A rice calciumdependent protein kinase ... - Wiley Online Library

The Plant Journal (2012) 69, 26–36

doi: 10.1111/j.1365-313X.2011.04766.x

A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance Takayuki Asano1,2,*, Nagao Hayashi1, Michie Kobayashi1,†, Naohiro Aoki2, Akio Miyao1, Ichiro Mitsuhara1, Hiroaki Ichikawa1, Setsuko Komatsu1,‡, Hirohiko Hirochika1, Shoshi Kikuchi1 and Ryu Ohsugi2,* 1 National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan, and 2 Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan Received 6 June 2011; revised 23 August 2011; accepted 24 August 2011; published online 10 October 2011. * For correspondence (fax +81 29 838 7007; e-mail [email protected], fax +81 3 5841 8048; e-mail [email protected]). † Present address: National Institute of Floricultural Science, Tsukuba 305-8519, Japan. ‡ Present address: National Institute of Crop Science, Tsukuba 305-8518, Japan.

SUMMARY Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant’s response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H2O2) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H2O2 accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance. Keywords: calcium-dependent protein kinase, salt stress, reactive oxygen species, abscisic acid, rice.

INTRODUCTION Soil salinity affects the growth, productivity and quality of crops in agricultural fields. High salinity stress induces a range of molecular, cellular and physiological responses in plants (Xiong et al., 2002; Vinocur and Altman, 2005). The phytohormone abscisic acid (ABA) regulates a range of physiological processes in plants, including seed germination, dormancy, seedling growth, stomatal movements and tolerance to various abiotic stresses (Finkelstein et al., 2002; Zhu, 2002; Cutler et al., 2010). ABA accumulates inside the plant under high salinity conditions, and plays pivotal roles in the plant’s stress responses by modulating the gene expression profile and cellular processes (Jia et al., 2002; Hirayama and Shinozaki, 2010; Raghavendra et al., 2010). ABA is also involved in the biotic stress response (Mauch26

Mani and Mauch, 2005; Ton et al., 2009). The exogenous application of ABA results in increased susceptibility to bacterial and fungal pathogens (Asselbergh et al., 2008; Fan et al., 2009; Jiang et al., 2010). ABA-deficient mutants show enhanced resistance to various pathogens (Audenaert et al., 2002; Anderson et al., 2004; Asselbergh et al., 2008; de Torres Zabala et al., 2009). The production of reactive oxygen species (ROS), including singlet oxygen, the superoxide radical ion and hydrogen peroxide (H2O2), is induced by ABA, and by biotic and abiotic stresses (Apel and Hirt, 2004; Miller et al., 2008, 2010). ROS function as signal transduction molecules in many biological processes, including plant growth and development, the cell cycle, programmed cell death, hormone signaling, and ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd

OsCPK12 modulates abiotic and biotic stress responses 27 biotic and abiotic stress responses (Apel and Hirt, 2004; Foyer and Noctor, 2005; Torres and Dangl, 2005; Miller et al., 2008). ABA-induced ROS are generated by NADPH oxidase (Kwak et al., 2003). A double loss of function of genes encoding the NADPH oxidase catalytic subunits AtrbohD/ AtrbohF impairs ABA-induced H2O2 production and ABAinduced stomatal closing (Kwak et al., 2003). Whereas ROS are important second messengers in ABA signaling in guard cells, ROS are toxic molecules that cause oxidative damage to cells (Apel and Hirt, 2004). To minimize and/or prevent oxidative damage to cells by ROS and to maintain cellular redox homeostasis, plants have evolved defense systems that include ROS scavenging enzymes, such as ascorbate peroxidase (APx), superoxide dismutase, catalase and glutathione peroxidase (Apel and Hirt, 2004; Miller et al., 2010). Various studies have shown that the ROS scavenging enzymes are involved in tolerance to abiotic stress in plants (Tsugane et al., 1999; Badawi et al., 2004; Kotchoni et al., 2006; Miller et al., 2007; Tseng et al., 2007). A gain-offunction mutant of Arabidopsis in which ASCORBATE PEROXIDASE 2 is constitutively overexpressed shows drought tolerance, and exhibits an improved efficiency of water use (Rossel et al., 2006). Transgenic Arabidopsis plants overexpressing OsAPXa or OsAPXb exhibit increased tolerance to salt stress (Lu et al., 2007). Although the delicate balance between ROS production and detoxification is modulated by a large and complicated network of genes, the molecular mechanisms underlying ROS production and detoxification in plants are largely unknown. Each calcium-dependent protein kinase (CDPK) consists of a variable N-terminal domain and several functional domains, including a protein kinase domain, an autoinhibitory region and a calmodulin-like domain (Harper et al., 1991; Harmon et al., 2000; Cheng et al., 2002; Hrabak et al., 2003). CDPKs are directly activated by the binding of Ca2+ to the calmodulin-like domain, and activated CDPKs regulate downstream components of calcium signaling (Harper et al., 2004; Harper and Harmon, 2005). CDPKs have been identified throughout the plant kingdom (Ludwig et al., 2004; Harper and Harmon, 2005) and in some protozoans (Ward et al., 2004), but not in animals (Cheng et al., 2002; Hrabak et al., 2003; Harper and Harmon, 2005). CDPKs constitute a large multigene family consisting of 34 genes in Arabidopsis (Cheng et al., 2002; Hrabak et al., 2003) and 29 genes in Oryza sativa (rice; Asano et al., 2005). CDPKs are involved in various physiological processes in plants. Analysis of the Arabidopsis cpk3 cpk6 double knock-out mutant indicated that CPK3 and CPK6 regulate ion channel activity in guard cells, and are involved in ABA-regulated stomatal signaling (Mori et al., 2006). A closely related pair of Arabidopsis CDPKs (CPK4 and CPK11) are positive regulators of ABA signaling processes, and of the salt and drought stress response (Zhu et al., 2007). The Arabidopsis cpk23 mutant shows markedly enhanced tolerance to drought and salt

