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First published online August 27, 2004; 10.1104/pp.104.046599 Plant Physiology 136:2734-2746 (2004) © 2004 American Society of Plant Biologists Arabidopsis Cys2/His2-Type Zinc-Finger Proteins Function as Transcription Repressors under Drought, Cold, and High-Salinity Stress Conditions1Biological Resources Division, Japan International Research Center for Agricultural Sciences, Tsukuba, Ibaraki 305–8686, Japan (H.S., K.M., Y.S., K.Y.-S.); Department of Botany, Graduate School of Science, Kyoto University, Kyoto 606–8502, Japan (H.S., T.M.); Laboratory of Plant Molecular Biology, Tsukuba Life Science Center, Institute of Physical and Chemical Research, Tsukuba, Ibaraki 305–0074, Japan (K.S.); Research Institute for Biological Sciences, Kayo-cho, Okayama 716–1241, Japan (M.I.); National Institute of Agrobiological Sciences, Tsukuba, Ibaraki 305–8602, Japan (T.M., M.I.); Laboratory of Plant Molecular Physiology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo 113–8657, Japan (K.Y.-S.); and Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi-shi, Saitama 332–0012, Japan (K.S., K.Y.-S.)
ZPT2-related proteins that have two canonical Cys-2/His-2-type zinc-finger motifs in their molecules are members of a family of plant transcription factors. To characterize the role of this type of protein, we analyzed the function of Arabidopsis L. Heynh. genes encoding four different ZPT2-related proteins (AZF1, AZF2, AZF3, and STZ). Gel-shift analysis showed that the AZFs and STZ bind to A(G/C)T repeats within an EP2 sequence, known as a target sequence of some petunia (Petunia hybrida) ZPT2 proteins. Transient expression analysis using synthetic green fluorescent protein fusion genes indicated that the AZFs and STZ are preferentially localized to the nucleus. These four ZPT2-related proteins were shown to act as transcriptional repressors that down-regulate the transactivation activity of other transcription factors. RNA gel-blot analysis showed that expression of AZF2 and STZ was strongly induced by dehydration, high-salt and cold stresses, and abscisic acid treatment. Histochemical analysis of  -glucuronidase activities driven by the AZF2 or STZ promoters revealed that both genes are induced in leaves rather than roots of rosette plants by the stresses. Transgenic Arabidopsis overexpressing STZ showed growth retardation and tolerance to drought stress. These results suggest that AZF2 and STZ function as transcriptional repressors to increase stress tolerance following growth retardation.
Drought, high salinity, and low temperature are adverse environmental conditions that limit the growth of plants. Plants respond and adapt to these stresses in order to survive. These stresses induce various biochemical and physiological changes, including growth inhibition, to acquire stress tolerance. A number of genes have been described that respond to stresses at the transcriptional level (Ingram and Bartels, 1996  ; Bray, 1997; Hasegawa et al., 2000; Zhu, 2002; Shinozaki et al., 2003). Although various genes are induced by these stresses, many stress-down-regulated genes are also reported (Seki et al., 2002). Analysis of stress-down-regulated as well as stress-up-regulated genes is important for understanding molecular responses to abiotic stresses. The plant hormone abscisic acid (ABA) is a key regulator mediating abiotic-stress-signaling pathways. ABA treatment also induces not only stress tolerance but also growth inhibition of plants.
