TY - JOUR
AU - Li, Chuanyou
AB - Abstract The molecular mechanism governing the response of plants to salinity stress, one of the most significant limiting factors for agriculture worldwide, has just started to be revealed. Here, we report AtSZF1 and AtSZF2, two closely related CCCH-type zinc finger proteins, involved in salt stress responses in Arabidopsis. The expression of AtSZF1 and AtSZF2 is quickly and transiently induced by NaCl treatment. Mutants disrupted in the expression of AtSZF1 or AtSZF2 exhibit increased expression of a group of salt stress-responsive genes in response to high salt. Significantly, the atszf1-1/atszf2-1 double mutant displays more sensitive responses to salt stress than the atszf1-1 or atszf2-1 single mutants and wild-type plants. On the other hand, transgenic plants overexpressing AtSZF1 show reduced induction of salt stress-responsive genes and are more tolerant to salt stress. We also showed that AtSZF1 is localized in the nucleus. Taken together, these results demonstrated that AtSZF1 and AtSZF2 negatively regulate the expression of salt-responsive genes and play important roles in modulating the tolerance of Arabidopsis plants to salt stress. Introduction High salinity has adverse effects on the growth of plants and the productivity of crops, and represents one of the most significant abiotic stresses facing plant agriculture worldwide (Zhu 2002, Ma et al. 2006). Increasing evidence indicated that high levels of sodium ions (Na+) are toxic to plants because of their inhibitory effects on cellular metabolism and ion homeostasis (Zhu 2001, Zhu 2002, Zhu 2003). One of the most important distinguishing features of plants is that they are sessile and thus have to endure environmental challenges. Plants have adapted to respond to high salinity stress at the molecular and cellular levels as well as at the physiological and biochemical levels, thus enabling them to survive (Hasegawa et al. 2000, Zhu 2002). Current studies on the molecular mechanism of the response of plants to salt stress have been focused on identification of components involved in the perception and signal transduction events leading from high salinity to the downstream plant response and adaptation (Hasegawa et al. 2000, Shinozaki and Yamaguchi-Shinozaki 2000, Xiong et al. 2002, Zhu 2002). Salt and drought stresses as well as ABA signaling pathways constitute a complex network and there is much overlap between their branches (Knight and Knight 2001, Zhu 2002, Shinozaki et al. 2003). For instance, microarray analyses show that more than half of the drought-inducible genes are also induced by high salinity and/or ABA, indicating the existence of significant cross-talk among the drought, high salinity and ABA responses (Seki et al. 2002a, Seki et al. 2002b, Shinozaki et al. 2003). ABA plays an important role in the regulation of the adaptation response of plants to high salinity and drought (Zhu 2001, Zhu 2002). Under salt stress conditions, many genes are induced at the transcriptional level in plants through ABA-dependent and/or ABA-independent pathways (Shinozaki and Yamaguchi-Shinozaki 2000, Xiong et al. 2002, Zhu 2002, Shinozaki et al. 2003). The products of these genes function not only in stress tolerance but also in the regulation of gene expression and signal transduction in stress responses (Yamaguchi-Shinozaki and Shinozaki 2006). Recently, cis- and trans-acting factors involved in stress-inducible gene expression have been analyzed extensively in Arabidopsis (Zhu 2002, Shinozaki et al. 2003). ABREs (ABA-responsive elements) and DREs (dehydration- or salt stress-responsive elements) are two major positive cis-acting elements in the promoters of many abiotic stress-inducible genes, which function in ABA-dependent and ABA-independent gene expression, respectively, in response to abiotic stress, (Yamaguchi-Shinozaki and Shinozaki 1994, Shinozaki et al. 2003, Yamaguchi-Shinozaki and Shinozaki 2005). Abiotic stress- or ABA-responsive marker genes such as RD29A contain both DREs and ABREs in their promoters and can be activated by ABA-dependent and ABA-independent abiotic stress pathways (Ishitani et al. 1997). A single ABRE cannot function independently, for instance, the stress-inducible gene RD29B has two copies of ABRE (Uno et al. 2000, Knight and Knight 2001). However, another stress-inducible gene RD29A has both an ABRE and a DRE, suggesting that the ABRE requires the DRE for ABA-induced gene expression (Knight et al. 1999, Knight and Knight 2001). The ERF/AP2 family transcription factors DREB2s (DRE-binding protein 2) that bind to the DRE cis-acting element have also been isolated, which are important transcriptional activators for salt stress- or drought-responsive gene expression in an ABA-independent manner (Liu et al. 1998, Nakashima et al. 2000). A family of basic leucine zipper (bZIP) transcription factors AREB/ABF (ABRE-binding protein/factor) and ABI5 can bind to the ABRE cis-acting element and activate ABA-dependent gene expression (Choi et al. 2000, Finkelstein and Lynch 2000, Lopez-Molina and Chua 2000, Uno et al. 2000, Kang et al. 2002). Salt stress signal transduction includes ionic and osmotic homeostasis signaling pathways (Zhu 2001, Xiong et al. 2002, Zhu 2002, Zhu 2003). So far, our knowledge about the high salt-induced osmotic stress signaling pathway, especially the ABA-independent pathway, is still largely restricted to individual genes, and the unifying picture remains hidden. However, for the ionic aspect of salt stress, a signaling pathway based on the SOS (salt overly sensitive) genes has been established by a conventional genetic screen, with well-defined signal transducers and signal output. SOS3, a calcineurin B-like protein, senses the rise in Ca2+ concentration caused by salt stress and interacts with SOS2, a protein kinase, which in turn activates SOS1, a plasma membrane Na+/H+ antiporter (Liu and Zhu 1998, Halfter et al. 2000, Liu et al. 2000, Shi et al. 2000, Qiu et al. 2002, Zhu 2003). In addition, SOS3–SOS2 regulates the expression of some salt-responsive genes and activates or suppresses the activities of other transporters involved in Na+ homeostasis (Zhu 2002, Zhu 2003). This system restores ion homeostasis of the cells under salt stress. However, due to the existence of complex cross-talk or overlap between salt stress and other abiotic stress as well as the ABA pathway, this type of conventional genetic screen has provided only limited understanding of the high salt-induced osmotic stress signaling pathway (Xiong et al. 2002, Zhu 2002, Yamaguchi-Shinozaki and Shinozaki 2006). In the present study, we described