stresses (Ma and Wu, 2007). Arabidopsis cpk5 cpk6, cpk5 cpk6 cpk11 and cpk5 cpk6 cpk11 cpk4 mutant plants show a diminished oxidative burst and reduced activation of the 22-amino-acid bacterial peptide flg22 responsive genes, as well as compromised pathogen defense (Boudsocq et al., 2010). In contrast to Arabidopsis CDPKs, little is known about the functions of rice CDPKs. Analysis of loss-of-function transgenic rice indicates that SPK (calcium-dependent seedspecific protein kinase) functions as a key regulator in seed development (Asano et al., 2002). OsCDPK7-overexpressing rice plants show enhanced tolerance to abiotic stresses, such as cold, drought and salinity (Saijo et al., 2000). We previously found that OsCPK12-overexpressing (OsCPK12OX) plants exhibit increased tolerance to low nitrogen stress (Asano et al., 2010). However, the biological function of OsCPK12 in planta is largely unknown. In the current work, we investigated the effects of OsCPK12 overexpression and disruption on the rice plant’s response to abiotic and biotic stresses. We show that OsCPK12-OX plants exhibit enhanced tolerance to salt stress, possibly as a result of decreased ROS accumulation, and also increased sensitivity to exogenous ABA and decreased resistance to blast disease. Therefore, OsCPK12 may oppositely modulate salt-stress tolerance and blast disease resistance. RESULTS Expression pattern of OsCPK12 OsCPK12 belongs to group IIa of the CDPK family (Asano et al., 2005) (Figure S1). The coding sequence of OsCPK12 consists of eight exons and seven introns. OsCPK12 encodes a predicted protein of 533 amino acids with an estimated molecular mass of 59.6 kDa, and possesses a structure typical of the CDPK family. OsCPK12 shares a high sequence similarity with Arabidopsis CPK29 (69% identity) and rice OsCPK19 (71%). OsCPK12 contains a potential N-terminal myristoylation site (MGNCFTKT), which has been shown to affect the subcellular localization of CDPK (Benetka et al., 2008). The N-terminus sequence shows high sequence similarity to plasma membrane-localized Arabidopsis CPK9 (MGNCFAKN). Previously, we showed that OsCPK12 is ubiquitously expressed in the roots, leaf blades, basal parts (including the meristems) and developing seeds of rice (Asano et al., 2010). To examine the expression of OsCPK12 in detail, a genomic region comprising 1862 bp of the OsCPK12 promoter 5¢ upstream of the start codon was ligated in frame with the b-glucuronidase (GUS) reporter gene, and the resultant POsCPK12:GUS construct was introduced into wild-type (WT) rice plants. GUS staining was predominantly detected in the vascular tissues of POsCPK12:GUS plants by histochemical analysis (Figure 1a–c), and was more strongly detected in the phloem tissue of the large vascular bundle (Figure 1a). To examine whether OsCPK12 is

ª 2011 The Authors The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2012), 69, 26–36

28 Takayuki Asano et al.

(a)

(b)

(c)

Figure 1. Spatial pattern of GUS expression. (a) Cross section of the third leaf blade of a POsCPK12:GUS plant. Scale bar: 100 lm. Inset, enlargement of boxed region, showing GUS expression in a large vascular bundle. (b) GUS staining of the root of a POsCPK12:GUS plant. Scale bar: 150 lm. (c) Cross section of the root of a POsCPK12:GUS plant. Scale bar: 50 lm.

transcriptionally regulated in response to salinity and ABA, we performed RT-PCR. No significant difference in the expression level of OsCPK12 was found in our experimental conditions (Figure S2a), suggesting that OsCPK12 is not regulated at the transcriptional level in response to salt stress. Generation of OsCPK12 transgenic plants and identification of the oscpk12 mutant To elucidate the biological function of OsCPK12 in planta, we investigated the phenotypes of OsCPK12-OX and loss-offunction lines under conditions of salt stress or biotic stress. RT-PCR analysis confirmed that the expression of OsCPK12 was significantly enhanced in the OsCPK12-OX plants in comparison with WT plants (Figure 2a). A Tos17 insertion mutant of OsCPK12 (line NE1534) was identified in the Rice Tos17 Insertion Mutant Database (http://tos.nias.affrc.go.jp/ index.html.en) (Miyao et al., 2003). The Tos17 insertion was located within exon 5 of OsCPK12 in the reverse orientation (Figure 2b). Therefore, the function of OsCPK12 was assumed to be incomplete in this line. As the progeny seeds of line NE1534 were distributed as a mixed population of heterozygotes and homozygotes for the OsCPK12 mutation, PCRbased screening was performed using two OsCPK12-specific primers and a Tos17-specific primer to select homozygous mutant lines. We identified homozygous oscpk12 mutant plants (Figure S3). RT-PCR analysis showed that OsCPK12 expression in the oscpk12 mutant was abolished by the insertion of Tos17 (Figure 2c). Because we could find only one Tos17 insertion mutant, OsCPK12 RNA interference (RNAi) plants were generated by introducing an RNAi construct. The expression of OsCPK12 was significantly reduced in the OsCPK12 RNAi plants compared with WT plants (Figure 2c). Opposite properties of OsCPK12-OX plants and loss-of-function mutants in salt-stress tolerance To characterize the high salinity tolerance of OsCPK12-OX plants and loss-of-function lines, 2-week-old rice plants were