Recently, cis- and trans-acting factors involved in stress-inducible gene expression have been analyzed extensively in Arabidopsis L. Heynh. (Zhu, 2002
We have previously reported a gene family of Cys-2/His-2-type zinc-finger proteins in Arabidopsis (Sakamoto et al., 2000
Plant ZPT-type proteins have one to four fingers each and can be classified according to the number of fingers. Two-fingered protein genes, here called ZPT2-related genes, constitute the major class of the ZPT-type family genes and include 14 ZPT2 genes in petunia (Takatsuji et al., 1992
To elucidate the functions of plant ZPT2-related proteins, we analyzed four Arabidopsis ZPT2-related genes, AZF1, AZF2, AZF3, and STZ, which had been cloned previously using PCR techniques (Sakamoto et al., 2000
Targeting of the AZF and STZ Proteins to the Nucleus
All of the putative AZF1, AZF2, AZF3, and STZ proteins contained a short stretch of basic amino acids near the N terminus. This region is conserved among most ZPT2-related proteins and might function as a potential nuclear localization signal. To investigate the nuclear localization of individual proteins, we performed an in vivo targeting experiment using the synthetic green fluorescent protein (sGFP) gene of the jellyfish Aequorea victoria (Chiu et al., 1996
Specific Binding of AZFs and STZ to the A(G/C)T Repeat within the EP2 Sequence
Since the AZFs and STZ contained the Cys-2/His-2-type zinc-finger motif that has been identified in many transcription factors as a DNA-binding motif, we observed whether they could have DNA-binding ability in vitro by gel-shift assay. We tested the EP2 sequence as a probe because it had already been reported that some of the petunia ZPT2 proteins bind to this probe in a sequence-specific manner (Takatsuji et al., 1992
To identify the target sequences within EP2 of the AZF and STZ proteins, we prepared a series of probes (M1–M6) by substitution of two or three bases at the same position in the tandemly repeated sequences (Fig. 2A). The binding ability of each fusion protein was examined by using these base-substituted probes (Fig. 2B). Each protein bound to M1 and M4 strongly but weakly to M2, M3, M5, and M6. Three petunia ZPT2-related proteins, ZPT2-1, ZPT2-2, and ZPT2-4, bind to two copies of the AGT core sequence separated by 10 bp in the EP2 sequence (Takatsuji et al., 1994 ). By the use of Southwestern assay, the wheat WZF1 protein has been shown to interact with a DNA fragment containing tandem copies of a CACTC sequence (ACT box; Sakamoto et al., 1993). As the ACT box has the AGT core sequence in the reverse orientation, we speculated on the importance of A(G/C)T in EP2 for the binding of the AZF and STZ proteins. The EP2 sequence contains three A(G/C)T sequences, two in the 13-bp EP1S sequence and the other at the junction of the tandem repeats. The M2, M3, M5, and M6 sequences lacked one or two of the three A(G/C)T sequences (Fig. 2A). We prepared some more probes by base substitution to identify which A(G/C)T is necessary in order for the AZFs and STZ to bind to EP2. These probes, M3-1, M3-2, M5-1, and M5-2, had a substitution in one of the two parts of M3 or M5. All four proteins showed similar binding specificities as each other to the base-substituted fragments (Fig. 2B). All bound to the M3-1 and M3-2 probes, in which the first and third AGT, respectively, were disrupted, though the shifted bands were weaker than that of the wild-type EP2 fragment. On the other hand, the proteins did not bind to the M5-1 probe except MBP-AZF1b, in which the second ACT was disrupted, just as they did not bind to M5. The substitution in M5-2, which did not disrupt any A(G/C)T, had little effect on the binding specificities of the proteins. These results indicate that the second ACT is most important for the binding and that the existence of either the first or the third AGT is necessary for the binding of the proteins to EP2. To confirm the importance of the A(G/C)T repeat sequences within EP2 for the binding of the AZFs and STZ, we used the AZF2 fusion protein and analyzed how it interacts with the M3-2 fragment. M3-2 retained the first AGT and second ACT but not the third ACT. M3-2-10 to M3-2-19 are the 1-base-substituted fragments of M3-2. The AGT and ACT repeats within M3-2 are important for AZF2 binding because AZF2 did not bind to M3-2-11, -12, -13, -16, -17, or -18 (Fig. 2C). Flanking sequences around the AGT and ACT are also important to AZF2 binding because the binding to M3-2-10, M3-2-14, and M3-2-15 was reduced in comparison with that to M3-2 (Fig. 2C). We expect that AZF2 interacts with the second ACT and third AGT of M3-1 directly in a similar way as it does with the first AGT and second ACT of M3-2. These results indicate that the binding of the AZF and STZ fusion proteins to the EP2 sequence is specific. Two pairs of the A(G/C)T repeats within the EP2 sequence seem to be the core target site for the AZF and STZ proteins, and sequences around the A(G/C)T repeats in this fragment might have an influence on binding.
