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. 2013 Jan 7;161(3):1202–1216. doi: 10.1104/pp.112.205385

OsTZF1, a CCCH-Tandem Zinc Finger Protein, Confers Delayed Senescence and Stress Tolerance in Rice by Regulating Stress-Related Genes1,[W],[OA]

Asad Jan 1,2, Kyonoshin Maruyama 1, Daisuke Todaka 1, Satoshi Kidokoro 1, Mitsuru Abo 1, Etsuro Yoshimura 1, Kazuo Shinozaki 1, Kazuo Nakashima 1,3, Kazuko Yamaguchi-Shinozaki 1,3,*
PMCID: PMC3585590  PMID: 23296688

OsTZF1, a CCCH-type zinc finger protein, acts as a negative regulator of leaf senescence in rice under stress conditions and confers abiotic stress tolerance by delaying stress-response phenotypes, possibly through the control of RNA metabolism of stress-responsive genes.

Abstract

OsTZF1 is a member of the CCCH-type zinc finger gene family in rice (Oryza sativa). Expression of OsTZF1 was induced by drought, high-salt stress, and hydrogen peroxide. OsTZF1 gene expression was also induced by abscisic acid, methyl jasmonate, and salicylic acid. Histochemical activity of β-glucuronidase in transgenic rice plants containing the promoter of OsTZF1 fused with β-glucuronidase was observed in callus, coleoptile, young leaf, and panicle tissues. Upon stress, OsTZF1-green fluorescent protein localization was observed in the cytoplasm and cytoplasmic foci. Transgenic rice plants overexpressing OsTZF1 driven by a maize (Zea mays) ubiquitin promoter (Ubi:OsTZF1-OX [for overexpression]) exhibited delayed seed germination, growth retardation at the seedling stage, and delayed leaf senescence. RNA interference (RNAi) knocked-down plants (OsTZF1-RNAi) showed early seed germination, enhanced seedling growth, and early leaf senescence compared with controls. Ubi:OsTZF1-OX plants showed improved tolerance to high-salt and drought stresses and vice versa for OsTZF1-RNAi plants. Microarray analysis revealed that genes related to stress, reactive oxygen species homeostasis, and metal homeostasis were regulated in the Ubi:OsTZF1-OX plants. RNA-binding assays indicated that OsTZF1 binds to U-rich regions in the 3′ untranslated region of messenger RNAs, suggesting that OsTZF1 might be associated with RNA metabolism of stress-responsive genes. OsTZF1 may serve as a useful biotechnological tool for the improvement of stress tolerance in various plants through the control of RNA metabolism of stress-responsive genes.

Plants encounter various abiotic stresses during their life cycle, such as cold, drought, and high salinity. Many genes induced by various abiotic stresses have been identified using genome-wide analysis techniques, including microarray analysis (Fowler and Thomashow, 2002; Seki et al., 2002; Rabbani et al., 2003; Bartels and Sunkar, 2005; Yamaguchi-Shinozaki and Shinozaki, 2006) and proteomics (Pechanova et al., 2010; Kosová et al., 2011). These induced genes either directly protect plants against stresses by the production of important metabolic proteins (functional proteins) or regulate the genes for signal transduction in the stress response (regulatory proteins). It is of immense importance to elucidate the exact function of genes involved in signal transduction and their regulation to understand their responses to different environmental stresses in plants (Cui et al., 2002; Ronald and Leung, 2002).

Zinc finger protein genes constitute a large and diverse gene family. They are involved in many aspects of plant growth and development and play important roles in many cellular functions, including transcriptional regulation, RNA binding, and protein-protein interactions (Ciftci-Yilmaz and Mittler, 2008). Zinc finger proteins have been classified into several different types, including C2H2, C2C2, C2HC, C2C2C2C2, C2HCC2C2, and CCCH, according to the number and order of the Cys (C) and His (H) residues binding the zinc ion in the secondary structure of the finger (Berg and Shi, 1996; Mackay and Crossley, 1998; Moore and Ullman, 2003; Schumann et al., 2007). A number of zinc finger proteins have been found to be involved in abiotic and biotic stress responses (Cui et al., 2002; Mukhopadhyay et al., 2004; Sakamoto et al., 2004). Zinc finger proteins containing a tandem zinc finger (TZF) domain, characterized by two CCCH zinc fingers separated by 18 amino acids, are well documented in humans, and a number of them have been associated with RNA metabolism (Varnum et al., 1991; Carballo et al., 1998, 2000; Shimada et al., 2002; Al-Souhibani et al., 2010; Jeong et al., 2010).

A genome-wide annotation analysis of CCCH zinc finger proteins found 67 genes in rice (Oryza sativa) and 68 genes in Arabidopsis (Arabidopsis thaliana; Wang et al., 2008). Analysis of zinc finger proteins in Arabidopsis and rice identified a plant-unique tandem CCCH zinc finger containing Cx7-8Cx5Cx3Hx16-Cx5Cx4Cx3H and an uncharacterized N-terminal side C-x5-H-x4-C-x3-H motif (Wang et al., 2008; Pomeranz et al., 2010). The functions of most of the genes in this unique subfamily are unknown, but recently, a few genes of Arabidopsis, PEI1 (an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis), AtSZF1/AtSZF2 (for salt-inducible zinc finger of Arabidopsis), and SOMNUS, were characterized to function in embryogenesis (Li and Thomas, 1998), salt stress response (Sun et al., 2007), and light-dependent seed germination (Kim et al., 2008), respectively. AtTZF1 was reported to shuttle between the nucleus and cytoplasmic foci and to be involved in abscisic acid (ABA)/GA-mediated growth and abiotic stress responses (Pomeranz et al., 2010; Lin et al., 2011). Rice OsDOS (for delay of the onset of senescence) was reported to be nucleus localized and involved in delaying leaf senescence (Kong et al., 2006). Here, we report the isolation and functional characterization of OsTZF1 (Os05g10670), which is induced under abiotic stress conditions and a homolog of AtTZF1 (Pomeranz et al., 2010) in rice. We show that OsTZF1 acts as a negative regulator of leaf senescence in rice under stress conditions and confers abiotic stress tolerance by delaying stress-response phenotypes, possibly through the control of RNA metabolism of stress-responsive genes.

RESULTS

Stress-Induced and Spatial Expression Profiles of OsTZF1 in Rice

To identify important genes under abiotic stress conditions, we monitored the expression profiles of rice ‘Nipponbare’ genes under drought conditions using a 44K rice microarray (Maruyama et al., 2012). The expression of OsTZF1 was induced by drought, high-salt stress, and ABA and was selected for further characterization. Expression of OsTZF1 in response to dehydration, ABA, NaCl, and hydrogen peroxide (H2O2) was analyzed by RNA gel-blot analysis to investigate time-dependent induction patterns. As shown in Figure 1, the OsTZF1 transcript accumulated in seedlings within 2 h following ABA, NaCl, and H2O2 treatments. In contrast, expression of the OsTZF1 gene induction peaked after 5 h and then decreased slightly over 24 h of dehydration stress treatment (Fig. 1A). There was no significant accumulation of OsTZF1 mRNA in seedlings treated with water only (Fig. 1A). The expression of OsTZF1 in rice seedlings was also up-regulated following treatment with exogenous salicylic acid (SA) or jasmonic acid (JA; Fig. 1B).

Figure 1.

Figure 1.

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Expression profiles of OsTZF1 under different stress and hormone treatments. A, RNA gel-blot analysis of OsTZF1 expression under different stress conditions. Hydroponically grown 2-week-old rice plants were dehydrated or transferred to solutions containing 10 µm ABA, 250 mm NaCl, 10 mm H2O2, or water (H2O) for the time periods indicated. B, Hormone-dependent expression analysis of OsTZF1. Two-week-old rice plants grown hydroponically were transferred to nutrient solution containing 10 µm ABA, 10 µm methyl jasmonate (MeJA), or 100 µm SA for the time periods indicated. C, Quantitative GUS analysis of control and POsTZF1-GUS transgenic rice plants under ABA and NaCl treatments. Plants were treated with ABA or NaCl as described. Cont, Control. D, Histochemical activity of POsTZF1:GUS transgenic rice plants under ABA and NaCl treatments. Hydroponically grown control and POsTZF1:GUS transgenic rice plants were transferred to nutrient solutions containing 10 µm ABA or 250 mm NaCl.

To assess the effect of the promoter region on the expression of OsTZF1 in rice seedlings under abiotic stresses, we generated transgenic rice plants containing a 1,417-bp OsTZF1 promoter fragment fused to the GUS reporter gene (POsTZF1:GUS). Quantitative analysis of GUS activity in the POsTZF1:GUS plants confirmed that the promoter region of OsTZF1 regulates the induction of this gene in response to ABA and NaCl (Fig. 1C). Histochemical GUS activity was mainly detected in the aerial parts of POsTZF1:GUS plants, and its intensity increased in response to ABA and NaCl (Fig. 1D).

To determine the organ-specific expression of the OsTZF1 gene, we isolated total RNA from callus, roots, shoot bases, seedling leaves, four expanded leaves at bolting stage, nodes, internodes, and two different stages of the rice panicle. RNA gel-blot analysis showed that the expression of OsTZF1 was high in callus, moderate in shoot bases, seedling leaves, internodes, and panicles after dehiscence, but no or low expression was observed in roots, expanded leaves, nodes, and panicles before dehiscence (Fig. 2A). Consistent with the results of RNA gel-blot analysis, histochemical GUS activity in the POsTZF1:GUS plants was observed in rice callus, germinating seedlings, seedling leaves, and panicles after dehiscence, while no or low histochemical GUS activity was observed in roots (Fig. 2, B–F).