exposed to 200 mM NaCl for 5 days. The OsCPK12-OX plants showed an increased tolerance to salt stress (Figure 2d). The dry weights of shoots and roots of the OsCPK12-OX plants were heavier than those of WT plants, and this difference was more significant in roots than in shoots (Figure 2e,f). In contrast to the OsCPK12-OX plants, oscpk12 and OsCPK12 RNAi plants showed decreased tolerance to salt stress (Figure 2g). The average dry weights of shoots and roots of the oscpk12 and OsCPK12 RNAi plants were less than those of WT plants under conditions of high salinity (Figure 2h,i). Interestingly, the reduction in biomass of the oscpk12 and OsCPK12 RNAi plants was more pronounced in roots than in shoots (Figure 2h,i). Thus, overexpression and knock-down of OsCPK12 expression showed opposite effects on saltstress tolerance, and this effect was more pronounced in the roots. Accumulation of ROS in OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants Abiotic stress induces the accumulation of ROS, which are toxic molecules that cause oxidative damage in plants (Apel and Hirt, 2004). To elucidate whether OsCPK12 plays an important role in ROS homeostasis, we investigated the accumulation of H2O2 by the precipitation of polymerized 3,3¢-diaminobenzidine (DAB) in OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants grown under conditions of high salinity. An intense brown precipitate was observed in the leaves of WT plants stained with DAB after 5 days of exposure to high salinity. Under conditions of high salinity the intensity of DAB staining was less in leaves of the OsCPK12OX plants than in those of WT plants (Figure 3a,b). The intensity of DAB staining was greater in the leaves of oscpk12 and OsCPK12 RNAi plants than in those of WT plants (Figure 3a,b). Thus, the OsCPK12-OX plants and the loss-of-function lines showed opposite trends in ROS accumulation. These results suggest that OsCPK12 is involved in the elimination of H2O2 produced under salt stress.


OsCPK12 modulates abiotic and biotic stress responses 29

Figure 2. The effects of OsCPK12-OX and loss-of-function mutants on salt-stress tolerance. (a) The level of OsCPK12 expression in OsCPK12-OX plants. Total RNA was isolated from the leaf blades of 2-week-old OsCPK12-OX (lines 13, 16 and 24) and wildtype (WT) plants grown under normal conditions. RT-PCR (25 cycles for OsCPK12 and 20 cycles for 18S rRNA) was performed with OsCPK12- and 18S rRNA-specific primers. (b) Intron–exon organization of OsCPK12 and the Tos17 insertion site. Solid boxes and lines indicate exons and introns, respectively. The position of the Tos17 insertion is indicated by a triangle. The arrows indicate the positions of OsCPK12-A, OsCPK12-X and Tos-com primers. (c) Expression of OsCPK12 in oscpk12 and OsCPK12 RNAi (lines 3 and 6) plants. RT-PCR (35 cycles for OsCPK12 and 20 cycles for 18S rRNA) was performed with OsCPK12- and 18S rRNA-specific primers. (d) Phenotypic comparison of rice plants grown under conditions of high salinity. Pot-grown, 2-week-old seedlings were transferred onto trays containing 200 mM NaCl solution for 5 days and then returned to normal conditions for 7 days. (e) Shoot dry weight of OsCPK12-OX plants after the salt-stress treatment described in (d). Error bars indicate the standard error of the mean (SEM) of three independent experiments. Between eight and 10 plants were used per line in each experiment; *P < 0.05. (f) Root dry weight of OsCPK12-OX plants after salt-stress treatment; *P < 0.05. (g) Phenotypic comparison between WT and loss-of-function lines after the salt-stress treatment shown in (d). (h) Shoot dry weight of oscpk12 and OsCPK12 RNAi plants after the salt-stress treatment. Error bars indicate the SEM (n = 8 or 9); *P < 0.05. (i) Root dry weight of oscpk12 and OsCPK12 RNAi plants after the salt-stress treatment. Error bars indicate the SEM (n = 8 or 9); *P < 0.05.

Expression of stress-inducible genes and ROS scavenging genes in OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants To investigate the downstream components of the OsCPK12-mediated salt-stress signaling pathways, we analyzed the expression levels of known stress-inducible genes in the leaves of OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants grown under high-salinity conditions. Because we thought that OsCPK12 might be involved in eliminating the H2O2 produced under salt stress, we examined the expression of the ROS-producing NADPH oxidase gene, OsrbohI (Wong et al., 2007), and the high salinity-inducible ascorbate peroxidase genes, OsAPx2 and OsAPx8 (Teixeira et al., 2006). The expression levels of OsAPx2 and OsAPx8 were higher in the leaves of the OsCPK12-OX plants than in those of the WT, whereas the expression level of OsrbohI was lower in OsCPK12-OX than in WT plants (Figure 3c). We

also analyzed the expression levels of the following ABAand stress-inducible genes: OsbZIP23, which encodes a basic leucine zipper (bZIP) transcription factor (Xiang et al., 2008); Rab21, which encodes a basic glycine-rich protein (Mundy and Chua, 1988); OsLEA3, which encodes a late embryogenesis abundant protein (Moons et al., 1997); OsP5CS, which encodes a D1-pyrroline-5-carboxylate synthetase involved in proline biosynthesis (Igarashi et al., 1997); OsNHX1, which encodes a vacuolar Na+/H+ antiporter (Fukuda et al., 2004); and salT, which encodes a glycine-rich protein (Claes et al., 1990). The expression levels of OsbZIP23, Rab21, OsLEA3, OsP5CS and OsNHX1 were similar in the leaves of salt-stressed OsCPK12-OX, loss-of-function and WT plants (Figure S2b). The expression of OsLEA3 and salT in roots was induced by high salinity in the OsCPK12OX plants to levels that were similar to those in WT plants (Figure S2c).