To determine whether the AZF and STZ proteins are capable of transactivating EP2-dependent transcription in plant cells, we performed transient analysis using protoplasts prepared from Arabidopsis leaves (Fig. 3). Protoplasts were cotransfected with a
Then we examined whether EP2 can function as a cis-acting element in chimeric promoters by using transient analysis (Fig. 4A). To construct these promoters, we used the 71-bp and 77-bp fragments of the rd29A and rd29B promoters, respectively, in addition to EP2. Both fragments are characterized as Arabidopsis promoters containing positive cis-elements—DRE and ABRE—for dehydration-responsive gene expression (Yamaguchi-Shinozaki and Shinozaki, 1994 ; Uno et al., 2000). When three copies of the 71-bp fragment of the rd29A promoter or five copies of the 77-bp fragment of the rd29B promoter were directly fused to the rd29A minimum promoter, reporter activities were 5 and 23 times higher than that of the minimum promoter. When three copies of EP2 were inserted between these positive fragments and the minimum promoter in both the rd29A and rd29B fragments, the reporter activities were negated. When three copies of EP2 were fused upstream of these positive fragments, reporter activity was induced in the case of rd29A but reduced in the case of rd29B, in comparison with the positive fragments only. These results suggest that the EP2 sequence functions as a negative cis-element in Arabidopsis cells. However, the effect of EP2 as a negative cis-element in the promoter is influenced by the position relations between other cis-elements, and it may be stronger when EP2 is downstream of the other positive cis-element.
Because EP2 was shown to be a negative cis-element, we speculated that the AZFs and STZ might act as transcriptional repressors mediated by binding to this element. Therefore, we analyzed the influences of coexpression of the AZF or STZ proteins on transactivation of transcriptional activators (Fig. 4, B and C). We used two different activators, DREB1A and AREB2. DREB1A and AREB2 transactivate via interaction with DRE and ABRE, respectively. The 71-bp fragment of the rd29A promoter contains one DRE, and the 77-bp fragment of the rd29B promoter contains two ABREs (Yamaguchi-Shinozaki and Shinozaki, 1994 ). Expression of DREB1A or AREB2 in protoplasts transactivated the expression of the GUS reporter gene driven by DRE and ABRE about 50 and 170 times, respectively. However, reduction of transactivation activities was observed in the cells in which the AZFs or STZ were coexpressed with these activators. In particular, the proteins effectively repressed the expression of the reporter gene transactivated by DREB1A. Thus, these results indicate that the AZFs and STZ function as transcriptional repressors via interaction with the EP2 sequence.
Previously, a fusion protein of STZ and the GAL4 DNA-binding domain was shown to function as an active repressor of transcription, and the DLN box in the C-terminal region was identified as a repression domain (Ohta et al., 2001
Expression patterns of the AZF and STZ genes were analyzed under various stress conditions by using RNA gel-blot analysis (Fig. 5A). Among the four genes, AZF2 and STZ exhibited strong induction by various stresses. Expression of both was induced by high salt and dehydration, and expression of STZ was induced also by cold. Expression of both was induced by ABA treatment. The level of AZF2 mRNA increased slowly and peaked within 24 h with high-salt, dehydration, and ABA treatments. On the other hand, the level of STZ mRNA increased rapidly and peaked within 10 min with high-salt stress. It decreased by 1 h and thereafter increased again by 24 h. With dehydration stress and ABA treatment, the level of STZ mRNA increased rapidly and peaked within 10 min, similar to that with high-salt stress, then it decreased slowly. The level of STZ mRNA by cold treatment increased from 20 min, peaked by 2 h, and thereafter decreased. By contrast, little expression of AZF1 and AZF3 was induced by the stresses. The expression levels of these genes were weaker than those of AZF2 and STZ. We analyzed the expression of AZFs and STZ by plant hormones other than ABA (Fig. 5B). AZF2 showed the strongest induction by ABA and a little induction by methyl jasmonate and salicylic acid. STZ showed similar induction by GA, methyl jasmonate, salicylic acid, and 2,4-dichlorophenoxyacetic acid to that by ABA. Expression of all four AZF and STZ genes was induced by ethephon treatment.