the identification and functional characterization of two novel CCCH-type zinc finger proteins designated as AtSZF1 and AtSZF2 (salt-inducible zinc finger) in Arabidopsis. We provide several lines of evidence showing that AtSZF1 and AtSZF2 play important roles in regulating salt stress responses in Arabidopsis. Results AtSZF1 and AtSZF2 encode CCCH-type zinc finger proteins AtSZF1 (At3g55980) and AtSZF2 (At2g40140), which encode putative CCCH (C-x8-C-x5-C-x3-H)-type zinc finger proteins, were identified as salt stress-inducible genes in the public microarray database (http://www.genevestigator.ethz.ch/at/). AtSZF1 and AtSZF2 share an overall identity of 68% at the amino acid level and show high similarity in structural features, which include two tandem CCCH-type zinc finger motifs and two ankyrin repeat domains in the N-terminal regions (Fig. 1A, B). The sequence and spacer length of the two tandem ankyrin repeats as well as the two zinc finger motifs were very similar (Fig. 1B). In Arabidopsis, there is a small subfamily of CCCH-type zinc finger proteins, which all have two tandem ankyrin repeats (Fig. 1C). The function of members of this protein family has yet to be determined. Zinc finger proteins are generally regarded as DNA-binding transcription factors (Laity et al. 2001). However, recent emerging evidence revealed that, as an unusual family of zinc finger proteins, the CCCH-type zinc finger proteins mainly bind to AU-rich elements of target mRNAs and, therefore, facilitate their metabolism or processing (Carballo et al. 2000, Lai and Blackshear 2001, Blackshear 2002, Lai et al. 2002, Carrick et al. 2004, Hudson et al. 2004, Brown 2005, Hall 2005). Fig. 1 View largeDownload slide Sequences and structural features of the AtSZF1 and AtSZF2 proteins. (A) ClustalW alignment of AtSZF1 and AtSZF2 protein sequences. The ankyrin repeat domains are shown in the boxes, and the CCCH-type zinc finger motifs are underlined. (B) Structural features of AtSZF1 and AtSZF2 proteins. ANK, ankyrin repeat domain; C3H-ZnF, CCCH-type zinc finger motif. (C) Phylogenetic tree of CCCH-type zinc finger proteins with two tandem ankyrin repeats from Arabidopsis. Fig. 1 View largeDownload slide Sequences and structural features of the AtSZF1 and AtSZF2 proteins. (A) ClustalW alignment of AtSZF1 and AtSZF2 protein sequences. The ankyrin repeat domains are shown in the boxes, and the CCCH-type zinc finger motifs are underlined. (B) Structural features of AtSZF1 and AtSZF2 proteins. ANK, ankyrin repeat domain; C3H-ZnF, CCCH-type zinc finger motif. (C) Phylogenetic tree of CCCH-type zinc finger proteins with two tandem ankyrin repeats from Arabidopsis. Expression analysis of AtSZF1 and AtSZF2 transcripts The salt-induced expression of AtSZF1 and AtSZF2 was confirmed by Northern blot analysis. As shown in Fig. 2A, the basal levels of AtSZF1 and AtSZF2 transcripts were relatively low in untreated wild-type plants. Upon NaCl treatment, however, the two transcripts increased quickly and transiently. At 15 min after treatment, AtSZF1 and AtSZF2 expression reached maximum. By 60 min after NaCl treatment, AtSZF1 and AtSZF2 expression recovered to pre-treatment levels (Fig. 2A). This expression pattern implies that AtSZF1 and AtSZF2 might play a role in the response of plants to salt stress. Fig. 2 View largeDownload slide Expression analysis of AtSZF1 and AtSZF2 transcripts. Ten-day-old wild-type or mutant Arabidopsis seedlings were sprayed with 200 mM NaCl, and then harvested at different time points for extraction of total RNA used for Northern blot analysis. The AtSZF1 and AtSZF2 cDNA fragments were used as probes. AtSZF1–GUS and AtSZF2–GUS reporter constructs (see Materials and Methods) were transformed into the wild type (Col-0), and the transgenic lines were used for GUS staining. Several independent transgenic lines were tested and similar results were obtained. Representative results are shown. (A) Northern blot analysis of AtSZF1 and AtSZF2 transcripts after NaCl treatment. (B) AtSZF1–GUS transgenic seedling grown on MS medium for 10 d. (C) Flowers of the AtSZF1–GUS transgenic seedling grown on soil for 6 weeks. (D) AtSZF2–GUS transgenic seedling grown on MS medium for 10 d. (E) Flowers of the AtSZF2–GUS transgenic seedling grown on soil for 6 weeks. (F) Two-week-old seedlings of transgenic lines were sprayed with H2O, 200 mM NaCl and 50 μM ABA for 30 min and stained for GUS activity assay. Fig. 2 View largeDownload slide Expression analysis of AtSZF1 and AtSZF2 transcripts. Ten-day-old wild-type or mutant Arabidopsis seedlings were sprayed with 200 mM NaCl, and then harvested at different time points for extraction of total RNA used for Northern blot analysis. The AtSZF1 and AtSZF2 cDNA fragments were used as probes. AtSZF1–GUS and AtSZF2–GUS reporter constructs (see Materials and Methods) were transformed into the wild type (Col-0), and the transgenic lines were used for GUS staining. Several independent transgenic lines were tested and similar results were obtained. Representative results are shown. (A) Northern blot analysis of AtSZF1 and AtSZF2 transcripts after NaCl treatment. (B) AtSZF1–GUS transgenic seedling grown on MS medium for 10 d. (C) Flowers of the AtSZF1–GUS transgenic seedling grown on soil for 6 weeks. (D) AtSZF2–GUS transgenic seedling grown on MS medium for 10 d. (E) Flowers of the AtSZF2–GUS transgenic seedling grown on soil for 6 weeks. (F) Two-week-old seedlings of transgenic lines were sprayed with H2O, 200 mM NaCl and 50 μM ABA for 30 min and stained for GUS activity assay. The spatial expression patterns of the two genes were determined by histochemical β-glucuronidase (GUS) staining of transgenic plants that harbored the constructs AtSZF1-GUS and AtSZF2-GUS, respectively. These experiments indicated that AtSZF1 and AtSZF2 exhibited very similar expression patterns. At the young seedling stage, AtSZF1 (Fig. 2B) and AtSZF2 (Fig. 2D) were mainly expressed in roots. In floral organs, AtSZF1 (Fig. 2C) and AtSZF2 (Fig. 2E) expression was particularly evident in mature anthers. Consistent with Northern blot analysis, treatment of the reporter lines with NaCl leads to a dramatic increase of GUS activity (Fig. 2F). However, the expression of AtSZF1 and AtSZF2 was not significantly affected by ABA treatment (Fig. 2F). AtSZF1 is a nuclear-localized protein To examine the subcellular localization of AtSZF1 in planta, a GFP (green fluorescent protein) reporter gene was fused in-frame to the N-terminus of AtSZF1 and transiently transformed into onion epidermal cells under the control of the 35S promoter. As shown in Fig. 3B, the GFP::AtSZF1 green fluorescent