Figure 3. Accumulation of H2O2 in the leaves of OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants exposed to salt stress. (a) In situ H2O2 accumulation was detected by 3,3¢-diaminobenzidine (DAB) staining. Two-week-old seedlings were either untreated or exposed to 200 mM NaCl for 5 days. Between five and 10 leaves were used for each line in this assay. These experiments were repeated twice, and a similar tendency was observed in each experiment. (b) Evaluation of DAB staining in the leaves of plants exposed, or not, to high salinity. The relative DAB staining intensities were calculated based on the stain intensity of wild-type plants. Error bars indicate the SEM (n = 4); *P < 0.05. (c) Relative expression of OsAPx2, OsAPx8 and OsrbohI in the leaves of rice plants exposed to high-salinity conditions using UBC as the internal control. Two-weekold seedlings were exposed to 200 mM NaCl for 5 days. Total RNA was extracted from the leaf blades of salt-stressed plants and subjected to quantitative RT-PCR analysis. The wild-type expression data are set as 1. Each value is the mean SEM of three independent experiments.

Response of OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants to ABA The phytohormone ABA plays important roles in the adaptation of plants to abiotic stresses, such as high salinity and

drought (Finkelstein et al., 2002; Cutler et al., 2010). To evaluate the role of OsCPK12 in the ABA response of plants, we analyzed the growth of seedlings of OsCPK12-OX, oscpk12 and OsCPK12 RNAi plants following the exogenous application of 0.5 lM ABA. The inhibition of seedling growth


OsCPK12 modulates abiotic and biotic stress responses 31 by ABA treatment was greater in the OsCPK12-OX plants than in WT plants (Figure 4a). The average shoot dry weight of the OsCPK12-OX plants was less than that of WT plants grown in the presence of ABA, whereas the growth of seedlings was similar in OsCPK12-OX and WT plants under normal conditions (Figure 4b). On the other hand, no significant difference in ABA response was found between the loss-of-function and WT plants, although the dry weights were slightly increased in the loss-of-function lines compared with WT plants upon treatment with ABA (Figure 4c,d). We examined the expression of OsAPx8, Rab21, OsLEA3 and salT in the leaves of OsCPK12-OX and WT plants grown in the presence of ABA. The expression levels of Rab21, OsLEA3 and salT were significantly higher in the OsCPK12-OX plants than in WT plants (Figure S2d). No significant difference in the expression of ABA-inducible OsAPx8 (Hong et al., 2007) was found between OsCPK12-OX and WT plants (Figure S2d). Susceptibility of OsCPK12-OX plants to blast fungus We examined the effects of overexpression and knock-down of OsCPK12 on resistance to fungal pathogens by inoculat-

ing the plants with compatible (virulent) or incompatible (avirulent) blast fungus (Figure 5a). OsCPK12-OX plants exhibited an increased susceptibility to both compatible and incompatible blast fungus compared with WT plants (Figures 5b and S4a), and the disease lesions in OsCPK12OX plants were larger than those in WT plants (Figures 5c and S4b,c). No significant difference in blast disease resistance was observed between the oscpk12 and WT plants (Figure S4c). The expression level of the pathogenesis-related (PR) genes OsPR1b (Agrawal et al., 2000) and PBZ1 (Midoh and Iwata, 1996) was lower in OsCPK12-OX lines than in WT plants (Figure S4d). DISCUSSION CDPKs constitute a large multigene family consisting of 34 genes in Arabidopsis (Cheng et al., 2002; Hrabak et al., 2003) and 29 genes in rice (Asano et al., 2005). Among rice and Arabidopsis CDPKs, rice OsCDPK7 (Saijo et al., 2000) and Arabidopsis CPK4 and CPK11 (Zhu et al., 2007), which belong to group I of the CDPK family (Figure S1), are involved in tolerance to abiotic stresses. In this study, we performed a functional analysis of OsCPK12, which belongs

Figure 4. Effect of abscisic acid (ABA) treatment on seedling growth. (a) The response of OsCPK12-OX plants to ABA. Five-day-old seedlings were transferred to Yoshida’s nutrient solution supplemented with 0.5 lM ABA and incubated in a growth chamber for 2 weeks. The ABA solution was changed once a week, and the seedlings were subsequently harvested for dry weight measurement. (b) Shoot dry weight of the OsCPK12-OX and WT plants grown in the presence or absence of ABA, as described in (a). Each value is the mean SEM of three independent experiments. Between four and 12 plants were used per line in each experiment; *P < 0.05. (c) The response of oscpk12 and OsCPK12 RNAi plants to ABA. Five-day-old seedlings were treated with 0.5 lM ABA for 2 weeks, as described in (a), and then harvested. (d) Shoot dry weight of oscpk12, OsCPK12 RNAi and WT plants grown in the presence or absence of ABA, as described in (a). Each value is the mean SEM of at least three independent experiments. Between five and 23 plants were used per line in each experiment.