Figure 2.

Figure 2.

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Expression profiles of OsTZF1 in different rice organs. A, RNA gel-blot analysis of OsTZF1 expression in different organs. For organ-specific expression, total RNA was extracted from callus, roots, shoot bases, seedling leaves, four leaves at bolting stage, nodes, internodes, panicles before dehiscence (PBD), and panicles after dehiscence (PAD). B to F, Histochemical analysis of POsTZF1:GUS gene activities in different tissues and organs of rice. Callus (B), 3-d-old seedlings (C), 10-d-old seedlings (D), panicles before dehiscence (E), and panicles after dehiscence (F) derived from the POsTZF1:GUS transgenic plants were incubated in GUS staining solution for 12 h.

Putative sequences of various cis-acting elements involved in the response to abiotic stresses were identified in the 1,417-bp promoter region of OsTZF1 (Supplemental Fig. S1) using the PLACE signal scan program (Higo et al., 1999). We found five ABA-responsive elements (ACGTG; Simpson et al., 2003), three MYB core sequences (CNGTTR; Urao et al., 1993), and four recognition sites for MYC (CANNTG; Abe et al., 2003). The OsTZF1 promoter also contained some putative cis-acting elements involved in the response to biotic stresses, including three WRKY71OS sequences (TGAC-containing W-box; Eulgem et al., 2000) and seven W-boxes of different types (data not shown), which are known as recognition sites for WRKY transcription factors.

Subcellular Localization of OsTZF1

To determine the subcellular localization of OsTZF1, transgenic rice plants expressing an OsTZF1-sGFP (for synthetic GFP) chimeric gene driven by either the native OsTZF1 promoter or by the maize (Zea mays) ubiquitin promoter were generated. Under normal growth conditions, OsTZF1 was predominantly localized in the cytoplasm of root meristem cells and was occasionally observed in cytoplasmic foci (Fig. 3A). OsTZF1-GFP was rarely observed in the nucleus of root cells at the young seedling stage (Fig. 3A). To examine whether these OsTZF1-associated cytoplasmic foci are stress related, seedlings were treated with 10 µm ABA or 50 mm NaCl for 12 h. As shown in Figure 3A, ABA and NaCl enhanced the formation of cytoplasmic foci in root cells. The size, shape, and number of these cytoplasmic foci varied depending on the condition of plants and the type of roots. These cytoplasmic foci resembled processing bodies (PBs) and stress granules (SGs) where mRNA turnover and translational repression take place (Parker and Sheth, 2007; Balagopal and Parker, 2009).

Figure 3.

Figure 3.

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Subcellular localization of OsTZF1 and colocalization with cytoplasmic foci marker genes. A, Subcellular localization of OsTZF1. Stably transformed transgenic rice plants expressing the OsTZF1-sGFP chimeric gene, driven by the native promoter, were analyzed by fluorescence microscopy. OsTZF1-sGFP transgenic rice plants were either untreated or treated with 10 µm ABA or 50 mm NaCl for 12 h. B, Colocalization of OsTZF1 with cytoplasmic foci marker genes. OsTZF1 is localized in cytoplasmic foci that resemble PBs and SGs. OsTZF1 colocalizes with PB marker DCP2 (top). OsTZF1 and SG marker PABP8 colocalize in SGs (bottom). CFP, Cyan fluorescent protein; DIC, differential interference contrast; YFP, yellow fluorescent protein.

To confirm whether OsTZF1 is associated with PBs and SGs, colocalization analysis using Arabidopsis marker genes for PBs and SGs was performed. OsTZF1 was fused in frame with yellow fluorescent protein, and the marker genes were fused with cyan fluorescent protein. DCP2 is an mRNA-decapping enzyme and localizes to PBs (Iwasaki et al., 2007; Weber et al., 2008). Both constructs were introduced into protoplasts prepared from rice mesophyll cells. OsTZF1-yellow fluorescent protein colocalized with DCP2 in cytoplasmic foci resembling PBs (Fig. 3B). Colocalization of OsTZF1 was also tested in SGs using the SG marker PABP8 (Newbury et al., 2006; Anderson and Kedersha, 2008). OsTZF1 and PABP8 colocalized in comparatively large cytoplasmic foci, considered to be SGs (Fig. 3B).

In onion (Allium cepa) epidermal cells, OsTZF1 was localized in the cytoplasm and cell membrane (Supplemental Fig. S2A). Furthermore, OsTZF1 localization could be observed in the nucleus in comparatively old OsTZF1-sGFP plants (Supplemental Fig. S2B). These results indicate that OsTZF1 has dynamic subcellular localization patterns in the cytoplasm and probably the nucleus.

In Vivo Functional Analysis of OsTZF1 Using OsTZF1-OX and OsTZF1-RNAi Plants

To analyze the function of OsTZF1 in rice, transgenic rice plants overexpressing OsTZF1 driven by the maize ubiquitin promoter (OsTZF1-OX [for overexpression]) were generated. Seven OsTZF1-OX transgenic rice plants (T2 generation) were analyzed by RNA gel-blot analysis, and two lines (OsTZF1-OX#6 and OsTZF1-OX#9) exhibiting moderate levels of transgene expression (Supplemental Fig. S3A) were selected for phenotypic and functional characterization. OsTZF1-RNA interference (RNAi) transgenic rice plants were also produced to further evaluate the function of OsTZF1. Seven OsTZF1-RNAi plants were analyzed by RNA gel-blot analysis, and two lines (OsTZF1-RNAi#7 and OsTZF1-RNAi#9) were selected for further functional analysis (Supplemental Fig. S3B).

The OsTZF1-OX plants showed a phenotype of repressed seed germination. The OsTZF1-OX seeds required more than 2 d after imbibition (DAI) for coleoptile plumule emergence compared with 24 to 30 h in control seeds, whereas OsTZF1-RNAi seeds showed an enhanced seed germination phenotype, with an elongated coleoptile plumule at 2 DAI (Fig. 4A). The differences in seed germination and growth became more obvious at 4 to 5 DAI (Fig. 4A). Differences in seed germination time affect subsequent growth; the growth of OsTZF1-OX seedlings was slow, while OsTZF1-RNAi seedlings showed enhanced growth compared with controls at 10 d after seed germination (Fig. 4B). These results indicated that OsTZF1 affects germination and subsequent rice seedling growth. The OsTZF1-OX seedlings planted in soil grew normally, and there was no difference in the growth of OsTZF1-OX, OsTZF1-RNAi, and control plants. At the bolting stage, the OsTZF1-OX and control plants were indistinguishable (Supplemental Fig. S3). The OsTZF1-OX plants exhibited delayed senescence of leaves and retained more photosynthetic activity (maximum photochemical efficiency of PSII in the dark-adapted state [Fv/Fm]) compared with controls after the start of seed setting (Supplemental Fig. S4). Interestingly, during the heading and seed-setting stages, OsTZF1-OX leaves were greener but developed a number of brown lesions that increased in frequency over time (Fig. 4C). The seeds of OsTZF1-OX were also brownish in appearance compared with controls at harvest (Fig. 4D). These results indicated that constitutive overexpression of OsTZF1 caused pleiotropic effects on the phenotype of rice plants.

Figure 4.

Figure 4.

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Phenotypic analysis of OsTZF1-OX and OsTZF1-RNAi rice plants. A, Seed germination phenotypes of control (Cont), OsTZF1-OX, and OsTZF1-RNAi plants during the 5 DAI. Graph curves indicate the growth of control, OsTZF1-OX, and OsTZF1-RNAi plants during a period of 5 DAI. B, Phenotypes of control, OsTZF1-OX, and OsTZF1-RNAi seedlings 10 d after germination. Bar graphs show the growth of control, OsTZF1-OX, and OsTZF1-RNAi seedlings 10 d after germination. C, Phenotypes of leaves from control and OsTZF1-OX plants at bolting stage. D, Typical phenotypes of seeds from control and OsTZF1-OX plants at harvest.

OsTZF1-OX Plants Exhibit High-Salt and Drought Stress Tolerance

The expression of OsTZF1 was induced by NaCl and dehydration stress (Fig. 1A); therefore, OsTZF1-OX and OsTZF1-RNAi plants were evaluated for high-salt stress and drought tolerance. Two-week-old rice seedlings grown in soil were treated with 250 mm NaCl for 3 d. After high-salt stress treatment, plants were allowed to recover under normal growth conditions (Fig. 5). OsTZF1-RNAi seedlings showed severe damage to leaves and ultimately died, whereas the OsTZF1-OX transgenic seedlings showed good tolerance to high-salt stress and continued to grow. More than 65% of the OsTZF1-OX plants survived, whereas only 33% of the control plants survived (Fig. 5, left panels). Conversely, the survival rate of the OsTZF1-RNAi plants was around 19% to 21% compared with 50% in the controls (Fig. 5, right panels). The OsTZF1-OX plants had higher photosynthetic activities than the control or OsTZF1-RNAi plants according to Fv/Fm measurements (Supplemental Fig. S5). Similarly, 2-week-old rice seedlings grown in soil were subjected to drought stress. Plants were allowed to recover under normal growth conditions after stress treatment (Supplemental Fig. S6). More than 60% of the OsTZF1-OX rice plants survived compared with only 38% in the controls. The survival rate of the OsTZF1-RNAi plants was around 21% to 23% compared with 46% in the controls (Supplemental Fig. S6). These results demonstrated that OsTZF1 positively regulates high-salt stress tolerance and drought stress tolerance in rice plants.