signal was detected in the nucleus of onion epidermal cells, whereas GFP alone was present throughout the cell (Fig. 3A). Taken together, these results indicate that AtSZF1 is a nuclear-localized protein. Given the high degree of similarity between AtSZF1 and AtSZF2 in terms of sequence and expression pattern, it is reasonable to predict that AtSZF2 may also be a nuclear-localized protein. Fig. 3 View largeDownload slide GFP::AtSZF1 is localized in the nucleus. The onion epidermal cells transiently expressing 35S-GFP or 35S-GFP::AtSZF1 (see Materials and Methods) were observed and photographed using a laser-scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany). (A) The GFP green fluorescence in onion epidermal cells transiently expressing 35S-GFP. (B) The GFP::AtSZF1 green fluorescence in onion epidermal cells transiently expressing 35S-GFP::AtSZF1. Bars in A and B = 20 μm. Fig. 3 View largeDownload slide GFP::AtSZF1 is localized in the nucleus. The onion epidermal cells transiently expressing 35S-GFP or 35S-GFP::AtSZF1 (see Materials and Methods) were observed and photographed using a laser-scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany). (A) The GFP green fluorescence in onion epidermal cells transiently expressing 35S-GFP. (B) The GFP::AtSZF1 green fluorescence in onion epidermal cells transiently expressing 35S-GFP::AtSZF1. Bars in A and B = 20 μm. Disruption of AtSZF1 or AtSZF2 expression leads to enhanced expression of a group of salt stress-responsive genes To explore the biological functions of AtSZF1 and AtSZF2, we identified homozygous T-DNA insertion lines that could disrupt the expression of AtSZF1 or AtSZF2. A T-DNA insertion line (SALK_141550) with a T-DNA inserted at about 300 bp upstream of the translation start codon of AtSZF1 (At3g55980) was named as atszf1-1 in this study (Fig. 4A). Gene expression analysis indicates that homozygous atszf1-1 mutants failed to accumulate AtSZF1 transcript (Fig. 4B). The T-DNA insertion line SALK_024800 (designated as atszf2-1), on the other hand, contains a T-DNA inserted in the coding region of AtSZF2 (At2g40140) (Fig. 4A) and disrupts the expression of AtSZF2 (Fig. 4B). The atszf1-1 and atszf2-1 mutants are indistinguishable from wild-type plants in appearance under normal growth conditions. Fig. 4 View largeDownload slide The atszf1-1 and atszf2-1 mutations cause enhanced expression of some stress-responsive genes upon salt stress treatment. (A) The schematic diagrams of the T-DNA insertions in AtSZF1 and AtSZF2. Filled boxes and lines represent exons and introns, respectively. Dashed lines denote the untranscribed regions. The positions of the T-DNA insertions are indicated by triangles. (B) Expression of AtSZF1 (left) and AtSZF2 (right) in atszf1-1 and atszf2-1 mutants was analyzed by Northern blot. Ten-day-old wild-type or mutant Arabidopsis seedlings grown on MS medium without any treatment were harvested for extraction of total RNA used for Northern blot analysis. The AtSZF1 and AtSZF2 cDNA fragments were used as probes. It took approximately a week of X-ray exposure to obtain the signals shown. (C) Expression analysis of some salt stress-responsive genes in atszf1-1 and atszf2-1 mutants in response to salt stress. Ten-day-old wild-type and mutant seedlings were sprayed with 200 mM NaCl or left untreated, and then harvested at different time points for extraction of total RNA used for Northern blot analysis. (D) RT–PCR analysis of AtSZF1 and AtSZF2 expression in the atszf1-1/atszf2-1 double mutant. The primer sequences used for RT–PCR are as described in Materials and Methods. (E) Expression analysis of several salt stress marker genes in the atszf1-1/atszf2-1 double mutant in response to salt stress. Ten-day-old wild-type and mutant seedlings were sprayed with 200 mM NaCl or left untreated, and then harvested after 3 h treatment for Northern blot analysis. Fig. 4 View largeDownload slide The atszf1-1 and atszf2-1 mutations cause enhanced expression of some stress-responsive genes upon salt stress treatment. (A) The schematic diagrams of the T-DNA insertions in AtSZF1 and AtSZF2. Filled boxes and lines represent exons and introns, respectively. Dashed lines denote the untranscribed regions. The positions of the T-DNA insertions are indicated by triangles. (B) Expression of AtSZF1 (left) and AtSZF2 (right) in atszf1-1 and atszf2-1 mutants was analyzed by Northern blot. Ten-day-old wild-type or mutant Arabidopsis seedlings grown on MS medium without any treatment were harvested for extraction of total RNA used for Northern blot analysis. The AtSZF1 and AtSZF2 cDNA fragments were used as probes. It took approximately a week of X-ray exposure to obtain the signals shown. (C) Expression analysis of some salt stress-responsive genes in atszf1-1 and atszf2-1 mutants in response to salt stress. Ten-day-old wild-type and mutant seedlings were sprayed with 200 mM NaCl or left untreated, and then harvested at different time points for extraction of total RNA used for Northern blot analysis. (D) RT–PCR analysis of AtSZF1 and AtSZF2 expression in the atszf1-1/atszf2-1 double mutant. The primer sequences used for RT–PCR are as described in Materials and Methods. (E) Expression analysis of several salt stress marker genes in the atszf1-1/atszf2-1 double mutant in response to salt stress. Ten-day-old wild-type and mutant seedlings were sprayed with 200 mM NaCl or left untreated, and then harvested after 3 h treatment for Northern blot analysis. To examine the effects of atszf1-1 and atszf2-1 mutations on the response of plants to salt stress, the expression levels of salt stress-responsive marker genes between mutants and the wild type were compared. It was found that, upon salt treatment, the expression levels of a group of salt-inducible genes, including RD29A, KIN1, COR15A and COR47, were significantly higher in atszf1-1 and atszf2-1 mutants than in the wild type (Fig. 4C). However, the transcriptional expression of DREB2A, a key regulator of salt stress-responsive gene expression, did not display a significant difference between mutants and wild-type plants (Fig. 4C). We also examined the ABA or cold-induced expression of some stress responsive genes in mutants and wild-type plants. As shown in Fig. 5, the ABA or cold-induced expression levels of KIN1 and COR15A did not exhibit a significant difference between mutants and wild-type plants (Fig. 5). Fig. 5 View largeDownload slide Expression analysis of some stress-responsive genes in atszf1-1 and atszf2-1 mutants in response to ABA and cold treatments, respectively. Ten-day-old wild-type or mutant seedlings were treated with 50 μM ABA on the