Figure 5. Effect of OsCPK12 overexpression on rice blast disease resistance. (a) Overexpression of OsCPK12 in OsCPK12-OX plants. Total RNA was isolated from the leaf blades of 2-week-old seedlings of OsCPK12-OX (lines 3 and 21) and WT plants grown under normal conditions. RT-PCR (25 cycles for OsCPK12 and 20 cycles for 18S rRNA) was performed with OsCPK12- and 18S rRNA-specific primers. (b) Disease symptoms at 11 days after inoculation with the virulent isolate of rice blast. Rice seedlings (22-days old) were inoculated with the compatible (virulent) blast fungus, Ina86-137 (race 007.0), by punch infection. (c) Disease lesion size in leaves at 11 days after inoculation. Error bars indicate the SEM (n = 8 or 12); *P < 0.05.

to group IIa of the CDPK family (Figure S1), and identified a role for CDPKs in abiotic and biotic stress responses. Our results suggest that OsCPK12 enhances tolerance to salt stress by reducing the accumulationof ROS (Figure 6). Furthermore, overexpression of OsCPK12 conferred increased sensitivity to exogenously applied ABA, and enhanced susceptibility to blast fungus. These results also suggest that OsCPK12 functions in multiple signaling pathways, and inversely modulates salt-stress tolerance and blast disease resistance. The opposite properties of OsCPK12-OX plants and the loss-of-function mutants on tolerance to salt stress were observed in both the shoots and roots, but were more

Figure 6. A model for OsCPK12-mediated pathogen and salt-stress signaling; PCD, programmed cell death.

pronounced in the roots (Figure 2d–i). These results suggest that OsCPK12 is an essential positive regulator of tolerance to salt stress. Although higher tolerance to salt stress was observed in the roots, the specific function of OsCPK12 in roots is unknown. Unexpectedly, the expression levels of stress-inducible genes under conditions of high salinity in the leaves or roots of OsCPK12-OX plants, and in the loss-offunction lines, were comparable with those in WT plants (Figure S2b,c). A recent study found that CPK3 is required for salt-stress acclimation in Arabidopsis. The expression of salt-inducible genes is induced by salt-stress treatment in both the cpk3 mutant and AtCPK3-overexpressing lines to levels that are similar to those of WT plants, whereas posttranslational protein phosphorylation patterns in the roots of cpk3 and WT plants revealed clear differences (Mehlmer et al., 2010). These results suggest that OsCPK12-mediated salt stress signaling in roots might be regulated posttranslationally. In contrast to OsCPK12, OsCDPK7 positively regulates the expression of rab16A, salT and OsLEA3 in roots under conditions of high salinity (Saijo et al., 2000). These results suggest that OsCPK12 and OsCDPK7 have different roles in salt-stress signaling (Figure 6). Abiotic stress induces ROS production in plant cells (Apel and Hirt, 2004; Miller et al., 2008, 2010). ROS scavenging systems detoxify ROS to minimize and/or prevent oxidative damage in cells. Indeed, an OsAPx2-overexpressing Arabidopsis plant exhibits reduced accumulation of ROS in planta, and thus enhanced tolerance to salt stress (Lu et al., 2007). Under conditions of high salinity, the accumulation of H2O2 in OsCPK12-OX plants was less than that in WT plants, whereas the accumulation was more in oscpk12 and OsCPK12 RNAi plants (Figure 3a,b). Therefore, oxidative damage to the cells of OsCPK12-OX plants might be reduced under high-salinity conditions. The levels of ROS accumulation were correlated with altered expression levels of OsAPx2 and OsAPx8 in the OsCPK12-OX and loss-of-function plants under conditions of high salinity (Figure 3c). These results suggest that OsCPK12 positively regulates ROS detoxification by controlling the expression of OsAPx2 and OsAPx8. ROS production is mediated by NADPH oxidase (Leshem et al., 2007). When Solanum tuberosum (potato) plants are attacked by pathogens, CDPK4 and CDPK5 regulate ROS production by phosphorylating NADPH oxidase and inducing the oxidative burst (Kobayashi et al., 2007). Furthermore, the induction of the oxidative burst is reduced in Arabidopsis cpk5 cpk6, cpk5 cpk6 cpk11 and cpk5 cpk6 cpk11 cpk4 mutants, suggesting that these CDPKs are involved in regulating ROS production (Boudsocq et al., 2010). The phosphorylation of the Arabidopsis NADPH oxidase, AtrbohD, and the production of ROS are enhanced by the protein phosphatase inhibitor, calyculin A (Ogasawara et al., 2008). Thus, CDPKs are reported to function as positive regulators of ROS production. In contrast, our results showed that OsCPK12-mediated signaling