Figure 5.

Figure 5.

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Analysis of high-salt stress tolerance in control (Cont), OsTZF1-OX, and OsTZF1-RNAi plants. Seeds in equivalent germination states were sown in soil and subjected to 250 mm NaCl stress as described in “Materials and Methods.” The results are averages of four independent experiments with 12 plants per experiment. Plants marked with asterisks had significantly higher survival rates than the controls as determined using the χ2 test (P < 0.01).

OsTZF1 Confers Increased Tolerance to Oxidative Stress

In plants, the primary symptoms of senescence are largely mediated by dramatic increases in reactive oxygen species (ROS; Thompson and Lake, 1987; Leshem, 1988). Using rice leaf fragments (4–5 cm in length), we tested the response of OsTZF1-OX, control, and OsTZF1-RNAi plants to dark-induced leaf senescence, UV-C exposure, and 10 mm H2O2. After 6 d of dark treatment, leaf yellowing was observed in the control, whereas the leaves of the OsTZF1-OX plants remained green (Fig. 6A). In contrast, the OsTZF1-RNAi leaf fragments exhibited more yellowing than controls (Fig. 6A). Under normal growth conditions, no diaminobenzidine (DAB) staining was observed in either controls or OsTZF1-OX, but enhanced DAB staining was observed in the OsTZF1-RNAi leaf fragments (Fig. 6A). Exposure to UV-C for 3 d caused significant yellowing and accumulation of DAB in the OsTZF1-RNAi leaves compared with controls, whereas less yellowing or DAB staining was observed in the OsTZF1-OX plants (Fig. 6B). The role of ROS in rice leaf senescence was confirmed directly by treating rice leaf fragments with 20 mm H2O2 solution for 3 d. There was less or no effect of H2O2 treatment on the OsTZF1-OX leaf fragments, while enhanced leaf yellowing and DAB staining was observed in OsTZF1-RNAi plants (Fig. 6C). These results suggested that the delayed leaf senescence phenotype of OsTZF1-OX plants was because of enhanced tolerance to oxidative stress.

Figure 6.

Figure 6.

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Analysis of delayed leaf senescence and ROS tolerance in control (Cont), OsTZF1-OX, and OsTZF1-RNAi plants. A, OsTZF1-OX plants showed a delay in dark-induced leaf senescence. Detached leaf fragments from control, OsTZF1-OX, and OsTZF1-RNAi plants were incubated in water for 6 d in the dark. The treated leaf fragments were stained with DAB (1 mg mL−1) solution to visually detect H2O2. B, OsTZF1-OX plants showed delayed UV-C-induced leaf senescence. Detached leaf fragments from control, OsTZF1-OX, and OsTZF1-RNAi plants were incubated in water for 3 d under UV-C. The treated leaf fragments were stained with DAB solution to visually detect H2O2. C, OsTZF1-OX plants showed delayed H2O2-induced leaf senescence. Detached leaf fragments from control, OsTZF1-OX, and OsTZF1-RNAi plants were incubated in 10 mm H2O2 solution for 3 d under dark conditions. The treated leaves were stained with DAB solution to visually detect H2O2. D, OsTZF1-OX plants showed high photosynthetic activities during dark-induced leaf senescence. The photosynthetic activities (Fv/Fm) of detached leaf fragments from control, OsTZF1-OX, and OsTZF1-RNAi plants were measured at 0, 2, 4, and 6 d during dark-induced leaf senescence. E, OsTZF1-OX plants showed reduced ion leakage during dark-induced leaf senescence. The ion leakage of detached leaf fragments from control, OsTZF1-OX, and OsTZF1-RNAi plants was measured at 0, 2, 4, and 6 d during dark-induced leaf senescence.

We evaluated the effect of darkness on Fv/Fm in detached leaf fragments. When leaf fragments were kept in the dark for 6 d, the photochemical efficiency of the OsTZF1-RNAi, control, and OsTZF1-OX dropped from approximately 0.8 to 0.18, 0.38, and 0.74, respectively (Fig. 6D). These data were in agreement with the phenotypes of leaves (Fig. 6, A–C).

The oxidation of lipids and proteins during membrane injury by ROS was evaluated by measuring plasma membrane electrolyte leakage (Fig. 6E). Senescing leaf fragments of OsTZF1-RNAi showed a marked increase in ion leakage, from an average of 11% to 40% and 42%, indicating membrane damage, whereas the increase in the ion leakage of controls was 12% to 36%. In contrast, the OsTZF1-OX leaf fragments maintained low electrolyte leakage (18% and 20%) even after 6 d of dark treatment. These results provided strong evidence of an enhanced tolerance of OsTZF1-OX plants to membrane damage during leaf senescence.

The effect of darkness alone or with JA, ABA, and NaCl on leaf senescence was also evaluated using leaf fragments from Ubi:OsTZF1-OX and POsTZF1:OsTZF1-OX plants (Supplemental Fig. S7). In each case, OsTZF1-OX leaves showed delayed senescence compared with controls. Surprisingly, the delayed senescence in POsTZF1:OsTZF1-OX plants under these conditions was more pronounced than in Ubi:OsTZF1-OX plants (Supplemental Fig. S7).

Global Gene Expression Changes Regulated by OsTZF1

To elucidate the molecular mechanism of stress tolerance conferred by OsTZF1 in rice, microarray analyses were carried out using the 44K rice oligo microarray (Agilent Technologies). Two independent OsTZF1-OX lines (OsTZF1-OX#6 and OsTZF1-OX#9) were used for the microarray analysis. A total of 198 genes showed 2.0-fold or greater change in OsTZF1-OX compared with the control. Among them, 119 genes were up-regulated and 79 genes were down-regulated in the OsTZF1-OX plants (Supplemental Table S1). Based on microarray analysis of stress-inducible genes (Maruyama et al., 2012), of the 119 up-regulated genes in OsTZF1-OX rice plants, 27 genes were induced by high-salt stress (Supplemental Table S1) and 55 genes were induced by dehydration stress (Supplemental Fig. S8; Supplemental Table S1). Of the 79 down-regulated genes in OsTZF1-OX, 28 and 32 genes were down-regulated by high-salt and dehydration stress, respectively (Supplemental Fig S8; Supplemental Table S1). Among the up-regulated genes in the OsTZF1-OX plants were genes encoding proteins with predicted functions in stress tolerance, such as dehydrins, detoxification (glutathione S-transferases [GSTs]), redox homeostasis (ferritin and metallothionin), and transcription factors (Supplemental Table S2). We also identified genes encoding proteins with predicted functions in biotic stress responses, such as pathogenesis-related proteins, chitinases, and peroxidases (Supplemental Table S2). Among the 79 down-regulated genes in OsTZF1-OX were genes involved in heavy metal and oligopeptide transport as well as polyamine and secondary metabolism (Supplemental Table S3). Quantitative reverse transcription (qRT)-PCR analysis confirmed the regulation of some of these genes in OsTZF1-OX plants (Fig. 7).

Figure 7.

Figure 7.

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qRT-PCR analysis of metal homeostasis-related genes identified in OsTZF1-OX plants by microarray analysis. Two-week-old, soil-grown OsTZF1-OX and control (Cont) plants were used to isolate RNA. The analyzed genes whose expression was up-regulated in OsTZF1-OX were Ferritin (Os11g0106700), MT-type1 (Os12g0568500), and ChaC-like (Os02g0465900). The down-regulated genes analyzed were OsNRAMP1 (Os07g0258400), Ferroportin1 (Os06g0560000), and OPT (Os03g0751100).

The OsTZF1-OX plants showed improved high-salt stress tolerance (Fig. 5). To examine the role of OsTZF1 in the transcriptional network in response to high-salt stress, we compared the expression profiles of OsTZF1-OX and control plants grown in soil for 2 weeks and then treated with 250 mm NaCl solution for 2 d using the 44K rice oligo microarray. After 2 d of high-salt stress treatment, 2,051 genes were up-regulated and 2,141 genes were down-regulated in the OsTZF1-OX plants (Fig. 8A; Supplemental Table S4). Surprisingly, out of 2,051 up-regulated genes in OsTZF1-OX plants, 844 and 1,459 genes were normally down-regulated by high-salt and dehydration stresses, respectively (Fig. 8A; Supplemental Fig. S8; Supplemental Table S4). Conversely, out of 2,141 down-regulated genes in OsTZF1-OX, 989 and 1,561 genes were normally up-regulated by high-salt and dehydration stresses, respectively (Fig. 8A; Supplemental Fig. S8; Supplemental Table S4). According to gene expression atlas data sources (http://signal.salk.edu/RiceGE/RiceGE_gAtlas_Source.html) and our unpublished microarray data (Maruyama et al., 2012), a significant number of genes showing decreased expression levels in the OsTZF1-OX plants are normally responsive to salt stress and dehydration, indicating that either OsTZF1-OX plants are impaired in stress-responsive gene expression or they exhibit an attenuated stress response and in turn attenuated stress-responsive gene expression.