Expression of both AZF2 and STZ was strongly induced by various stresses and ABA treatment. To analyze the role of ABA in the expression of these two genes, we used the abi1, abi2, abi3, and aba1 mutants of Arabidopsis (Fig. 5C). The aba mutant is deficient in ABA biosynthesis, and the three abi mutants have an impaired response to ABA. ABI1 and ABI2 encode PP2C-type protein phosphatases and are thought to be essential for negative regulation of ABA signaling in both vegetative tissues and seeds (Leung et al., 1994 , 1997; Meyer et al., 1994). ABI3 encodes a homolog of the maize (Zea mays) VP-1 protein and a seed-specific transcription factor (Giraudat et al., 1992). Compared with the wild type, AZF2 induction in the aba1 and abi1 mutants was reduced by dehydration but not by NaCl stress. No changes of STZ expression between the wild-type and mutant plants were observed under any stress conditions. These results suggest that a part of the AZF2 expression induced by dehydration is induced via the ABA-dependent pathway and that this ABA-dependent expression is mediated by the ABI1 protein. However, the high-salt-responsive expression of AZF2 and the expression of STZ were mainly induced via an ABA-independent pathway.
To study the tissue specificity of AZF and STZ expression, we constructed chimeric genes consisting of the AZF or STZ promoter regions fused to the GUS reporter gene and introduced them into Arabidopsis. We used a 1.6-kb DNA fragment for AZF1, 2.2-kb for AZF2, 1.3-kb for AZF3, and 2.6-kb for STZ upstream sequences to make these chimeric gene constructs. We examined the localization of the GUS reporter gene expression under the control of the AZF and STZ promoters in transgenic plants. Histochemical analysis of GUS activity in rosette plants revealed that all AZF and STZ genes were commonly expressed in roots but at different levels in each root tissue under normal growth conditions (Fig. 6A). Expression of AZF1 and AZF3 was restricted to roots at a low level, whereas expression of AZF2 and STZ was detected not only in roots but also in aerial parts. This result is in agreement with the pattern of RNA gel-blot analysis previously reported for STZ (Lippuner et al., 1996
To determine where AZF2 and STZ expression is induced under stress conditions, we analyzed the expression of these genes in transgenic Arabidopsis containing the promoter::GUS fusion genes. First, we checked the level of induction of the GUS gene driven by the AZF2 and STZ promoters by using RNA gel-blot analysis (Fig. 6B). The expression patterns of the GUS gene under stress conditions were almost the same as those of the endogenous AZF2 and STZ genes. This result confirms that both the AZF2 and STZ promoters used here contain cis-acting elements involved in stress-responsive gene expression. Then we determined the spatial pattern of AZF2 and STZ expression under stress conditions by using GUS staining (Fig. 6C). Both AZF2 and STZ were up-regulated mainly in rosette leaves of salt-treated plants.
To examine the role of STZ and AZF2 in plants under stress conditions, we tried to generate transgenic Arabidopsis plants that constitutively overexpress AZF2 or STZ under the control of the CaMV 35S promoter. The transformation efficiency of these plants was very low, and we repeated the transformation again and again. Finally, we generated 49 lines of AZF2 transformants and 26 lines of STZ. Transgenic plant lines overexpressing AZF2 or STZ were selected by RNA gel-blot analysis. However, no AZF2 overexpressors were obtained, and STZ was overexpressed in only two independent transgenic lines. The growth of the STZ transgenic plants on germination medium (GM) agar plates or on soil was compared with that of the wild-type plants at 2 or 5 weeks, respectively, after sowing. Both transgenic lines on both GM agar plates and soil showed growth retardation compared with the wild-type plants (Fig. 7, A and B), and the level of the growth retardation was correlated with that of STZ expression in the transgenic plants (Fig. 7C).