roots for 3 h or incubated in 4°C under light conditions for 12 h, and then harvested for extraction of total RNA used for Northern blot analysis. Fig. 5 View largeDownload slide Expression analysis of some stress-responsive genes in atszf1-1 and atszf2-1 mutants in response to ABA and cold treatments, respectively. Ten-day-old wild-type or mutant seedlings were treated with 50 μM ABA on the roots for 3 h or incubated in 4°C under light conditions for 12 h, and then harvested for extraction of total RNA used for Northern blot analysis. Genomic DNA of the AtSZF1 gene was introduced into the atszf1-1 mutant under the control of its putative native promoter. Positive transformants were verified by measuring the expression of AtSZF1 (Fig. 6A). The transgene restored the expression of salt stress-responsive genes of the atszf1-1 mutant to wild-type levels (Fig. 6 and data not shown). These results demonstrated that the enhanced expression of salt stress-responsive genes in the atszf1-1 mutant resulted from the disrupted expression of AtSZF1. Fig. 6 View largeDownload slide Genetic complementation of the atszf1-1 mutant. (A) RNA gel blot analysis of the expression of the AtSZF1 gene of the pC1300-AtSZF1 transgenic plants in the atszf1-1 background. (B) RNA gel blot analysis of the expression of the KIN1 gene of the pC1300-AtSZF1 transgenic plants in the atszf1-1 background. Ten-day-old wild-type, mutant and transgenic seedlings were sprayed with 200 mM NaCl or left untreated as control; tissues were harvested at the indicated times for RNA extraction. Fig. 6 View largeDownload slide Genetic complementation of the atszf1-1 mutant. (A) RNA gel blot analysis of the expression of the AtSZF1 gene of the pC1300-AtSZF1 transgenic plants in the atszf1-1 background. (B) RNA gel blot analysis of the expression of the KIN1 gene of the pC1300-AtSZF1 transgenic plants in the atszf1-1 background. Ten-day-old wild-type, mutant and transgenic seedlings were sprayed with 200 mM NaCl or left untreated as control; tissues were harvested at the indicated times for RNA extraction. The atszf1-1/atszf2-1 double mutant displays increased sensitivity to salt stress Since the atszf1-1 and atszf2-1 mutants show enhanced expression of salt-responsive genes, we asked whether the two mutants display altered responses to salt stress in other aspects. Phenotypic comparison was conducted among atszf1-1, atszf2-1 and the wild type in the presence of high salt (NaCl) at different growth stages including seed germination, early seedling growth and soil-grown adult plants. In these experiments, the atszf1-1 and atszf2-1 mutants showed a relatively subtle phenotypic difference compared with the wild type (Fig. 7 and data not shown). These results might suggest the existence of functional redundancy between AtSZF1 and AtSZF2. We therefore constructed an atszf1-1/atszf2-1 double mutant line, in which the expression of AtSZF1 and AtSZF2 was simultaneously disrupted (Fig. 4D). RNA blot analysis revealed that the NaCl-induced expression levels of salt-responsive genes, such as RD29A and KIN1, were higher in the atszf1-1/atszf2-1 double mutant than those in the single mutants and wild-type plants (Fig. 4E). Fig. 7 View largeDownload slide The atszf1-1/atszf2-1 double mutant shows increased sensitivity to salt stress in seed germination and early seedling growth. Wild-type, atszf1-1, atszf2-1 and atszf1-1/atszf2-1 double mutant plants were sown on MS medium plates with (B, D) or without (A, C) 100 mM NaCl and stratified at 4°C for 2 d, and then transferred to growth conditions (16 h light/8 h dark cycle at 22°C) for phenotypic analysis. The seedlings with open cotyledons were scored for seed germination phenotype during different days after stratification. The seedlings with green cotyledons were scored for cotyledon greening rate during different days after stratification. All assays were repeated at least three times with similar results. More than 50 seeds were used in each experiment. Fig. 7 View largeDownload slide The atszf1-1/atszf2-1 double mutant shows increased sensitivity to salt stress in seed germination and early seedling growth. Wild-type, atszf1-1, atszf2-1 and atszf1-1/atszf2-1 double mutant plants were sown on MS medium plates with (B, D) or without (A, C) 100 mM NaCl and stratified at 4°C for 2 d, and then transferred to growth conditions (16 h light/8 h dark cycle at 22°C) for phenotypic analysis. The seedlings with open cotyledons were scored for seed germination phenotype during different days after stratification. The seedlings with green cotyledons were scored for cotyledon greening rate during different days after stratification. All assays were repeated at least three times with similar results. More than 50 seeds were used in each experiment. The atszf1-1/atszf2-1 double mutant, together with the two single mutants, was further compared with the wild type for their response to NaCl-mediated stress in seed germination and early seedling growth. As shown in Fig. 7B and D, in the presence of 100 mM NaCl, the double mutant was more inhibited than the single mutants and wild-type plants, as indicated by a lower seed germination rate and cotyledon greening rate. These four genotypes did not show significant differences when grown on MS medium (Fig. 7A, C). In the root bending assay (Liu and Zhu 1998, Halfter et al. 2000, Liu et al. 2000, Shi et al. 2000, Qiu et al. 2002, Zhu 2003), we also found that the root growth of the atszf1-1/atszf2-1 double mutant was more inhibited than that of the wild-type or single mutant plants (Fig. 8). The salt stress response of the atszf1-1/atszf2-1 double mutant was also examined in soil. For this experiment, the atszf1-1/atszf2-1 double mutant, the atszf1-1 or atszf2-1 single mutants and wild-type plants were grown in soil for 3 weeks under normal growth conditions and were then irrigated with 200 mM NaCl solution. After 2-week treatment, it was found that the leaves of the atszf1-1/atszf2-1 double mutant showed serious wilting or died, and the upper parts of the stems of atszf1-1/atszf2-1 plants also showed severely wilting, as compared with wild-type and atszf1-1 or atszf2-1 mutant plants (Fig. 10 and data not shown). These plants grew well in soil under normal growth conditions (data not shown). Fig. 8 View largeDownload slide The atszf1-1/atszf2-1 double mutant shows increased sensitivity to salt stress in the root bending assay. Six-day-old seedlings were transferred from normal MS medium to MS medium supplemented or not with 100 mM NaCl (with roots upside down). The pictures were taken 10 d after transfer. The experiment was repeated at least