Figure 8.

Figure 8.

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Transcriptome analysis of genes regulated in OsTZF1-OX under high-salt stress. A, Venn diagram showing the relationship between genes regulated in OsTZF1-OX treated with 250 mm NaCl for 2 d and NaCl stress-inducible genes. In total, 2,051 and 2,141 genes were up- and down-regulated, respectively, in OsTZF1-OX plants treated with 250 mm NaCl for 2 d in relation to similarly treated controls. B, RNA gel-blot analysis of genes regulated in OsTZF1-OX identified by microarray analysis. Two-week-old rice plants treated with 250 mm NaCl for 0, 1, 2, and 3 d or subjected to dark-induced senescence for 0, 3, 6, and 9 d were used to isolate RNA. Probes were prepared from the 3′ UTRs of isocitrate lyase (Os07g0529000), (1,3;1,4)-β-glucanase (Os05g0375400), RD22 (Os10g0409400), YSL6 (Os08g0508000), N-amino transporter (Os02g0191300), akin-β (Os05g0491200), and AOS (Os03g0225900) as described in “Materials and Methods.”

To confirm these results, the expression of genes encoding representative stress-related proteins identified by the microarray analysis, including isocitrate lyase, β-glucanase, RD22, YSL6, an N-amino transporter, the β-subunit of SnRK1 (akin-β), and allene oxide synthase (AOS), was analyzed during high-salt stress in OsTZF1-OX and control plants (Fig. 8B). Soil-grown 2-week-old plants were subjected to 250 mm NaCl stress for 3 d. The stress-responsive expression of the above genes was delayed or reduced in OsTZF1-OX compared with control plants (Fig. 8B, left panel). The OsTZF1-OX plants exhibited a delayed-senescence phenotype; therefore, the expression of the above genes was also examined in relation to dark-induced senescence. Two-week-old OsTZF1-OX and control plants grown in soil were subjected to dark-induced senescence, and the expression of the above genes was examined using RNA gel-blot analysis. Interestingly, reduced senescence-induced expression of the above genes was observed in OsTZF1-OX plants compared with controls (Fig. 8B, right panel). Similar results were obtained when the expression of these genes was analyzed in UV-C-exposed OsTZF1-OX and control plants (data not shown).

Consistent with the above results, the induction of native OsTZF1 expression in response to high-salt stress was also lower in OsTZF1-OX than control plants treated with 250 mm NaCl stress for 3 d; however, positive up-regulation of some stress-related genes was also observed (Supplemental Fig. S9). Of the 2,051 up-regulated genes in OsTZF1-OX treated with NaCl for 2 d, only 51 and 86 genes are normally up-regulated by high-salt and dehydration stresses, respectively (Fig. 8A; Supplemental Fig. S8; Supplemental Table S3). Expression analysis of the representative up-regulated stress-responsive genes, including Tic32, RNS4, and WRKY45, showed positive regulation by high-salt stress in OsTZF1-OX plants (Supplemental Fig. S9). These results reveal that the majority of stress-related genes show an inverse relationship in the microarray analysis of OsTZF1-OX and control plants treated with 250 mm NaCl.

OsTZF1 Regulates Metal and Oligopeptide Transport-Related Genes

The microarray analysis revealed that many genes related to biotic stress response were up-regulated by OsTZF1 (Supplemental Table S1). Furthermore, genes like Ferritin, MT-type1, and ChaC-like were up-regulated and OsNRAMP1, Feroportin1, and Oligopeptide Transporter (OPT) were down-regulated (Fig. 7; Supplemental Tables S2 and S3). The OsTZF1-OX plants consistently showed a lesion-mimicking phenotype after heading and during seed setting. To test if the lesion-mimicking phenotype of the OsTZF1-OX plants was from nutrient deficiency or toxicity, we grew OsTZF1-OX and control plants in two sets of soil. One set was watered with tap water and the other set was maintained with water that had been pH adjusted to 9.5, as the availability of many nutrients, including phosphorus, nitrogen, manganese, iron, zinc, and copper, depends on optimal soil pH (Islam et al., 1980). The OsTZF1-OX plants watered with tap water developed lesions upon entering the heading and seed-setting stages. The other set of OsTZF1-OX plants, maintained with water at pH 9.5, developed no or few lesions (Supplemental Fig. S10A). We determined the contents of iron, manganese, zinc, and metalloid B in the leaves of controls and OsTZF1-OX plants at the heading stage. We found that OsTZF1-OX had slightly reduced iron accumulation and greater accumulation of manganese; however, these differences were not significant between the OsTZF1-OX and control plants (Supplemental Fig. S10B).

OsTZF1 Binds RNA in Vitro

Previously, it was shown that plant TZFs bind DNA or RNA through in vitro bead-binding assays (Pomeranz et al., 2010). We performed in vitro binding assays on recombinant OsTZF1-GST protein and homopolymers of A, U, C, and G using the RNA gel electrophoresis mobility shift assay (REMSA). We observed that OsTZF1 could only bind to U homopolymers, whereas GST alone could not bind to any of the homopolymers (Fig. 9A).

Figure 9.

Figure 9.

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RNA-binding studies of OsTZF1. A, OsTZF1 binds U homopolymers. REMSA showed that OsTZF1 forms a complex with U homopolymers. GST alone did not bind any homopolymers. B, OsTZF1 binds U-rich ARE-like sequences in the 3′ UTR of akin-β and AOS. An ARE sequence was used as a positive control (left). C, OsTZF1 protein gradient and competition assays. For the protein gradient, the OsTZF1 protein was increased 4-fold. For competition assays, corresponding cold probes were used. OsTZF1-RNA complexes are marked with arrowheads.

Down-regulated genes with U-rich sequences in their 3′ untranslated region (UTR) were selected from the microarray analysis of OsTZF1-OX. Two such genes were akin-β and AOS. The expression of these genes was increased under high-salt stress in control plants; however, their expression remained relatively low in OsTZF1-OX plants compared with controls (Fig. 8B). Because akin-β and AOS were down-regulated in OsTZF1-OX plants and contain U-rich and AU-rich elements (AREs; akin-β, 5′-UUUUUUUUUAAUUUCUCUCUUUGUUUCUU-3′; AOS-I, 5′-GUUCGAUUCCAUAUUUGUAAUUUUGUUGUUAUU-3′; AOS-II, 5′-UUGUUUGCUGUUGAAUUACGAUUGUGUAUUUUA-3′) in their 3′ UTRs, we performed REMSA to determine whether OsTZF1 binds ARE and ARE-like motifs. As shown in Figure 9B, OsTZF1 formed a complex with an ARE sequence used as a positive control, which caused a band shift. A band shift was also observed with the ARE-like motifs of akin-β and AOS (Fig. 9B). No band shift was observed when GST protein was used (Fig. 9B). Using a protein gradient of OsTZF1, an increase in the band shift was observed for the ARE-like motifs of akin-β and AOS (Fig. 9C, left panel). Furthermore, this shift was abolished when the respective competitors of akin-β and AOS were included in the experiment (Fig. 9C, right panel). Together, these results demonstrated that OsTZF1 binds to U-rich regions, AREs, and ARE-like motifs.

DISCUSSION

In humans, TZFs control the expression of cytokines, whereas in yeast (Saccharomyces cerevisiae), TZF homologs (Cth1 and Cth2) promote the reprogramming of iron-dependent metabolism and iron storage through the control of target mRNA half-lives (Blackshear et al., 2005; Puig et al., 2008). The characterization of plant-unique TZF genes has revealed their importance in development and different environmental responses (Li and Thomas, 1998; Kim et al., 2008; Grabowska et al., 2009; Guo et al., 2009; Lin et al., 2011; Lee et al., 2012), but their exact molecular mechanisms are still unknown. To determine the function of OsTZF1, we examined its expression profiles and investigated the gain- and loss-of-function phenotypes. Our results indicated that OsTZF1 is involved in seed germination, seedling growth, leaf senescence, and oxidative stress tolerance. The OsTZF1-OX plants exhibited phenotypes of reduced and delayed seed germination (Fig. 4A). Young rice seedlings were smaller but grew normally once transplanted into soil (Fig. 4B; Supplemental Fig. S3). In contrast, OsTZF1-RNAi plants showed early and enhanced seed germination and grew faster than controls (Fig. 4, A and B), suggesting that OsTZF1 plays a negative regulatory role during seed germination and seedling growth. Arabidopsis SOMNUS (AtC3H2), a homolog of OsTZF1, was reported to be a negative regulator of phytochrome-mediated seed germination and a positive regulator of ABA accumulation/negative regulator of GA accumulation through its effects on ABA/GA metabolic genes (Kim et al., 2008). AtTZF1 was also considered to affect ABA/GA-mediated growth and stress responses (Lin et al., 2011). Since ABA/GA metabolic or signaling-related genes were not detected in our microarray analysis, it is not clear at this stage whether OsTZF1 is involved in ABA/GA-mediated growth responses.