To examine whether overexpression of STZ affects the tolerance to drought stress, we grew the wild-type and transgenic plants in pots for 3 weeks and then left them unwatered for 2 weeks. Both STZ transgenic lines showed growth retardation, but these 3-week-old soil plants did not show big difference between the wild-type (pBI121) and STZ transgenic plants. We measured the soil water contents for all three lines during the drought stress experiment and found that they are not so different among three plans (data not shown). Almost all the wild-type plants died within this 2-week period, whereas nearly all the transgenic plants of both STZ lines survived this level of drought stress and continued to grow when rewatered (Fig. 7D). We also tried to explore the differences in recovery after desiccation using plants grown on the agar plates. Seventeen-day-old wild-type and transgenic plants were removed from the agar plates and were kept on plastic plates for 4 h (20% ± 10% relative humidity) followed by rehydration overnight. The wild-type plants had wilted and crinkled leaves, whereas the transgenic plants had green leaves that were spread out and appeared healthier as shown in Figure 7E. Then, we calculated the survival rates of the wild-type and transgenic plants. Only 5.5% of the wild-type plants (pBI121) survived, whereas 88.9% and 63.9% of the 35S:STZa and 35S:STZb survived, respectively (Fig. 7E).
The leakage of electrolytes is a sensitive measure of loss-of-membrane integrity and it is commonly used to assay osmolality injury (Gilmour et al., 1998
We characterized four Arabidopsis genes, AZF1, AZF2, AZF3, and STZ, encoding plant-specific transcription factors with two Cys-2/His-2-type zinc-finger motifs (ZPT2-related proteins). The ZPT2-related proteins form a relatively large family of transcription factors in plants (Takatsuji, 1999 ). We searched the completed Arabidopsis genome sequence and found 18 genes encoding ZPT2-related proteins. These 18 genes were divided into two groups on the basis of similarities in their DNA-binding domains. The first group contains 6 genes, including AZF1, AZF2, AZF3, and STZ, and the second group contains the other 12 genes. The six genes of the first group have high homology not only in their DNA-binding domains but also in their N-terminal basic regions (B box); have regions consisting of three acidic residues followed by hydrophobic residues rich in Leu (L box); and have C-terminal short hydrophobic regions containing DLNL sequences (DLN box). The B box is a potential nuclear localization signal, and the L box and DLN box are thought to play roles in protein-protein interactions or in maintaining the folded structure (Sakamoto et al., 2000). All six genes may function similarly as transcription factors in Arabidopsis plants.
As the zinc-finger motifs of the AZFs and STZ have high structural similarity to those of the ZPT2-related proteins, we thought that these proteins would bind two tandemly repeated AGT cores separated by 10 bp in the EP2 sequence (Takatsuji et al., 1994
Using transient analysis we showed that the EP2 sequence is a negative cis-element, and the AZFs and STZ act as transcriptional repressors mediated by binding to this negative cis-element in Arabidopsis protoplasts. Ohta et al. (2001)
Using RNA gel-blot analysis, we showed that AZF2 and STZ were clearly induced at the transcription level by abiotic stresses such as drought, cold, and high salt. These genes were also induced by ABA. These results indicate that these proteins function under abiotic stress conditions. The expression of all four AZF and STZ genes was induced by ethephon. Ethylene is one of the key regulators that mediate a plant's response to biotic and abiotic stresses, such as pathogen infection, wounding, and UV irradiation (Alonso and Ecker, 2001
Both AZF2 and STZ bound to the same cis-acting element, the A(G/C)T repeat, and repressed gene expression, but the different manner of response to each stress suggested that these two proteins function in different signal transduction pathways under stress conditions. Analysis of gene expression in transgenic Arabidopsis plants containing the promoter::GUS fusion genes showed that both the AZF2 and STZ promoter regions are sufficient for drought-stress-responsive gene expression. We searched both promoters to find some putative cis-acting elements involved in gene expression associated with drought, high-salt, and cold stresses. The cis-acting elements we found—DRE (CCGAC), ABRE (RACGTGGC), MYCRS (CANNTG), and MYBRS (RAACYR)—are well known (Shinozaki et al., 2003
We also found many MYBRS and MYCRS in both STZ and AZF2 promoters. In plants, typical examples of the cooperation of MYB and MYC proteins, which bind to MYBRS and MYCRS respectively, are maize C1 and R for anthocyanin biosynthesis (Tuerck and Fromm, 1994 For further understanding of the function of AZF2 and STZ under abiotic stress conditions, we tried to generate transgenic Arabidopsis plants overexpressing AZF2 and STZ under the control of the constitutive CaMV 35S promoter. However, transformants were rare. Moreover, in almost all of the obtained transformants, the expression of transgenes was not enhanced (data not shown). These results suggest that overexpression or ectopic expression of each of AZF2 and STZ causes serious damage to plant development or growth. We obtained two independent lines of STZ overexpressors, but we could not get any overexpressors for AZF2. This may be due to stronger repression activity of AZF2 than of STZ, as shown in the transient assay using Arabidopsis protoplasts.