three times with similar results. Fig. 8 View largeDownload slide The atszf1-1/atszf2-1 double mutant shows increased sensitivity to salt stress in the root bending assay. Six-day-old seedlings were transferred from normal MS medium to MS medium supplemented or not with 100 mM NaCl (with roots upside down). The pictures were taken 10 d after transfer. The experiment was repeated at least three times with similar results. Overexpression of AtSZF1 enhances salt stress tolerance of transgenic lines The increased susceptibility of the atszf-1/atszf2-1 double mutant to salt stress prompted us to evaluate the effect, if any, of AtSZF1 overexpression on the salt stress response. The open reading frame of AtSZF1 under the control of the 35S promoter was transformed into wild-type plants. Based on Northern blot analysis, several independent transgenic plants showing increased expression of the AtSZF1 transgene were selected for further investigation (Fig. 9A). Fig. 9 View largeDownload slide The AtSZF1-overexpressing plants are more tolerant to salt stress than wild-type plants. (A) Expression of the AtSZF1 transgene in AtSZF1-overexpressing lines. (B) Expression of several stress-responsive genes in AtSZF1-overexpressing lines. (C) The root bending assay of AtSZF1-overexpressing lines. Seedlings of 35S-GFP::AtSZF1 transgenic lines and wild-type plants were grown first on MS agar plates for 6d and then transferred to MS agar plates with or without 125 mM NaCl (with roots upside down). The pictures were taken 7 d after transfer. The experiment was repeated at least three times with similar results. Fig. 9 View largeDownload slide The AtSZF1-overexpressing plants are more tolerant to salt stress than wild-type plants. (A) Expression of the AtSZF1 transgene in AtSZF1-overexpressing lines. (B) Expression of several stress-responsive genes in AtSZF1-overexpressing lines. (C) The root bending assay of AtSZF1-overexpressing lines. Seedlings of 35S-GFP::AtSZF1 transgenic lines and wild-type plants were grown first on MS agar plates for 6d and then transferred to MS agar plates with or without 125 mM NaCl (with roots upside down). The pictures were taken 7 d after transfer. The experiment was repeated at least three times with similar results. The AtSZF1-overexpressing plants were then used to determine whether increased expression of AtSZF1 would affect the expression of salt-inducible marker genes and, therefore, modulate the tolerance of plants to salt stress. As shown in Fig. 9B, overexpression of AtSZF1 leads to reduced expression of RD29A and KIN1. Furthermore, transgenic plants overexpressing AtSZF1 are more tolerant to salt stress than wild-type plants, as shown in the root bending assay on high salt-containing medium (Fig. 9C) and in response to salt stress when grown in soil (Fig. 10). Taken together, these results show that overexpression of AtSZF1 leads the transgenic plants to become more tolerant to salt stress. Fig. 10 View largeDownload slide Salt tolerance analysis of the AtSZF1-overexpressing line, the atszf1-1/atszf2-1 double mutant and wild-type plants. Plants were grown in soil for 3 weeks under normal growth conditions and then were irrigated with 200 mM NaCl solution. The pictures were taken after 2-week treatment. Fig. 10 View largeDownload slide Salt tolerance analysis of the AtSZF1-overexpressing line, the atszf1-1/atszf2-1 double mutant and wild-type plants. Plants were grown in soil for 3 weeks under normal growth conditions and then were irrigated with 200 mM NaCl solution. The pictures were taken after 2-week treatment. Discussion A wealth of evidence indicates that salt stress, drought stress and ABA signaling pathways constitute a complex network and there is much overlap between their branches (Knight and Knight 2001, Zhu 2002, Shinozaki et al. 2003). The response of plants to salt stress is a complex trait that is still not well understood. So far, only a limited number of regulators in the salt stress response pathway in plants have been identified. We have demonstrated that the transcriptional expression of AtSZF1 and AtSZF2, which encode two CCCH-type zinc finger proteins, was quickly and transiently induced by salt stress, implying that the AtSZF1 and AtSZF2 proteins may be involved in the salt stress response in Arabidopsis. Indeed, our genetic and molecular analyses provided several lines of evidence showing that AtSZF1 and AtSZF2 negatively regulate salt-responsive gene expression and modulate the tolerance to salt stress in Arabidopsis. Firstly, the atszf1-1 and atszf2-1 mutants, showing disrupted expression of AtSZF1 and AtSZF2, respectively, exhibit enhanced expression of a group of salt stress-inducible marker genes in response to salt stress (Fig. 4C). Secondly, the atszf1-1/atszf2-1 double mutant displays an increased sensitive response to salt stress in seed germination and growth inhibition compared with wild-type and atszf1-1 or atszf2-1 mutant plants (Fig. 7, 8). Thirdly, transgenic lines overexpressing AtSZF1 are more tolerant to salt stress in seedling growth inhibition than wild-type plants (Fig. 9C). Significantly, in our salt tolerance assay of soil-grown adult plants, the atszf1-1/atszf2-1 double mutant plants are more susceptible to salt stress, while transgenic lines overexpressing AtSZF1 are more tolerant to salt stress than wild-type plants (Fig. 10). Of interest is the finding that disruption of AtSZF1 and AtSZF2 expression not only increased the salt-induced expression of stress-responsive genes, but also rendered the atszf1/atszf2 double mutant more sensitive to damage by salt stress. The salt-induced gene expression patterns and salt response phenotype of the atszf mutants showed a high level of similarity to the so-called hos (high expression of osmotic responsive genes) mutants identified by the Zhu laboratory (Ishitani et al. 1997). Like atszf1-1 and atszf2-1, the hos5 mutant also exhibited enhanced expression of a group of stress-responsive genes in response to high salinity, which suggested that HOS5 represents a negative regulator of salt stress-induced gene expression in Arabidopsis (Xiong et al. 1999). Molecular identification of the HOS5 gene will help to clarify its nature and aid our understanding of its function in the salt stress response. Another example came from the Arabidopsis mutant fiery1 (fry1), which also displayed superinduction of ABA- and stress-responsive genes. Seed germination and post-embryonic development of fry1 were more sensitive to ABA or stress inhibition. The mutant plants are also compromised in tolerance to freezing, drought and salt stresses (Xiong et al. 2001). We