The delayed senescence of OsTZF1-OX rice was supported by the testing of different senescence-inducing factors (Fig. 6; Supplemental Fig. S7). The degrees of leaf yellowing and the rates of chlorophyll loss induced by darkness, UV-C, and H2O2 in detached OsTZF1-OX leaves were significantly lower than in the controls (Fig. 6). Senescence is affected not only by age but also by multiple other factors (Lim et al., 2007). JA-, ABA-, and stress (high-salt)-induced senescence was also delayed in OsTZF1-OX plants (Supplemental Fig. S7), indicating that the delayed senescence might be due to a common factor like tolerance to oxidative stress.

The expression of OsTZF1 was induced by ABA, JA, SA, H2O2, and several abiotic stresses (Fig. 1, A and B), suggesting that it is involved in multiple stress responses. High-salt stress at the seedling stage caused OsTZF1-RNAi plants to exhibit chlorosis and damage to leaves, whereas OsTZF1-OX plants showed enhanced tolerance (Fig. 5; Supplemental Fig. S7). DAB staining revealed higher H2O2 accumulation in control and OsTZF1-RNAi plants compared with OsTZF1-OX (Fig. 6C). Furthermore, OsTZF1-OX seedlings showed enhanced tolerance to drought stress compared with control or OsTZF1-RNAi plants (Supplemental Fig. S6). Analysis of Arabidopsis mutants with delayed-senescence phenotypes has revealed that extended longevity results in increased stress tolerance; for example, gigantea, ore1, ore3, ore9, and jub1 mutants showed increased stress tolerance (Kurepa et al., 1998; Woo et al., 2004; Wu et al., 2012). Due to an imbalance between the production and scavenging of ROS, senescence is triggered by oxidative stress and delayed senescence in plants is correlated with increased tolerance to oxidative stress (Muller et al., 2007; Wu et al., 2012). Our results suggest that OsTZF1 confers tolerance to abiotic stresses in rice by enhancing tolerance to oxidative stress.

Transcriptome analysis of OsTZF1-OX plants revealed the up-regulation of many biotic and abiotic stress-related genes, including pathogenesis-related proteins and transcription factors, peroxidases, dehydrins, metal-detoxifying proteins, and ROS homeostasis genes (Supplemental Table S2). On the other hand, genes involved in metal and oligopeptide transport as well as polyamine and secondary metabolism were down-regulated in the OsTZF1-OX plants (Supplemental Table S3). Our results indicate that OsTZF1 functions in conferring tolerance to high-salt, drought, and oxidative stress in addition to delaying leaf senescence in rice. At present, it is not known precisely how TZFs regulate gene expression changes in plants. We showed that OsTZF1 binds to the RNA of some down-regulated genes containing U-rich and ARE-like motifs in their 3′ UTR (Fig. 9). It is possible that OsTZF1 is involved in RNA turnover processes, leading to changes in gene expression.

The analysis of transcriptome expression profiles of OsTZF1-OX and control plants treated with 250 mm NaCl for 2 d revealed that out of the 2,051 up-regulated genes in OsTZF1-OX, 844 and 1,459 genes are normally down-regulated by high-salt stress and dehydration stress, respectively (Fig. 8A; Supplemental Fig. S8B). Interestingly, among the 2,141 down-regulated genes in the OsTZF1-OX rice plants, 989 and 1,561 genes are normally up-regulated by high-salt stress and dehydration stress, respectively. The inverse relationships of these stress-inducible genes upon high-salt stress emphasize that OsTZF1-OX exhibits an attenuated stress response and, in turn, attenuated stress-responsive gene expression (Fig. 8; Supplemental Fig. S8B). In other words, OsTZF1-OX and control plants exhibited different induction ratios of stress-inducible genes under 2-d salt stress (Fig. 8B). The atszf1-1 atszf2-1 double mutant is sensitive to high salt but exhibits higher induction of stress-inducible genes than controls (Sun et al., 2007). In Arabidopsis, a series of hos (for high expression of osmotic responsive genes) mutants, including hos1 (Lee et al., 2001), hos5 (Xiong et al., 1999), and hos9 (Zhu et al., 2004), higher expression of stress-responsive genes than controls in response to abiotic stresses. The Arabidopsis mutants fiery1 and fiery2 displayed induction of ABA- and stress-responsive genes (Xiong et al., 2001, 2002). Surprisingly, all of these mutants were compromised in their tolerance to the tested abiotic stress conditions. These mutants are reminiscent of our microarray data showing an inverse relationship between the induction of stress-responsive genes and stress tolerance.

Leaves at different stages of development respond differently to identical treatments; for example, older leaves respond to ethylene but younger leaves do not (Grbic and Bleecker, 1995; Weaver et al., 1998). The chloroplast biogenesis protein Tic32 was up-regulated in OsTZF1-OX plants under both normal and stress conditions (Supplemental Fig. S9). This might lead to differential chloroplast development in OsTZF1-OX and control plants and subsequently differential retrograde responses. Taking into consideration the delayed-senescence phenotype and tolerance to ROS of the OsTZF1-OX plants, their stress-hyposensitive response is understandable.

The OsTZF1-OX plants displayed a lesion-mimicking phenotype during heading and seed setting (Fig. 4C). Microarray analysis revealed many genes related to biotic stress responses (Supplemental Tables S2 and S3). The products of these genes might be responsible for the lesion-mimicking phenotype. Furthermore, the up-regulation of Ferritin, MT-type1, and ChaC-like proteins and the down-regulation of OsNRAMP1, Feroportin1, and OPT points toward a role for metal ions in the lesion-mimicking phenotype of OsTZF1-OX plants (Fig. 7; Supplemental Tables S2 and S3). High-pH (9.5) water rescued the lesion-mimicking phenotype in OsTZF1-OX plants (Supplemental Fig. S10A). The toxicities of iron and manganese produce necrotic phenotypes, and the absorption of iron and manganese are particularly perturbed by high pH. To address the question directly, we determined the metal contents of iron, manganese, zinc, and metalloid B in the leaves of control and OsTZF1-OX plants at the heading stage. The results showed that OsTZF1-OX plants had slightly reduced iron accumulation and higher accumulation of manganese; however, these differences were not significant between OsTZF1-OX and control plants (Supplemental Fig. S10B). Furthermore, high-pH treatment lowered the overall content of the above metals, but there was little difference between OsTZF1-OX and control plants (data not shown). This inconsistency might be due to the differential accumulation of metal ions in different tissues, organs, and cells of rice plants. It is also possible that other undetermined metal ions, unknown xenobiotics, or oligopeptides are responsible for the lesion-mimicking phenotype of OsTZF1-OX plants. For example, rice Sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis (Fujiwara et al., 2010). Expression of the SL gene (Os12g0268000) was down-regulated in OsTZF1-OX plants treated with NaCl for 2 d (Supplemental Table S4). Further work is required to determine the precise function of OsTZF1 in relation to this lesion-mimicking phenotype in rice.

Most of the CCCH finger proteins reported to date (e.g. OsDOS, HUA1, PEI1, AtSZF1, and SOMNUS) are localized in the nucleus and function as transcriptional regulators, except HUA1, which is an RNA-binding protein (Li and Thomas, 1998, Li et al., 2001; Kong et al., 2006; Sun et al., 2007; Kim et al., 2008). We showed that OsTZF1 is predominantly localized in the cytoplasm and cytoplasmic foci under stress conditions (Fig. 3). Although OsTZF1 was occasionally observed in the nucleus (Supplemental Fig. S2), no transcriptional activation was observed in relation to OsTZF1 (data not shown), and it probably does not function as a transcriptional regulator. Some studies have revealed that CCCH zinc finger proteins affect mRNA stability, as mentioned earlier (Addepalli and Hunt, 2008; Pomeranz et al., 2010); thus, it can be speculated that OsTZF1 participates in RNA metabolism. To address whether OsTZF1 has RNA-binding activity, we carried out REMSA. The results showed that OsTZF1 binds U-rich, ARE, and ARE-like motifs (Fig. 9). This observation suggests that the OsTZF1 protein is likely to be involved in RNA metabolism. During the preparation of this paper, Lee et al. (2012) reported that the Arabidopsis CCCH-type zinc finger proteins AtC3H49/AtTZF3 and AtC3H20/AtTZF2 are involved in ABA and JA responses and that their recombinant proteins displayed RNase activity in vitro, suggesting that they might be involved in the mRNA turnover process. Although OsTZF1 could bind to U-rich and ARE-like motifs in the 3′ UTR of akin-β and AOS, it remains to be established whether they are the real target mRNAs of OsTZF1 and how it affects their metabolism.

Based on the available experimental data showing that OsTZF1 transcription is activated by ABA, JA, SA, H2O2, and abiotic stresses such as high-salt or drought stress and that it confers stress tolerance, we suggest that OsTZF1 assists in regulating cellular ROS homeostasis. OsTZF1 expression lowers ROS concentration in plant tissues through redox homeostasis genes like Ferritins/MTs and ROS-scavenging enzymes such as peroxidases and GSTs (Supplemental Table S1), while the opposite effect may be speculated in the OsTZF1-RNAi plants. Concomitant with enhanced OsTZF1 expression and reduced ROS accumulation, the induction ratio of significant numbers of stress-related genes was compromised in 2-d NaCl-treated OsTZF1-OX plants. We propose that OsTZF1 lowers the cellular ROS level, thereby minimizing the stimulatory effect on stress-related genes. In addition, there is the possibility that OsTZF1 regulates stress tolerance and senescence through other target genes of currently unknown molecular function. Future work will be required to address the intricacies of the underlying regulatory network in greater detail.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Rice (Oryza sativa ssp. japonica ‘Nipponbare’) was used for various experiments in this study, including expression analysis, transformation, and hormone treatments, as described previously (Ito et al., 2006; Nakashima et al., 2007). Seedlings of rice were grown in basal nutrient solution (Makino et al., 1988). Two-week-old rice plants were dehydrated in a plastic box for dehydration treatment. Two-week-old rice plants were transferred from the basal nutrient solution to nutrient solutions containing 250 mm NaCl, 10 mm H2O2, 10 μm ABA, 10 μm methyl jasmonate, or 100 μm SA for stress treatments.