As both independent lines of the STZ overexpressors showed growth retardation and tolerance to drought stress, the target down-regulated genes might promote plant tolerance and inhibit plant growth. Because the expression of AZF2 was also induced by drought stress, AZF2 might regulate similar target genes to those of STZ. By using microarray analysis, we have shown that many photosynthesis-related genes and genes for carbohydrate metabolism are down-regulated under drought, high-salt, and cold stress conditions (Seki et al., 2002 Independently, we tried to generate transgenic Arabidopsis plants that overexpress AZF1 and AZF3 under the control of the CaMV 35S promoter. However, the transformation efficiency of these plants was very low and we could not obtain any transgenic plants overexpressing AZF1 or AZF3 like AZF2. Both AZF1 and AZF3 were expressed specifically in roots at a low level under the control condition and were induced only weakly by stress. As both AZF1 and AZF3 bind to the same cis-acting element as that of STZ and function as repressors, these two proteins may repress the expression of some specific genes in roots such as some kinds of photosynthesis-related genes or genes for carbohydrate metabolism. We are now generating transgenic plants overexpressing AZF1 and AZF3 using a chemical induction system for transcription. The result of this experiment will show us the real target genes of AZF1 and AZF3, and the function of these proteins will be elucidated in roots.
Plant transcription factors were classified on the basis of the conserved DNA-binding domains (Riechman et al., 2000
Plant Materials and Treatments
Plants (Arabidopsis L. Heynh. ecotype Columbia or Landsberg erecta) were grown on GM agar plates for 3 weeks, as described previously (Yamaguchi-Shinozaki and Shinozaki, 1994
The termination codon of each AZF and STZ coding region was removed with the following primers: 5'-CGGGATCCGAAATCACATCTCACAG-3' plus 5'-CGGGATCCGAAGTCGTCACTGAGAC-3' for AZF1, 5'-GCTCTAGAATGGCCCTCGAAGCGATGAAC-3' plus 5'-CGGATCCAAGATAAATCTTCTTTCTTGATGACTTGG-3' for AZF2, 5'-GCTCTAGATTTTCTATAGCAATGGCGC-3' plus 5'-GCTCTAGATTCAGGCGAGGCTTCTTA-3' for AZF3, and 5'-CGGGATCCCTCAGAATCTTTAACTT-3' plus 5'-CGGGATCCAGTTGAAGTTTGACCGG-3' for STZ.
The PCR-amplified fragments were cut with BamHI (AZF1 and STZ), BamHI and XbaI (AZF2), or XbaI (AZF3) and filled in 35S-sGFP to fuse in frame to the GFP gene (Nakashima et al., 1998
Fragments encoding the truncated proteins containing the two canonical zinc-finger motifs of each AZF and STZ were PCR amplified with the following primers: 5'-CGGGATCCTCACCGTCCGATCACCGAG-3' plus 5'-GCTCTAGATACCGTTGTTGCCACCGTC-3' for AZF1, 5'-GCTCTAGAACGCCGCCGCCAGAATCAAAG-3' plus 5'-GCTCTAGAGGTTGCCTTCGTAGTGACAAC-3' for AZF2, 5'-CGGGATCCACGGTTGCGGAGAAGCCG-3' plus 5'-GCTCTAGAAGTTCGAAACGCCACCATC-3' for AZF3, and 5'-CGGGATCCCCGGCGGTGGAGAAGTTG-3' plus 5'-GCTCTAGATGTTGTTGTTTCCTTCGT-3' for STZ. The PCR fragments were cut with BamHI (AZF1, AZF3, and STZ) or XbaI (AZF2) and filled in pMAL-c2 (New England Biolabs, Beverly, MA). The resulting plasmids were transformed to Escherichia coli BL21-Gold. Production and purification of the MBP and gel-shift assays were performed essentially according to the method described by Yoshioka et al. (2001)
The 35S:AZFs/STZ effector plasmids were constructed with DNA fragments containing each AZF or STZ coding region cloned into the SmaI site of the plant expression vector pBI35S
RNA gel-blot hybridization was performed as described (Yamaguchi-Shinozaki and Shinozaki, 1994
