same time period in half-strength MS medium. Heat shock was performed at 37°C for 3 h in a humid chamber, while control plants were kept under similar conditions at 22°C for the same time. To induce transcription of the inserted cDNAs, plants were sprayed with 4 µM 17-β-estradiol (prepared in dimethyl sulfoxide [DMSO] as 4 mM stock and then diluted in water; Sigma) and harvested at a defined time point following the treatment. Control plants were sprayed with 0.1% DMSO in water. For comparative analysis of transcript levels in plant tissues and organs, the samples were harvested either at the same time or within the same light period of the day. Leaves were collected from 4-week-old greenhouse-grown plants. Siliques were removed from flowering plants 4 and 10 d after pollination. Wilted leaves were collected from 4-week-old greenhouse-grown plants, which were kept without watering for 5 d. Roots samples were collected from 4-week-old plants grown in the greenhouse.
Total RNA was isolated from plant tissues using the Tri-reagent method (Chomczynski and Sacchi, 1987
Detection of ADH1 enzyme by histochemical staining was performed as described (Baud and Graham, 2006
Sequence homology searches were performed using the TAIR BLAST service (http://www.Arabidopsis.org/Blast/index.jsp). PCR primers were designed with the Primer3 software (http://biotools.umassmed.edu/bioapps/primer3_www.cgi). Multiple sequence alignments were generated using the ClustalW program (http://www.ebi.ac.uk/clustalw/index.html). Protein domain analyses were performed using the SMART service (http://smart.embl-heidelberg.de/). Analysis of publicly available transcript profiling data was performed using the Genevestigator service and database (http://www.genevestigator.ethz.ch/).
The following materials are available in the online version of this article.
The authors thank Annamária Király for her technical assistance, Mihály Dobó for growing the plants, and Imre Sommsich and Bekir Ülker for providing the pER8GW vector. Received January 27, 2008; accepted April 18, 2008; published April 25, 2008.
1 This work was supported by the EU FP5 (no. QLRT–2001–00841), Marie-Curie Action (no. 020232), OTKA grants (nos. K–68226 and F–68598), and joint research project DFG–436UNG–13/172/01 between the Deutsche Forschungsgemeinschaft and the Hungarian Academy of Sciences. 
2 These authors contributed equally to the article.
3 Present address: Department of Biochemistry, University of Szeged, 6726–Szeged, Hungary. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: László Szabados (szabados{at}brc.hu).
[W] The online version of this article contains Web-only data.
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.108.116897 * Corresponding author; e-mail szabados{at}brc.hu.
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