speculate that, like the hos5 and fry1 mutations, disruption of AtSZF1 or AtSZF2 expression might significantly reduce the excitatory threshold of gene induction by salt stress and, as a consequence, lead to enhanced expression of salt-responsive genes and render the mutant plants more susceptible to salt stress. Given the fact that the biochemical functions of the salt-responsive RD29A, KIN1 and others are not known, it is difficult so far to correlate their expression levels with the response of the plant to salt stress. Previous studies have demonstrated that some CCCH-type zinc finger proteins play important regulatory roles in different biological processes by modulating the mRNA processing or degradation of key components in various organisms (Lai et al. 2000, Lai and Blackshear 2001, Li et al. 2001, Blackshear 2002, Cheng et al. 2003, Carrick et al. 2004, Hudson et al. 2004). In the plant kingdom, the best known CCCH-type zinc finger protein showing RNA-binding activity is HUA1 from Arabidopsis. HUA1 functions in floral reproductive organ identity by binding AG pre-mRNA and facilitating its processing (Li et al. 2001, Cheng et al. 2003). Kong et al. (2006) reported that a novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. However, it is unclear how OsDOS acts during leaf senescence, because the target of OsDOS remains unidentified. Recently, several lines of evidence have shown that RNA metabolism plays important roles in abiotic stress responses (Sunkar and Zhu 2004, Borsani et al. 2005, Gong et al. 2005, Lee et al. 2006). Clearly, the identification of the genes targeted by AtSZF1 and AtSZF2 will help to understand how AtSZF1 and AtSZF2 act in the regulation of the salt stress response. Materials and Methods Plant materials and growth conditions Arabidopsis thaliana ecotype Columbia (Col-0) was used in this study. The atszf1-1 (SALK_141550) and atszf2-1 (SALK_024800) mutants were obtained from the Arabidopsis Biological Resources Center (Alonso et al. 2003). Plants were grown under a 16 h light/8 h dark cycle at 22°C in soil or on Murashige–Skoog (MS) medium (1×MS salts, 3% sucrose, 0.8% agar). Plasmid construction and plant transformation All molecular manipulations were performed according to standard methods (Sambrook and Russell 2001). A genomic clone of AtSZF1 was obtained by PCR using LA Taq DNA polymerase (TAKARA SHUZO CO., LTD, Ohtsu, Japan), which included a 2 kb promoter sequence, the entire coding region and 0.5 kb of 3′-untranslated sequence. The genomic DNA fragment was digested by BamHI and KpnI, and inserted into the same sites of the binary vector pCAMBIA1300 to generate the pC1300-AtSZF1g construct. The coding sequence of AtSZF1 cDNA was amplified by PCR, digested by XhoI and SpeI, and inserted into the same site of binary vector p35S-GFP to generate the 35S-GFP::AtSZF1 construct. The 2 kb genomic fragments upstream of the translation start codon of the AtSZF1and AtSZF2 genes were amplified by PCR, digested by HindIII plus BamHI, or by PstI plus BamHI, and fused with the GUS reporter gene into the binary vector pCAMBIA1391-Z. The above constructs were then transformed into Agrobacterium tumefaciens strain GV3101 (pMP90), and were used for transformation of Arabidopsis plants by vacuum infiltration (Bechtold and Pelletier 1998). The following primers were used for PCR. For the complementation test, the primers used for the pC1300-AtSZF1g construct are: At3g55980F4, 5′-CCGGATCCcttccctttacattgggaggca-3′ (BamHI) plus At3g55980B4, 5′-CCGGTACCtgtatactttattatgactcca-3′ (KpnI). For the overexpression and subcellular localization of AtSZF1 protein, the primers used are: At3g55980F, 5′-GGCTCGAGATGTGCAGTGGACCAAAGAGCAA-3′ (XhoI) plus At3g55980R, 5′-GGACTAGTgtgtgtgtTTACACCACAGTCTG-3′ (SpeI). The primers used for the AtSZF1 and AtSZF2 promoters are: At3g55980proF, 5′-GGAAGCTTgcatttgtctcccgatccattacc-3′ (HindIII) plus At3g55980proR, 5′-GGGGATCCaatactatcctgtagattcaagaaa-3′ (BamHI); and At2g40140ProF, 5′-GGCTGCAGgtggaggttacctgctaagtc-3′ (PstI) plus At2g40140ProR, 5′-GGGGATCCatctcttcctgtgattcaaaagtg-3′ (BamHI). For atszf1-1 and atszf2-1 genotyping, the primers used are: SALK_141550LP, 5′-AGAAGAGTCAGCACAAGAGCG-3′ plus SALK_141550RP, 5′-TTCCAGTGGAAACGATGAAAG-3′; and SALK_024800LP, 5′-AGATATGTGCGGTGCAAAGAG-3′ plus SALK_024800RP, 5′-GTGAAACCATTGCAGAACCAG-3′. RT–PCR analysis A 2 μg aliquot of total RNA extracted from wild-type or mutant seedlings was used for reverse transcription primed by oligo(dT). SuperScript II (Invitrogen) was used for the reverse transcription reaction according to the manufacturer's instructions. A 0.5 μl aliquot of the reaction mixture was used for subsequent PCR. The primers used for the AtSZF1 gene were: At3g55980F2, 5′-GGCAACAATGTGGAAGAGACGT-3′ and At3g55980B1, 5′-GTGTGTGTTTACACCACAGTCTG-3′; those for the AtSZF2 gene were SALK_024800LP and SALK_024800RP (see above); and those for ACTIN8 as control were: ACTIN8F, 5′-CCTTGCTGGTCGTGACCTTACTGA-3′ and ACTIN8R, 5′-CTCTCAGCACCGATCGTGATCACT-3′. Seed germination and cotyledon greening assay For the seed germination and cotyledon greening assay, wild-type, atszf1-1, atszf2-1 and atszf1-1/atszf2-1 double mutant plants were sown on MS medium plates with or without 100 mM NaCl and stratified at 4°C for 2 d, and then transferred to growth conditions (16 h light/8 h dark cycle at 22°C). The seedlings with open or green cotyledons were scored during different days after stratification for the seed germination phenotype and cotyledon greening rate, respectively. At least 50 seeds for each genotype were grown horizontally on one medium. The seeds of different genotype were collected at the same time. The experiment was repeated at least three times. Subcellular localization The p35S-GFP and p35S-GFP::AtSZF1 constructs were introduced into onion epidermal cells for transient expression with a Biolistic Particle Delivery System (Bio-Rad, Hercules, CA, USA), and the fluorescence of GFP was visualized with a laser scanning confocal microscope (LSM 510, Zeiss, Oberkochen, Germany). Northern blot analysis Ten-day-old Arabidopsis seedlings were treated or not with 200 mM NaCl or 50 μM ABA for different times as indicated. Total RNA was prepared by a guanidine thiocyanate extraction method, and RNA gel blot analysis was performed as previously described (Zheng et al. 2006). A 10 μg aliquot of total RNA was separated in an agarose gel containing 10% formaldehyde, blotted onto a Hybond N+ membrane (Amersham), and probed with the PCR-amplified DNA fragments using the following primers: AtSZF1F, 5′-GGCTCGAGATGTGCAGTGGACCAAAGAGCAA-3′ plus AtSZF1R, 5′-GGACTAGTgtgtgtgtTTACACCACAGTCTG-3′; AtSZF2F, 