RNA Gel-Blot Analysis and Quantitative PCR Analysis

Isolation of total RNA, RNA gel-blot hybridization, and qRT-PCR analysis were performed as described previously (Nakashima et al., 2007). The 5′ end-specific DNA fragment (approximately 300 bp) of the OsTZF1 full-length complementary DNA was used as a probe for hybridization. Gene-specific probes were used for the other genes in the RNA gel-blot analysis (Supplemental Table S5). The primers used in qRT-PCR are listed in Supplemental Table S5.

Analysis of Transgenic Rice Plants Containing OsTZF1 promoter:GUS fusions

The promoter region of OsTZF1, 1,417 bp upstream of ATG (POsTZF1), was amplified from the rice genome by PCR using KOD DNA polymerase (Toyobo; http://www.toyobo.co.jp/bio). The primers used are listed in Supplemental Table S5. The OsTZF1 promoter fragment was inserted into the SmaI site of the GUS expression vector pGreenII 0129 (Hellens et al., 2000), generating POsTZF1:GUS. Confirmation of the fusion constructs, transformation of rice, and growth of the transgenic plants were performed as described previously (Ito et al., 2006; Nakashima et al., 2007). Histochemical activity of GUS in POsTZF1:GUS transgenic plant materials was detected according to Jefferson et al. (1987). Quantitative analysis of GUS activity was performed as described previously (Nakashima and Yamaguchi-Shinozaki, 2002).

Transient and Stable Expression of the sGFP Proteins

The OsTZF1 coding region was fused to sGFP (Chiu et al., 1996; Nakashima et al., 1997) using the pGreenII 0129 vector (Hellens et al., 2000) either with a ubiquitin or OsTZF1 native promoter. These constructs were used for transient and stable expression of sGFP proteins. For transient expression, the DNA constructs were introduced into onion (Allium cepa) epidermal cells as described previously (Fujita et al., 2004). After incubation at 22°C for 12 h, GFP fluorescence was observed using a confocal laser scanning microscope (LSM5 PASAL; Carl Zeiss; http://www.zeiss.com/). Stable transgenic rice plants expressing OsTZF1-sGFP under the control of the native OsTZF1 promoter were generated as described above. Cellular localization of the OsTZF1-sGFP proteins in the lateral root cells of transgenic rice plants was observed as described above. For colocalization, the OsTZF1 coding region was fused with yellow fluorescent protein, and the DCP2 and PABP8 coding regions were fused with cyan fluorescent protein in frame in the pGreenII vector. These constructs were introduced into rice mesophyll protoplasts and observed as described above.

Analysis of OsTZF1-OX and OsTZF1-RNAi Plants

To generate transgenic rice plants overexpressing OsTZF1 (OsTZF1-OX), we used the pBIG-ubi vector (Becker, 1990; Ito et al., 2006). For the generation of RNA knockdown plants (OsTZF1-RNAi), the pANDA vector was used (Miki et al., 2005). The primers used are listed in Supplemental Table S5. We introduced the constructs into wild-type rice ‘Nipponbare’ by Agrobacterium tumefaciens-mediated transformation (Goto et al., 1999), and T2 and T3 seeds were used in further experiments. Transgenic plants (OsTZF1-OX and OsTZF1-RNAi) and vector controls were grown in the isolation greenhouse under normal growth conditions (14 h of light at 28°C/10 h of dark at 25°C).

Stress Tolerance of the Transgenic Rice Plants

For high-salt stress treatment, 2-week-old rice seedlings grown in soil were transferred to 250 mm NaCl solution for 3 d. After the stress treatment, plants were allowed to recover under normal growth conditions for 2 weeks. The number of plants that survived and continued to grow were counted. The statistical significance of the values was determined using the χ2 test. Photosynthetic activity (Fv/Fm) was measured according to the method described by Kasuga et al. (2004). For drought stress treatment, 2-week-old rice seedlings grown in soil were dehydrated. After the stress treatment, plants were allowed to recover under normal growth conditions for 2 weeks. The number of plants that survived and continued to grow was counted. The statistical significance of the values was determined using the χ2 test.

Senescence Testing Using Leaf Fragments

Leaf fragments from 6-week-old OsTZF1-OX and OsTZF1-RNAi rice plants were used in experiments with dark, UV-C, and H2O2 treatments. For UV-C treatment, leaf fragments on petri plates covered with lids were incubated under 15-W UV-C tube light (254 nm) for 3 d. For H2O2 treatment, detached leaf fragments were incubated in water (mock) and solution containing 10 mm H2O2 for the time periods indicated. The respective leaf fragments were used for the detection of ROS by DAB staining (Thordal-Christensen et al., 1997). For dark treatment, leaf fragments from 6-week-old OsTZF1-OX plants were incubated in water (mock) or solutions containing 10 μm ABA, 10 μm methyl jasmonate, or 250 mm NaCl in the dark for 5 d.

Microarray Analysis

Two-week-old seedlings of OsTZF1-OX and control rice grown in soil, untreated or treated with 250 mm NaCl for 2 d, were harvested, and total RNA was isolated by the TRIzol method (Invitrogen; http://www.invitrogen.com/). Total RNA was used for the preparation of Cy5- and Cy3-labeled complementary RNA, and these were subjected to microarray experiments using the 44K rice oligo microarray (Agilent Technologies; http://www.home.agilent.com). All microarray experiments, including data analysis, were carried out as described previously (Nakashima et al., 2007). The microarray data for “OsTZF1_untreated” were assigned accession number E-MEXP-3700, and the microarray data for “OsTZF1_NaCl 2days” were assigned accession number E-MEXP-3701 in MIAMExpress (www.ebi.ac.uk/miamexpress/).

Measurement of Iron, Manganese, Zinc, and Metalloid B in OsTZF1-OX and Control Plants

Metal contents in OsTZF1-OX and control plants were measured by inductively coupled plasma optical emission spectrometer (SPS-3500, SII; Chiba). Shoots were dried for 24 h and dissected into pieces by a pair of ceramic scissors. The dried samples (approximately 100 mg) were carefully weighed in Teflon cups and decomposed by nitric acid in Teflon bombs heated at 150°C for 10 h. The sample solutions were made to 10 mL with 0.1 m nitric acid and filtrated by polytetrafluoroethylene membranes (0.45 mm) before analysis.

RNA-Binding Assay

REMSA was conducted as described by Brewer et al. (2004). RNA probes were synthesized by cloning probe sequences into pBluescript KS+ containing an upstream T7 promoter. The primers used are listed in Supplemental Table S5. Plasmid vectors were linearized using the KpnI restriction enzyme and then used for RNA synthesis labeled with [α-32P]UTP using the Promega T7 Riboprobe Kit. Binding reactions consisted of each protein being incubated with 1 × 106 cpm of various radiolabeled RNA probes. Each reaction was assembled in a final volume of 20 μL containing 10 mm Tris-HCl (pH 8.0), 40 mm KCl, 2 mm dithiothreitol, 3 mm MgCl2, 50 μm ZnCl2, 10 mm HEPES, 10% glycerol, and 20 μg μL−1 heparin. Binding reactions were incubated at room temperature for 10 min and then separated using electrophoresis through a 6% (40:1 acrylamide:bis-acrylamide) native gel at 4°C. After electrophoresis, gels were exposed using phosphorescence imaging (Fuji Film).

Supplemental Data

The following materials are available in the online version of this article.

Acknowledgments

We are grateful for the technical support provided by Kaoru Amano, Emiko Kishi, Kyoko Murai, and Kyouko Yoshiwara of the Japan International Research Center for Agricultural Sciences. We thank Masami Toyoshima of the Japan International Research Center for Agricultural Sciences for her editorial assistance. We thank Masaki Mori of the National Institute of Agrobiological Sciences, Japan, for helpful comments.