OsCPK12 modulates abiotic and biotic stress responses 33 negatively regulates the expression of OsrbohI. A recent study revealed that Arabidopsis mitogen-activated protein kinase 8 (MPK8) directly binds to calmodulins in a Ca2+dependent manner, and that activated MPK8 negatively regulates ROS accumulation by controlling the expression of RbohD (Takahashi et al., 2011). This result suggests that the regulation of Rboh genes at the mRNA level is important for the suitable feedback amplification of ROS, as well as for the activation of Rboh. Collectively, our observations and the above results suggest that OsCPK12-mediated signaling contributes to the maintenance of ROS homeostasis under conditions of high salinity by controlling the expression of OsAPx2, OsAPx8 and OsrbohI. In our study, OsCPK12-OX plants showed increased sensitivity to exogenous ABA (Figure 4a,b). Furthermore, the expression of Rab21, OsLEA3 and salT was elevated in OsCPK12-OX plants upon the exogenous application of ABA (Figure S2d). On the contrary, the responses of oscpk12 and OsCPK12 RNAi plants to ABA were similar to those of WT plants, indicating that the suppression of OsCPK12 expression in rice does not affect the response to ABA (Figure 4c,d). These results suggest that OsCPK12 positively regulates the ABA signaling pathway, and that functional redundancy amongst OsCPK12 and other rice CDPK(s) exists in the ABA signaling pathway of rice. The difference in ABA response between the OsCPK12-OX and loss-of-function lines implies that OsCPK12 confers tolerance to salt stress by repressing ROS accumulation rather than by effecting ABA-mediated salt signaling (Figure 6). Our current study suggests that OsCPK12-mediated signaling negatively regulates the expression of OsrbohI under conditions of high salinity. Previous studies indicated that NADPH oxidase has a pivotal role in ROS-mediated defense responses (Yoshioka et al., 2009). Virus-induced gene silencing of Nicotiana benthamiana RBOHA and RBOHB attenuates ROS production and resistance to Phytophthora infestans (Yoshioka et al., 2003). OsCPK12-OX plants were susceptible to both compatible and incompatible races of blast fungus (Figures 5c and S4b,c). This result suggests that OsCPK12 is involved in the negative regulation of both basal and resistance (R) gene-mediated defense pathways in rice, probably via the repression of ROS production. Another hypothesis is that OsCPK12-mediated ABA signaling is involved in the defense system against blast fungus. ABA serves as a key signaling molecule in plant–pathogen interactions (Mauch-Mani and Mauch, 2005; Asselbergh et al., 2008). In fact, ABA-treated rice seedlings show dramatically enhanced disease susceptibility not only to compatible but also to incompatible blast fungus strains (Jiang et al., 2010). Therefore, ABA might negatively regulate both basal and R gene-mediated defense pathways to blast fungus. The OsCPK12-OX plants showed increased sensitivity to exogenous ABA, and were susceptible to both compatible and incompatible races of blast fungus

(Figures 4,5 and S4). The altered pathogen response in OsCPK12-OX plants may be caused by an enhanced response to ABA following overexpression of OsCPK12. On the other hand, the oscpk12 mutant showed similar levels of blast resistance and ABA response as WT plants (Figures 4d and S4c), suggesting that resistance to blast fungus is unaffected by the knock down of OsCPK12 expression. The ABA-inducible rice mitogen-activated protein kinase (MAPK) OsMAPK5 has been reported to positively regulate tolerance to drought, salt and cold, and to negatively modulate PR gene expression and broad-spectrum disease resistance (Xiong and Yang, 2003). Suppression of OsMAPK5 expression and its kinase activity results in the constitutive expression of PR genes in OsMAPK5 RNAi lines, and significantly enhances resistance to blast fungus, whereas control and OsMAPK5-OX plants exhibit the same level of disease susceptibility to blast infection. Thus, OsCPK12 and OsMAPK5 show similar properties in both abiotic and disease-resistance signaling. Further studies are needed to elucidate the relationship between OsCPK12 and OsMAPK5 in the abiotic stress and pathogen responses. In conclusion, OsCPK12-OX plants showed enhanced tolerance to salt stress by repressing ROS accumulation under conditions of high salinity. In contrast, the OsCPK12OX plants were susceptible to blast fungus. Our results suggest that OsCPK12 positively regulates salt tolerance and negatively modulates blast disease resistance. Identifying the downstream component(s) of OsCPK12 may elucidate the complex CDPK-mediated signaling pathways underlying the plant’s response to ABA and salt stress. EXPERIMENTAL PROCEDURES Growth conditions and stress treatment Rice (O. sativa L. cv. Nipponbare) plants were grown in a growth chamber under 60% relative humidity with a 14-h light (28C)/10-h dark (25C) cycle, which was referred to as the normal condition. For expression analysis of OsCPK12 in response to ABA and salt stress, rice seeds were surface sterilized and germinated hydroponically in deionized water in a growth chamber. Twelve-day-old seedlings were transferred to deionized water containing 100 lM ABA (SigmaAldrich, http://www.sigmaaldrich.com) or 200 mM NaCl for up to 24 h. For the salt-stress tolerance test, high-salinity treatment was performed as described previously (Xiong and Yang, 2003). Briefly, T1 and T2 transgenic seeds were surface sterilized and germinated on filter paper soaked in 50 mg L)1 hygromycin for 5 days. Hygromycin-resistant seedlings were transplanted into soil. Two-weekold seedlings were transferred onto trays containing 200 mM NaCl solution for up to 5 days, and then returned to normal conditions for 7 days. The level of salt tolerance was evaluated from dry weights after the recovery period. To evaluate the effects of ABA on rice seedlings, sterilized seeds were grown as described above for 5 days in a growth chamber. The five-day-old seedlings were transferred to Yoshida’s nutrient solution (Yoshida et al., 1976) supplemented with 0.5 lM ABA for 2 weeks. The ABA solution was changed once a week, and the seedlings were harvested for dryweight measurement.