5′-ATGTGCGGTGCAAAGAGCAACCT-3′ plus AtSZF2R, 5′-CTGCTTCTTATGCCACAATCTGC-3′; RD29AF, 5′-GGAGGAGTACCGGAGATTGCT-3′ plus RD29AR, 5′-CTCCGCCACATAATCTCTACC-3′; KIN1F, 5′-CCATTAAGCCCACATCTCTTC-3′ plus KIN1R, 5′-CGGATCGACTTATGTATCGTG-3′; COR15AF, 5′-TGCTAACATGAGCTGTTCTCA-3′ plus COR15AR, 5′-GTGACGGTGACTGTGGATACC-3′; COR47F, 5′-CCATCTTAAAGCAACTACACA-3′ plus COR47R, 5′-CGTAAGAGTGAGTATACGATG-3′; and DREB2AF, 5′-ATGGCAGTTTATGATCAGAGT-3′ plus DREB2AR,5′-CCATTACCATCCTTTCCCTCG-3′. Analysis of β-glucuronidase (GUS) activity Histochemical staining for GUS activity in transgenic plants was performed as previously described (Jefferson et al. 1987). Acknowledgments We would like to thank the Arabidopsis Biological Resources Center for providing the atszf1-1 (SALK_141550) and atszf2-1 (SALK_024800) mutant seeds. This work was supported by grants from the National Natural Science Foundation of China (grant Nos. 30425033 and 30530440 to C.L.) and the Chinese Academy of Sciences (grant No. CXTD-S2005-2 to C.L.). References Alonso JM,  Stepanova AN,  Leisse TJ,  Kim CJ,  Chen H, et al.  Genome-wide insertional mutagenesis of Arabidopsis thaliana,  Science ,  2003, vol.  301 (pg.  653- 657) Google Scholar CrossRef Search ADS PubMed  Bechtold N,  Pelletier G.  In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration,  Methods Mol. Biol ,  1998, vol.  82 (pg.  259- 266) Google Scholar PubMed  Blackshear PJ.  Tristetraprolin and other CCCH tandem zinc-finger proteins in the regulation of mRNA turnover,  Biochem. Soc. Trans ,  2002, vol.  30 (pg.  945- 952) Google Scholar CrossRef Search ADS PubMed  Borsani O,  Zhu J,  Verslues PE,  Sunkar R,  Zhu J.-K.  Endogenous siRNAs derived from a pair of natural cis-antisense transcripts regulate salt tolerance in Arabidopsis,  Cell ,  2005, vol.  123 (pg.  1279- 1291) Google Scholar CrossRef Search ADS PubMed  Brown RS.  Zinc finger proteins: getting a grip on RNA,  Curr. Opin. Struct. Biol ,  2005, vol.  15 (pg.  94- 98) Google Scholar CrossRef Search ADS PubMed  Carballo E,  Lai WS,  Blackshear PJ.  Evidence that tristetraprolin is a physiological regulator of granulocyte–macrophage colony-stimulating factor messenger RNA deadenylation and stability,  Blood ,  2000, vol.  95 (pg.  1891- 1899) Google Scholar PubMed  Carrick DM,  Lai WS,  Blackshear PJ.  The tandem CCCH zinc finger protein tristetraprolin and its relevance to cytokine mRNA turnover and arthritis,  Arthritis Res. Ther ,  2004, vol.  6 (pg.  248- 264) Google Scholar CrossRef Search ADS PubMed  Cheng Y,  Kato N,  Wang W,  Li J,  Chen X.  Two RNA binding proteins, HEN4 and HUA1, act in the processing of AGAMOUS pre-mRNA in Arabidopsis thaliana,  Dev. Cell ,  2003, vol.  4 (pg.  53- 66) Google Scholar CrossRef Search ADS PubMed  Choi H,  Hong J,  Ha J,  Kang J,  Kim SY.  ABFs, a family of ABA-responsive element binding factors,  J. Biol. Chem ,  2000, vol.  275 (pg.  1723- 1730) Google Scholar CrossRef Search ADS PubMed  Finkelstein RR,  Lynch TJ.  The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor,  Plant Cell ,  2000, vol.  12 (pg.  599- 609) Google Scholar CrossRef Search ADS PubMed  Gong Z,  Dong C. -H,  Lee H,  Zhu J,  Xiong L,  Gong D,  Stevenson B,  Zhu J.-K.  A DEAD box RNA helicase is essential for mRNA export and important for development and stress responses in Arabidopsis,  Plant Cell ,  2005, vol.  17 (pg.  256- 267) Google Scholar CrossRef Search ADS PubMed  Halfter U,  Ishitani M,  Zhu J.-K.  The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3,  Proc. Natl Acad. Sci. USA ,  2000, vol.  97 (pg.  3735- 3740) Google Scholar CrossRef Search ADS   Hall TM.  Multiple modes of RNA recognition by zinc finger proteins,  Curr. Opin. Struct. Biol ,  2005, vol.  15 (pg.  367- 373) Google Scholar CrossRef Search ADS PubMed  Hasegawa PM,  Bressan RA,  Zhu J.-K,  Bohnert HJ.  Plant cellular and molecular responses to high salinity,  Annu. Rev. Plant Physiol. Plant Mol. Biol ,  2000, vol.  51 (pg.  463- 499) Google Scholar CrossRef Search ADS PubMed  Hudson BP,  Martinez-Yamout MA,  Dyson HJ,  Wright PE.  Recognition of the mRNA AU-rich element by the zinc finger domain of TIS11d,  Nat. Struct. Mol. Biol ,  2004, vol.  11 (pg.  257- 264) Google Scholar CrossRef Search ADS PubMed  Ishitani M,  Xiong L,  Stevenson B,  Zhu J.-K.  Genetic analysis of osmotic and cold stress signal transduction in Arabidopsis: interactions and convergence of abscisic acid-dependent and abscisic acid-independent pathways,  Plant Cell ,  1997, vol.  9 (pg.  1935- 1949) Google Scholar CrossRef Search ADS PubMed  Jefferson RA,  Kavanagh TA,  Bevan MW.  GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants,  EMBO J ,  1987, vol.  6 (pg.  3901- 3907) Google Scholar PubMed  Kang JY,  Choi HI,  Im MY,  Kim SY.  Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling,  Plant Cell ,  2002, vol.  14 (pg.  343- 357) Google Scholar CrossRef Search ADS PubMed  Knight H,  Knight MR.  Abiotic stress signalling pathways: specificity and cross-talk,  Trends Plant Sci ,  2001, vol.  6 (pg.  262- 267) Google Scholar CrossRef Search ADS PubMed  Knight H,  Veale EL,  Warren GJ,  Knight MR.  The sfr6 mutation in Arabidopsis suppresses low-temperature induction of genes dependent on the CRT/DRE sequence motif,  Plant Cell ,  1999, vol.  11 (pg.  875- 886) Google Scholar PubMed  Kong Z,  Li M,  Yang W,  Xu W,  Xue Y.  A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice,  Plant Physiol ,  2006, vol.  141 (pg.  1376- 1388) Google Scholar CrossRef Search ADS PubMed  Lai WS,  Blackshear PJ.  Interactions of CCCH zinc finger proteins with mRNA: tristetraprolin-mediated AU-rich element-dependent mRNA degradation can occur in the absence of a poly(A) tail,  J. Biol. Chem ,  2001, vol.  276 (pg.  23144- 23154) Google Scholar CrossRef Search ADS PubMed  Lai WS,  Carballo E,  Thorn JM,  Kennington EA,  Blackshear PJ.  Interactions of CCCH zinc finger proteins with mRNA. Binding of tristetraprolin-related zinc finger proteins to AU-rich elements and destabilization of mRNA,  J. Biol. Chem ,  2000, vol.  275 (pg.  17827- 17837) Google Scholar CrossRef Search ADS PubMed  Lai WS,  Kennington EA,  Blackshear PJ.  Interactions of CCCH zinc finger proteins with mRNA: non-binding tristetraprolin mutants exert an inhibitory effect on degradation of AU-rich element-containing mRNAs,  J. Biol. Chem ,  2002, vol.  277 (pg.  9606- 9613) Google Scholar CrossRef Search ADS PubMed  Laity JH,  Lee BM,  Wright PE.  Zinc finger proteins: new insights into structural and functional diversity,  Curr. Opin. Struct. Biol ,  2001, vol.  11 (pg.  39- 46) Google Scholar CrossRef Search ADS PubMed  Lee BH,  Kapoor A,  Zhu J,  Zhu J.-K.  