Glossary

ABA

abscisic acid

H2O2

hydrogen peroxide

SA

salicylic acid

JA

jasmonic acid

PB

P body

SG

stress granule

OX

overexpression

RNAi

RNA interference

DAI

days after imbibition

Fv/Fm

maximum photochemical efficiency of PSII in the dark-adapted state

ROS

reactive oxygen species

DAB

diaminobenzidine

GST

glutathione S-transferase

qRT

quantitative reverse transcription

REMSA

RNA gel electrophoresis mobility shift assay

UTR

untranslated region

ARE

AU-rich element

References

  1. Abe H, Urao T, Ito T, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. (2003) Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15: 63–78 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Addepalli B, Hunt AG. (2008) Ribonuclease activity is a common property of Arabidopsis CCCH-containing zinc-finger proteins. FEBS Lett 582: 2577–2582 [DOI] [PubMed] [Google Scholar]
  3. Al-Souhibani N, Al-Ahmadi W, Hesketh JE, Blackshear PJ, Khabar KS. (2010) The RNA-binding zinc-finger protein tristetraprolin regulates AU-rich mRNAs involved in breast cancer-related processes. Oncogene 29: 4205–4215 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson P, Kedersha N. (2008) Stress granules: the Tao of RNA triage. Trends Biochem Sci 33: 141–150 [DOI] [PubMed] [Google Scholar]
  5. Balagopal V, Parker R. (2009) Polysomes, P bodies and stress granules: states and fates of eukaryotic mRNAs. Curr Opin Cell Biol 21: 403–408 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bartels D, Sunkar R. (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24: 23–58 [Google Scholar]
  7. Becker D. (1990) Binary vectors which allow the exchange of plant selectable markers and reporter genes. Nucleic Acids Res 18: 203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Berg JM, Shi Y. (1996) The galvanization of biology: a growing appreciation for the roles of zinc. Science 271: 1081–1085 [DOI] [PubMed] [Google Scholar]
  9. Blackshear PJ, Phillips RS, Ghosh S, Ramos SVB, Richfield EK, Lai WS. (2005) Zfp3613, a rodent X chromosome gene encoding a placenta-specific member of the tristetraprolin family of CCCH tandem zinc finger proteins. Biol Reprod 73: 1072. [DOI] [PubMed] [Google Scholar]
  10. Brewer BY, Malicka J, Blackshear PJ, Wilson GM. (2004) RNA sequence elements required for high affinity binding by the zinc finger domain of tristetraprolin: conformational changes coupled to the bipartite nature of Au-rich mRNA-destabilizing motifs. J Biol Chem 279: 27870–27877 [DOI] [PubMed] [Google Scholar]
  11. Carballo E, Lai WS, Blackshear PJ. (1998) Feedback inhibition of macrophage tumor necrosis factor-alpha production by tristetraprolin. Science 281: 1001–1005 [DOI] [PubMed] [Google Scholar]
  12. Carballo E, Lai WS, Blackshear PJ. (2000) Evidence that tristetraprolin is a physiological regulator of granulocyte-macrophage colony-stimulating factor messenger RNA deadenylation and stability. Blood 95: 1891–1899 [PubMed] [Google Scholar]
  13. Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J. (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6: 325–330 [DOI] [PubMed] [Google Scholar]
  14. Ciftci-Yilmaz S, Mittler R. (2008) The zinc finger network of plants. Cell Mol Life Sci 65: 1150–1160 [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Cui J, Jander G, Racki LR, Kim PD, Pierce NE, Ausubel FM. (2002) Signals involved in Arabidopsis resistance to Trichoplusia ni caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae. Plant Physiol 129: 551–564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Eulgem T, Rushton PJ, Robatzek S, Somssich IE. (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5: 199–206 [DOI] [PubMed] [Google Scholar]
  17. Fowler S, Thomashow MF. (2002) Arabidopsis transcriptome profiling indicates that multiple regulatory pathways are activated during cold acclimation in addition to the CBF cold response pathway. Plant Cell 14: 1675–1690 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Fujita M, Fujita Y, Maruyama K, Seki M, Hiratsu K, Ohme-Takagi M, Tran LSP, Yamaguchi-Shinozaki K, Shinozaki K. (2004) A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway. Plant J 39: 863–876 [DOI] [PubMed] [Google Scholar]
  19. Fujiwara T, Maisonneuve S, Isshiki M, Mizutani M, Chen L, Wong HL, Kawasaki T, Shimamoto K. (2010) Sekiguchi lesion gene encodes a cytochrome P450 monooxygenase that catalyzes conversion of tryptamine to serotonin in rice. J Biol Chem 285: 11308–11313 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Goto F, Yoshihara T, Shigemoto N, Toki S, Takaiwa F. (1999) Iron fortification of rice seed by the soybean ferritin gene. Nat Biotechnol 17: 282–286 [DOI] [PubMed] [Google Scholar]
  21. Grabowska A, Wisniewska A, Tagashira N, Malepszy S, Filipecki M. (2009) Characterization of CsSEF1 gene encoding putative CCCH-type zinc finger protein expressed during cucumber somatic embryogenesis. J Plant Physiol 166: 310–323 [DOI] [PubMed] [Google Scholar]
  22. Grbic V, Bleecker AB. (1995) Ethylene regulates the timing of leaf senescence in Arabidopsis. Plant J 8: 595–602 [Google Scholar]
  23. Guo YH, Yu YP, Wang D, Wu CA, Yang GD, Huang JG, Zheng CC. (2009) GhZFP1, a novel CCCH-type zinc finger protein from cotton, enhances salt stress tolerance and fungal disease resistance in transgenic tobacco by interacting with GZIRD21A and GZIPR5. New Phytol 183: 62–75 [DOI] [PubMed] [Google Scholar]
  24. Hellens RP, Edwards EA, Leyland NR, Bean S, Mullineaux PM. (2000) pGreen: a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation. Plant Mol Biol 42: 819–832 [DOI] [PubMed] [Google Scholar]
  25. Higo K, Ugawa Y, Iwamoto M, Korenaga T. (1999) Plant cis-acting regulatory DNA elements (PLACE) database: 1999. Nucleic Acids Res 27: 297–300 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Islam AKMS, Edwards DG, Asher CJ. (1980) pH optima for crop growth: results of a flowing solution culture experiment with six species. Plant Soil 54: 339–357 [Google Scholar]
  27. Ito Y, Katsura K, Maruyama K, Taji T, Kobayashi M, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. (2006) Functional analysis of rice DREB1/CBF-type transcription factors involved in cold-responsive gene expression in transgenic rice. Plant Cell Physiol 47: 141–153 [DOI] [PubMed] [Google Scholar]
  28. Iwasaki S, Takeda A, Motose H, Watanabe Y. (2007) Characterization of Arabidopsis decapping proteins AtDCP1 and AtDCP2, which are essential for post-embryonic development. FEBS Lett 581: 2455–2459 [DOI] [PubMed] [Google Scholar]
  29. Jefferson RA, Kavanagh TA, Bevan MW. (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Jeong MS, Kim EJ, Jang SB. (2010) Expression and RNA-binding of human zinc-finger antiviral protein. Biochem Biophys Res Commun 396: 696–702 [DOI] [PubMed] [Google Scholar]
  31. Kasuga M, Miura S, Shinozaki K, Yamaguchi-Shinozaki K. (2004) A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer. Plant Cell Physiol 45: 346–350 [DOI] [PubMed] [Google Scholar]
  32. Kim DH, Yamaguchi S, Lim S, Oh E, Park J, Hanada A, Kamiya Y, Choi G. (2008) SOMNUS, a CCCH-type zinc finger protein in Arabidopsis, negatively regulates light-dependent seed germination downstream of PIL5. Plant Cell 20: 1260–1277 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Kong Z, Li M, Yang W, Xu W, Xue Y. (2006) A novel nuclear-localized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant Physiol 141: 1376–1388 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kosová K, Vítámvás P, Prášil IT, Renaut J. (2011) Plant proteome changes under abiotic stress: contribution of proteomics studies to understanding plant stress response. J Proteomics 74: 1301–1322 [DOI] [PubMed] [Google Scholar]
  35. Kurepa J, Smalle J, Van Montagu M, Inzé D. (1998) Oxidative stress tolerance and longevity in Arabidopsis: the late-flowering mutant gigantea is tolerant to paraquat. Plant J 14: 759–764 [DOI] [PubMed] [Google Scholar]
  36. Lee HJ, Xiong LM, Gong ZZ, Ishitani M, Stevenson B, Zhu JK. (2001) The Arabidopsis HOS1 gene negatively regulates cold signal transduction and encodes a RING finger protein that displays cold-regulated nucleo-cytoplasmic partitioning. Genes Dev 15: 912–924 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Lee SJ, Jung HJ, Kang H, Kim SY. (2012) Arabidopsis zinc finger proteins AtC3H49/AtTZF3 and AtC3H20/AtTZF2 are involved in ABA and JA responses. Plant Cell Physiol 53: 673–686 [DOI] [PubMed] [Google Scholar]
  38. Leshem YY. (1988) Plant senescence processes and free radicals. Free Radic Biol Med 5: 39–49 [DOI] [PubMed] [Google Scholar]
  39. Li J, Jia D, Chen X. (2001) HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein. Plant Cell 13: 2269–2281 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Li Z, Thomas TL. (1998) PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis. Plant Cell 10: 383–398 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Lim PO, Kim HJ, Nam HG. (2007) Leaf senescence. Annu Rev Plant Biol 58: 115–136 [DOI] [PubMed] [Google Scholar]