34 Takayuki Asano et al. Construction of binary vectors and rice transformation

Detection of ROS

The PCR primers used are listed in Table S1. PCR was performed using KOD-Plus (Toyobo, http://www.toyobo.co.jp/e), according to the manufacturer’s instructions. The OsCPK12 overexpression construct was produced as follows. A fragment bearing the 3¢ portion of OsCPK12 was amplified by RT-PCR using the OsCPK12-A and OsCPK12-BstEII primers. The amplified fragment was digested with NcoI and BstEII (Toyobo) and then inserted 3¢ downstream of the cauliflower mosaic virus (CaMV) 35S promoter in the binary vector pCAMBIA1301. Next, a fragment spanning the 5¢ coding region of OsCPK12 was amplified by RTPCR with the OsCPK12-NcoI and OsCPK12-X primers, and the PCR fragment was digested with NcoI and inserted between the 35S promoter and the 3¢ region of OsCPK12 in the pCAMBIA1301 plasmid (Figure S5a). The direction of the inserted fragment was confirmed by PCR using the OsCPK12-A and 3¢-Tnos primers. The cloned OsCPK12 fragment was confirmed by nucleotide sequence analysis with the OsCPK12-A, OsCPK12-C, OsCPK12-4, OsCPK12-6 and OsCPK12-8 primers (Table S1). An OsCPK12-specific fragment containing the 3¢-coding and the adjacent 3¢-untranslated regions was amplified to generate the double-stranded RNAi construct by RT-PCR using the OsCPK12-A-entry and OsCPK12-X primers. The amplified fragment was cloned into the Gateway entry vector, pENTR/SD/D-TOPO (Invitrogen, http://www.invitrogen.com), and subsequently into a binary vector, pANDA (Miki et al., 2005) (Figure S5b). To produce the POsCPK12:GUS construct, a fragment containing the 5¢-upstream region of OsCPK12 (nucleotide positions: )1862 to )1 bp from the translation initiation codon) was cloned as follows. The first round of genomic PCR was performed using the OsCPK12-f1 and OsCPK12-X primers (Table S1). A 1-ll volume of the PCR products was diluted to 100 ll with H2O, and 1 ll of the diluted DNA was used as a template for the second round of PCR. The fragment corresponding to the promoter of OsCPK12 was amplified by PCR using the OsCPK12-f3 and OsCPK12-BglII primers (Table S1). The amplified fragment was digested with BglII and XbaI, and then ligated in frame 5¢-upstream of gusA in pCAMBIA1301. The three aforementioned binary vectors were introduced individually into Agrobacterium tumefaciens strain EHA105 (Hood et al., 1993) by electroporation. Transgenic rice plants were produced by Agrobacterium-mediated transformation as described previously (Toki et al., 2006).

3,3¢-Diaminobenzidine (DAB) staining was performed for the in situ detection of H2O2 as previously described (Fitzgerald et al., 2004). Two-week-old seedlings were exposed to 200 mM NaCl for 5 days, and the salt-stressed leaves were detached and incubated in 1 mg ml)1 DAB solution containing 0.01% Triton X-100. This solution was infiltrated under low vacuum pressure for 45 min, and the leaves were incubated overnight at 25C. The leaves were cleared twice in 70% ethanol for 15 min and fixed with an ethanol/acetic acid/glycerol (3:1:1) mixture. DAB-stained leaves were scanned, and the pixel intensity of the DAB stain was quantified using Adobe PHOTOSHOP CS4 software.

Analysis of GUS gene expression GUS staining was conducted as described (Tsugeki et al., 2009). Seedlings were soaked in ice-cold 90% acetone for 15 min and then in GUS staining solution containing 0.5 mg ml)1 X-Gluc, 50 mM sodium phosphate buffer (pH 7.0), 3 mM potassium ferricyanide, 3 mM potassium ferrocyanide, 5% methanol and 0.1% Triton X-100. The solution was infiltrated under low vacuum pressure for 45 min, and then the seedlings were incubated overnight at 37C. The seedlings were fixed in ethanol/acetic acid (6:1) and washed several times with 70% ethanol.

Screening of the oscpk12 mutant Homozygous oscpk12 mutant plants were identified by genomic PCR with the OsCPK12-A, OsCPK12-X (Table S1) and Tos-com (5¢-TAGCTGAGACCGATGCTTCA-3¢) primers. The PCR analysis was performed using TaKaRa LA Taq (Takara Bio, http://www.takarabio.com) under the following conditions: 94C for 2 min, followed by 30 cycles of 94C for 30 s, 58C for 1 min and 72C for 2 min, with a final extension at 72C for 2 min.

Gene expression analysis Isolation of RNA and RT-PCR assays were performed as described previously (Asano et al., 2002, 2005). Rice total RNA was extracted from roots and leaf blades using an RNeasy Plant Mini Kit (Qiagen, http://www.qiagen.com). The isolated RNA was treated with TURBO DNase (Applied Biosystems/Ambion, http://www.ambion.com). cDNA synthesis was performed using a SuperScript VILO cDNA Synthesis Kit (Invitrogen). Quantitative RT-PCR analysis was performed using the iCycler iQ system (Bio-Rad, http://www.bio-rad. com) with iQ SYBR Green Supermix (Bio-Rad) in a total volume of 25 ll. The reactions were performed in biological triplicate using RNA samples extracted from three independent plant materials. Expression values were normalized with those of the ubiquitinconjugating enzyme (UBC) gene (Jain et al., 2006). For RT-PCR, PCR analysis was performed using 0.5 U of EX Taq Hot Start Version (TaKaRa Bio), under the following conditions: 94C for 2 min, followed by 26–35 cycles of 94C for 30 s, 58C for 30 s and 72C for 1 min. The primers used for RT-PCR are listed in Table S2.