STABILIZED1, a stress-upregulated nuclear protein, is required for pre-mRNA splicing, mRNA turnover, and stress tolerance in Arabidopsis,  Plant Cell ,  2006, vol.  18 (pg.  1736- 1749) Google Scholar CrossRef Search ADS PubMed  Li J,  Jia D,  Chen X.  HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein,  Plant Cell ,  2001, vol.  13 (pg.  2269- 2281) Google Scholar CrossRef Search ADS PubMed  Liu J,  Ishitani M,  Halfter U,  Kim CS,  Zhu J.-K.  The Arabidopsis thaliana SOS2 gene encodes a protein kinase that is required for salt tolerance,  Proc. Natl Acad. Sci. USA ,  2000, vol.  97 (pg.  3730- 3734) Google Scholar CrossRef Search ADS   Liu J,  Zhu J.-K.  A calcium sensor homolog required for plant salt tolerance,  Science ,  1998, vol.  280 (pg.  1943- 1945) Google Scholar CrossRef Search ADS PubMed  Liu Q,  Kasuga M,  Sakuma Y,  Abe H,  Miura S,  Yamaguchi-Shinozaki K,  Shinozaki K.  Two transcription factors, DREB1 and DREB2, with an EREBP/AP2 DNA binding domain separate two cellular signal transduction pathways in drought- and low-temperature-responsive gene expression, respectively, in Arabidopsis,  Plant Cell ,  1998, vol.  10 (pg.  1391- 1406) Google Scholar CrossRef Search ADS PubMed  Lopez-Molina L,  Chua N.-H.  A null mutation in a bZIP factor confers ABA-insensitivity in Arabidopsis thaliana,  Plant Cell Physiol ,  2000, vol.  41 (pg.  541- 547) Google Scholar CrossRef Search ADS PubMed  Ma S,  Gong Q,  Bohnert HJ.  Dissecting salt stress pathways,  J. Exp. Bot ,  2006, vol.  57 (pg.  1097- 1107) Google Scholar CrossRef Search ADS PubMed  Nakashima K,  Shinwari ZK,  Sakuma Y,  Seki M,  Miura S,  Shinozaki K,  Yamaguchi-Shinozaki K.  Organization and expression of two Arabidopsis DREB2 genes encoding DRE-binding proteins involved in dehydration- and high-salinity-responsive gene expression,  Plant Mol. Biol ,  2000, vol.  42 (pg.  657- 665) Google Scholar CrossRef Search ADS PubMed  Qiu Q.-S,  Guo Y,  Dietrich MA,  Schumaker KS,  Zhu J.-K.  Regulation of SOS1, a plasma membrane Na+/H+ exchanger in Arabidopsis thaliana, by SOS2 and SOS3,  Proc. Natl Acad. Sci. USA ,  2002, vol.  99 (pg.  8436- 8441) Google Scholar CrossRef Search ADS   Sambrook J,  Russell DW. ,  Molecular Cloning: A Laboratory Manual ,  2001 3rd Cold Spring Harbor, NY Cold Spring Harbor Laboratory Press Seki M,  Ishida J,  Narusaka M,  Fujita M,  Nanjo T, et al.  Monitoring the expression pattern of around 7,000 Arabidopsis genes under ABA treatments using a full-length cDNA microarray,  Funct. Integr. Genomics ,  2002, vol.  2 (pg.  282- 291) Google Scholar CrossRef Search ADS PubMed  Seki M,  Narusaka M,  Ishida J,  Nanjo T,  Fujita M, et al.  Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray,  Plant J ,  2002, vol.  31 (pg.  279- 292) Google Scholar CrossRef Search ADS PubMed  Shi H,  Ishitani M,  Kim C,  Zhu J.-K.  The Arabidopsis thaliana salt tolerance gene SOS1 encodes a putative Na+/H+ antiporter,  Proc. Natl Acad. Sci. USA ,  2000, vol.  97 (pg.  6896- 6901) Google Scholar CrossRef Search ADS   Shinozaki K,  Yamaguchi-Shinozaki K.  Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways,  Curr. Opin. Plant Biol ,  2000, vol.  3 (pg.  217- 223) Google Scholar CrossRef Search ADS PubMed  Shinozaki K,  Yamaguchi-Shinozaki K,  Seki M.  Regulatory network of gene expression in the drought and cold stress responses,  Curr. Opin. Plant Biol ,  2003, vol.  6 (pg.  410- 417) Google Scholar CrossRef Search ADS PubMed  Sunkar R,  Zhu J.-K.  Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis,  Plant Cell ,  2004, vol.  16 (pg.  2001- 2019) Google Scholar CrossRef Search ADS PubMed  Uno Y,  Furihata T,  Abe H,  Yoshida R,  Shinozaki K,  Yamaguchi-Shinozaki K.  Arabidopsis basic leucine zipper transcription factors involved in an abscisic acid-dependent signal transduction pathway under drought and high-salinity conditions,  Proc. Natl Acad. Sci. USA ,  2000, vol.  97 (pg.  11632- 11637) Google Scholar CrossRef Search ADS   Xiong L,  Ishitani M,  Lee H,  Zhu J.-K.  HOS5—a negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana,  Plant J ,  1999, vol.  19 (pg.  569- 578) Google Scholar CrossRef Search ADS PubMed  Xiong L,  Lee B,  Ishitani M,  Lee H,  Zhang C,  Zhu J.-K.  FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis,  Genes Dev ,  2001, vol.  15 (pg.  1971- 1984) Google Scholar CrossRef Search ADS PubMed  Xiong L,  Schumaker KS,  Zhu J.-K.  Cell signaling during cold, drought, and salt stress,  Plant Cell ,  2002, vol.  14 Suppl. (pg.  S165- S183) Google Scholar PubMed  Yamaguchi-Shinozaki K,  Shinozaki K.  A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to drought, low-temperature, or high-salt stress,  Plant Cell ,  1994, vol.  6 (pg.  251- 264) Google Scholar CrossRef Search ADS PubMed  Yamaguchi-Shinozaki K,  Shinozaki K.  Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters,  Trends Plant Sci ,  2005, vol.  10 (pg.  88- 94) Google Scholar CrossRef Search ADS PubMed  Yamaguchi-Shinozaki K,  Shinozaki K.  Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses,  Annu. Rev. Plant Biol ,  2006, vol.  57 (pg.  781- 803) Google Scholar CrossRef Search ADS PubMed  Zheng W,  Zhai Q,  Sun J,  Li C.-B,  Zhang L, et al.  Bestatin, an inhibitor of aminopeptidases, provides a chemical genetics approach to dissect jasmonate signaling in Arabidopsis,  Plant Physiol ,  2006, vol.  141 (pg.  1400- 1413) Google Scholar CrossRef Search ADS PubMed  Zhu J.-K.  Cell signaling under salt, water and cold stresses,  Curr. Opin. Plant Biol ,  2001, vol.  4 (pg.  401- 406) Google Scholar CrossRef Search ADS PubMed  Zhu J.-K.  Salt and drought stress signal transduction in plants,  Annu. Rev. Plant Biol ,  2002, vol.  53 (pg.  247- 273) Google Scholar CrossRef Search ADS PubMed  Zhu J.-K.  Regulation of ion homeostasis under salt stress,  Curr. Opin. Plant Biol ,  2003, vol.  6 (pg.  441- 445) Google Scholar CrossRef Search ADS PubMed  Abbreviations: Abbreviations: ABRE ABA-responsive element C3H-ZnF CCCH-type zinc finger motif CCCH C-x8-C-x5-C-x3-H DRE dehydration- or salt stress-responsive element GUS β-glucuronidase GFP green fluorescent protein RT–PCR reverse transcription–PCR 35S cauliflower mosaic virus 35S RNA promoter © The Author 2007. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org
TI - The CCCH-Type Zinc Finger Proteins AtSZF1 and AtSZF2 Regulate Salt Stress Responses in Arabidopsis
JF - Plant and Cell Physiology
DO - 10.1093/pcp/pcm088
DA - 2007-08-01
UR - https://www.deepdyve.com/lp/oxford-university-press/the-ccch-type-zinc-finger-proteins-atszf1-and-atszf2-regulate-salt-cgW4AehUvq
SP - 1148
EP - 1158
VL - 48
IS - 8
DP - DeepDyve
ER -