  42. Lin PC, Pomeranz MC, Jikumaru Y, Kang SG, Hah C, Fujioka S, Kamiya Y, Jang JC. (2011) The Arabidopsis tandem zinc finger protein AtTZF1 affects ABA- and GA-mediated growth, stress and gene expression responses. Plant J 65: 253–268 [DOI] [PubMed] [Google Scholar]
  43. Mackay JP, Crossley M. (1998) Zinc fingers are sticking together. Trends Biochem Sci 23: 1–4 [DOI] [PubMed] [Google Scholar]
  44. Makino A, Mae T, Ohira K. (1988) Differences between wheat and rice in the enzymic properties of ribulose-1,5-bisphosphate carboxylase oxygenase and the relationship to photosynthetic gas-exchange. Planta 174: 30–38 [DOI] [PubMed] [Google Scholar]
  45. Maruyama K, Todaka D, Mizoi J, Yoshida T, Kidokoro S, Matsukura S, Takasaki H, Sakurai T, Yamamoto YY, Yoshiwara K, et al. (2012) Identification of cis-acting promoter elements in cold- and dehydration-induced transcriptional pathways in Arabidopsis, rice and soybean. DNA Res 9: 37–49 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Miki D, Itoh R, Shimamoto K. (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol 138: 1903–1913 [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Moore M, Ullman C. (2003) Recent developments in the engineering of zinc finger proteins. Brief Funct Genomics Proteomics 1: 342–355 [DOI] [PubMed] [Google Scholar]
  48. Mukhopadhyay A, Vij S, Tyagi AK. (2004) Overexpression of a zinc-finger protein gene from rice confers tolerance to cold, dehydration, and salt stress in transgenic tobacco. Proc Natl Acad Sci USA 101: 6309–6314 [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Muller FL, Lustgarten MS, Jang Y, Richardson A, Van Remmen H. (2007) Trends in oxidative aging theories. Free Radic Biol Med 43: 477–503 [DOI] [PubMed] [Google Scholar]
  50. Nakashima K, Kiyosue T, Yamaguchi-Shinozaki K, Shinozaki K. (1997) A nuclear gene, erd1, encoding a chloroplast-targeted Clp protease regulatory subunit homolog is not only induced by water stress but also developmentally up-regulated during senescence in Arabidopsis thaliana. Plant J 12: 851–861 [DOI] [PubMed] [Google Scholar]
  51. Nakashima K, Tran LS, Van Nguyen D, Fujita M, Maruyama K, Todaka D, Ito Y, Hayashi N, Shinozaki K, Yamaguchi-Shinozaki K. (2007) Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J 51: 617–630 [DOI] [PubMed] [Google Scholar]
  52. Nakashima K, Yamaguchi-Shinozaki K (2002) Use of β-glucuronidase to show dehydration and high-salt gene expression. In JF Jackson, HF Linskens, eds, Molecular Methods of Plant Analysis, Vol 22: Testing for Genetic Manipulation in Plants. Springer-Verlag, Berlin, pp 37–61 [Google Scholar]
  53. Newbury SF, Mühlemann O, Stoecklin G. (2006) Turnover in the Alps: an mRNA perspective. Workshops on mechanisms and regulation of mRNA turnover. EMBO Rep 7: 143–148 [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Parker R, Sheth U. (2007) P bodies and the control of mRNA translation and degradation. Mol Cell 25: 635–646 [DOI] [PubMed] [Google Scholar]
  55. Pechanova O, Hsu CY, Adams JP, Pechan T, Vandervelde L, Drnevich J, Jawdy S, Adeli A, Suttle JC, Lawrence AM, et al. (2010) Apoplast proteome reveals that extracellular matrix contributes to multistress response in poplar. BMC Genomics 11: 674. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Pomeranz MC, Hah C, Lin PC, Kang SG, Finer JJ, Blackshear PJ, Jang JC. (2010) The Arabidopsis tandem zinc finger protein AtTZF1 traffics between the nucleus and cytoplasmic foci and binds both DNA and RNA. Plant Physiol 152: 151–165 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Puig S, Vergara SV, Thiele DJ. (2008) Cooperation of two mRNA-binding proteins drives metabolic adaptation to iron deficiency. Cell Metab 7: 555–564 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Rabbani MA, Maruyama K, Abe H, Khan MA, Katsura K, Ito Y, Yoshiwara K, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. (2003) Monitoring expression profiles of rice genes under cold, drought, and high-salinity stresses and abscisic acid application using cDNA microarray and RNA gel-blot analyses. Plant Physiol 133: 1755–1767 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Ronald P, Leung H. (2002) The rice genome: the most precious things are not jade and pearls. Science 296: 58–59 [DOI] [PubMed] [Google Scholar]
  60. Sakamoto H, Maruyama K, Sakuma Y, Meshi T, Iwabuchi M, Shinozaki K, Yamaguchi-Shinozaki K. (2004) Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. Plant Physiol 136: 2734–2746 [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Schumann U, Prestele J, O’Geen H, Brueggeman R, Wanner G, Gietl C. (2007) Requirement of the C3HC4 zinc RING finger of the Arabidopsis PEX10 for photorespiration and leaf peroxisome contact with chloroplasts. Proc Natl Acad Sci USA 104: 1069–1074 [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Seki M, Narusaka M, Ishida J, Nanjo T, Fujita M, Oono Y, Kamiya A, Nakajima M, Enju A, Sakurai T, et al. (2002) Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray. Plant J 31: 279–292 [DOI] [PubMed] [Google Scholar]
  63. Shimada M, Kawahara H, Doi H. (2002) Novel family of CCCH-type zinc-finger proteins, MOE-1, -2 and -3, participates in C. elegans oocyte maturation. Genes Cells 7: 933–947 [DOI] [PubMed] [Google Scholar]
  64. Simpson SD, Nakashima K, Narusaka Y, Seki M, Shinozaki K, Yamaguchi-Shinozaki K. (2003) Two different novel cis-acting elements of erd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence. Plant J 33: 259–270 [DOI] [PubMed] [Google Scholar]
  65. Sun J, Jiang H, Xu Y, Li H, Wu X, Xie Q, Li C. (2007) The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis. Plant Cell Physiol 48: 1148–1158 [DOI] [PubMed] [Google Scholar]
  66. Thompson BG, Lake BH. (1987) The effect of radiation on the long term productivity of a plant based CELSS. Adv Space Res 7: 133–140 [DOI] [PubMed] [Google Scholar]
  67. Thordal-Christensen H, Zhang ZG, Wei YD, Collinge DB. (1997) Subcellular localization of H2O2 in plants: H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11: 1187–1194 [Google Scholar]
  68. Urao T, Yamaguchi-Shinozaki K, Urao S, Shinozaki K. (1993) An Arabidopsis myb homolog is induced by dehydration stress and its gene product binds to the conserved MYB recognition sequence. Plant Cell 5: 1529–1539 [DOI] [PMC free article] [PubMed] [Google Scholar]
  69. Varnum BC, Ma QF, Chi TH, Fletcher B, Herschman HR. (1991) The TIS11 primary response gene is a member of a gene family that encodes proteins with a highly conserved sequence containing an unusual Cys-His repeat. Mol Cell Biol 11: 1754–1758 [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Wang D, Guo Y, Wu C, Yang G, Li Y, Zheng C. (2008) Genome-wide analysis of CCCH zinc finger family in Arabidopsis and rice. BMC Genomics 9: 44. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Weaver LM, Gan S, Quirino B, Amasino RM. (1998) A comparison of the expression patterns of several senescence-associated genes in response to stress and hormone treatment. Plant Mol Biol 37: 455–469 [DOI] [PubMed] [Google Scholar]
  72. Weber C, Nover L, Fauth M. (2008) Plant stress granules and mRNA processing bodies are distinct from heat stress granules. Plant J 56: 517–530 [DOI] [PubMed] [Google Scholar]
  73. Woo HR, Kim JH, Nam HG, Lim PO. (2004) The delayed leaf senescence mutants of Arabidopsis, ore1, ore3, and ore9 are tolerant to oxidative stress. Plant Cell Physiol 45: 923–932 [DOI] [PubMed] [Google Scholar]
  74. Wu AH, Allu AD, Garapati P, Siddiqui H, Dortay H, Zanor MI, Asensi-Fabado MA, Munné-Bosch S, Antonio C, Tohge T, et al. (2012) JUNGBRUNNEN1, a reactive oxygen species-responsive NAC transcription factor, regulates longevity in Arabidopsis. Plant Cell 24: 482–506 [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Xiong L, Ishitani M, Lee H, Zhu JK. (1999) HOS5: a negative regulator of osmotic stress-induced gene expression in Arabidopsis thaliana. Plant J 19: 569–578 [DOI] [PubMed] [Google Scholar]
  76. Xiong L, Lee Bh, Ishitani M, Lee H, Zhang C, Zhu JK. (2001) FIERY1 encoding an inositol polyphosphate 1-phosphatase is a negative regulator of abscisic acid and stress signaling in Arabidopsis. Genes Dev 15: 1971–1984 [DOI] [PMC free article] [PubMed] [Google Scholar]
  77. Xiong LM, Lee H, Ishitani M, Tanaka Y, Stevenson B, Koiwa H, Bressan RA, Hasegawa PM, Zhu JK. (2002) Repression of stress-responsive genes by FIERY2, a novel transcriptional regulator in Arabidopsis. Proc Natl Acad Sci USA 99: 10899–10904 [DOI] [PMC free article] [PubMed] [Google Scholar]
  78. Yamaguchi-Shinozaki K, Shinozaki K. (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57: 781–803 [DOI] [PubMed] [Google Scholar]
  79. Zhu JH, Shi HZ, Lee BH, Damsz B, Cheng S, Stirm V, Zhu JK, Hasegawa PM, Bressan RA. (2004) An Arabidopsis homeodomain transcription factor gene, HOS9, mediates cold tolerance through a CBF-independent pathway. Proc Natl Acad Sci USA 101: 9873–9878 [DOI] [PMC free article] [PubMed] [Google Scholar]

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