The fragments containing the upstream region of each of the AZF and STZ genes were PCR-amplified with the following primers: 5'-ACGCGTCGACATAAGTTGCATAACGACAGC-3' plus 5'-CGGGATCCAAGATTTAATTCTGTGAGATG-3' for AZF1, 5'-ACGCGTCGACGATTCCATAGCCGTCACAGTG-3' plus 5'-CGGGATCCGATCAGATGAATCTTCTTCTA-3' for AZF2, 5'-ACGCGTCGACAGGTCCCCCTTCCGCTTGTGA-3' plus 5'-CGGGATCCCTTCAAGCGCCATTGCTATAG-3' for AZF3, and 5'-CCCAAGCTTGCATGCACACACAGAGGAGAG-3' plus 5'-CGGGATCCTAAGTTAAAGATTCTGAGG-3' for STZ. The resulting fragments were cut with SalI and BamHI (AZF1, AZF2, and AZF3) or HindIII and BamHI (STZ) and filled in pBI101 (CLONTECH, Palo Alto, CA). These constructs were used to transform Arabidopsis plants by using the vacuum infiltration method with Agrobacterium, as described by Bechtold and Pelletier (1998)
To construct the 35S::STZ plasmid, the SmaI fragment of the STZ coding region was cloned into the SmaI site of a binary vector, pBE2113Not, which was constructed as described (Liu et al., 1998
Arabidopsis plants were germinated and grown on GM plates containing 20 mg L–1 kanamycin for 10 d. The plants were transferred to 9-cm pots filled with 1:1 perlite:vermiculite. They were grown under continuous illumination of approximately 2,500 lux at 22°C. Drought stress was imposed by withholding water for 2 weeks. They were photographed, and the numbers of plants that survived and continued to grow were counted. The statistical significance of the values was determined by using the
We thank Drs. H. Takatsuji and K. Yoshioka for their helpful advice on the gel-shift assay. We thank Dr. M.M. Parvez for critically reading this manuscript. We are also grateful to Mss. E. Ohgawara, K. Murai, M. Yamamoto, and F. Saito for their excellent technical assistance. Received May 17, 2004; returned for revision May 31, 2004; accepted June 1, 2004.
1 This work was supported by the Program for the Promotion of Basic Research Activities for Innovative Biosciences and by a project grant from the Ministry of Agriculture, Forestry and Fisheries, Japan. 
2 Present address: Franz-Volhalrd-Klinik/MDC, Wiltberg str. 50, 13125 Berlin, Germany. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.104.046599. * Corresponding author; e-mail kazukoys{at}jircas.affrc.go.jp; fax 81 29 838 6643.
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ABSTRACT
RESULTS
, and NOS-T represent the CaMV 35S promoter, the translational enhancer of tobacco mosaic virus, and the polyadenylation signal of the nopaline synthase gene, respectively. The reporter gene (GUS) was driven by the rd29A minimal promoter (mp) with or without three repeats of EP2 [(EP2)3]. B, Transient assay using (EP2)3-mp:GUS, 35S:AZFs, and 35S:STZ. The reporter gene was transfected with each effector plasmid and the vector as control treatments. Each transfection contained 30 µg each of the reporter and effector plasmids. Transactivation of (29A)3-mp:GUS by DREB1A was used as a positive control (Liu et al., 1998
2 test). Number codes = number of surviving plants out of total number. E, Difference in recovery after rehydration among the wild-type, 35S::STZa, and 35S::STZb plants. Photographs show the plants dehydrated on dry plastic plates in air for 4 h and then rehydrated overnight. F, Electrolyte leakage was evaluated after dehydration treatment. A 17-d-old plant was used in each experiment. Plants were removed from the agar plates and dehydrated on dry plastic plates for 4 h. The values are means of 15 independent samples. Statistical significance compared with the value of the control plants was determined by Welch's test (P < 0.005).