Evaluation of blast resistance Rice cv. Nipponbare has blast-resistance genes Pish (Imbe and Matsumoto, 1985) and Pi19 (Hayashi et al., 1998). Ina86-137 (race 007.0), a compatible (virulent) race, and Kyu77-07A (avrPish; race 102.0), an incompatible (avirulent) race, of the blast fungus Magnaporthe grisea were used for blast-resistance tests. The blast fungus was grown on an oatmeal agar plate at 25C in dark for 10–12 days and the surface of the fungal growth was scraped with a toothbrush. The scraped plates were left under a fluorescent light for 3–4 days to induce sporulation. The agar slice with attached conidia was placed on a rice leaf wounded by a hole puncher of 2–4week-old seedlings. Inoculated rice seedlings were placed in a dew chamber for 24 h at 25C and then moved to a glasshouse. The plants were examined for lesion formation 6–12 days after inoculation. Rice blast resistance was evaluated by measuring the length of the disease lesions on leaves after punch inoculation with blast fungus.

ACKNOWLEDGEMENTS We thank Naho Hara and Akemi Tagiri for the production of transgenic rice plants, Setsuko Kimura for technical assistance, Dr Noritoshi Inagaki for technical advice on the production of leaf cross sections, Dr Fumiyoshi Myouga for technical advice on DAB staining, and Drs Daisuke Miki and Ko Shimamoto for providing the pAND vector. This work was supported by grants from the Japan Society for the Promotion of Science (Grant-in-Aid No. 17780013 for Young Scientists to TA) and the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN to RO).


OsCPK12 modulates abiotic and biotic stress responses 35 SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article: Figure S1. Phylogenetic relationships amongst CDPKs from rice and Arabidopsis. Figure S2. Expression analysis of OsCPK12 and ABA- and stressinducible genes. Figure S3. Identification of homozygous oscpk12 mutants. Figure S4. Disease lesion size in oscpk12 and OsCPK12-OX plants. Figure S5. Binary vectors used in the production of OsCPK12 transgenic plants. Table S1. PCR primers for OsCPK12. Table S2. PCR primers used in gene expression analyses. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.

REFERENCES Agrawal, G.K., Rakwal, R. and Jwa, N.S. (2000) Rice (Oryza sativa L.) OsPR1b gene is phytohormonally regulated in close interaction with light signals. Biochem. Biophys. Res. Commun. 278, 290–298. Anderson, J.P., Badruzsaufari, E., Schenk, P.M., Manners, J.M., Desmond, O.J., Ehlert, C., Maclean, D.J., Ebert, P.R. and Kazan, K. (2004) Antagonistic interaction between abscisic acid and jasmonate-ethylene signaling pathways modulates defense gene expression and disease resistance in Arabidopsis. Plant Cell, 16, 3460–3479. Apel, K. and Hirt, H. (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu. Rev. Plant Biol. 55, 373– 399. Asano, T., Kunieda, N., Omura, Y. et al. (2002) Rice SPK, a calmodulin-like domain protein kinase, is required for storage product accumulation during seed development: phosphorylation of sucrose synthase is a possible factor. Plant Cell, 14, 619–628. Asano, T., Tanaka, N., Yang, G., Hayashi, N. and Komatsu, S. (2005) Genomewide identification of the rice calcium-dependent protein kinase and its closely related kinase gene families: comprehensive analysis of the CDPKs gene family in rice. Plant Cell Physiol. 46, 356–366. Asano, T., Wakayama, M., Aoki, N., Komatsu, S., Ichikawa, H., Hirochika, H. and Ohsugi, R. (2010) Overexpression of a calcium-dependent protein kinase gene enhances growth of rice under low-nitrogen conditions. Plant Biotechnol. 27, 369–373. Asselbergh, B., Achuo, A.E., Hofte, M. and Van Gijsegem, F. (2008) Abscisic acid deficiency leads to rapid activation of tomato defence responses upon infection with Erwinia chrysanthemi. Mol. Plant Pathol. 9, 11–24. Audenaert, K., De Meyer, G.B. and Hofte, M.M. (2002) Abscisic acid determines basal susceptibility of tomato to Botrytis cinerea and suppresses salicylic acid-dependent signaling mechanisms. Plant Physiol. 128, 491– 501. Badawi, G.H., Kawano, N., Yamauchi, Y., Shimada, E., Sasaki, R., Kubo, A. and Tanaka, K. (2004) Over-expression of ascorbate peroxidase in tobacco chloroplasts enhances the tolerance to salt stress and water deficit. Physiol. Plant. 121, 231–238. Benetka, W., Mehlmer, N., Maurer-Stroh, S., Sammer, M., Koranda, M., Neumuller, R., Betschinger, J., Knoblich, J.A., Teige, M. and Eisenhaber, F. (2008) Experimental testing of predicted myristoylation targets involved in asymmetric cell division and calcium-dependent signalling. Cell Cycle, 7, 3709–3719. Boudsocq, M., Willmann, M.R., McCormack, M., Lee, H., Shan, L., He, P., Bush, J., Cheng, S.H. and Sheen, J. (2010) Differential innate immune signalling via Ca2+ sensor protein kinases. Nature, 464, 418–422. Cheng, S.H., Willmann, M.R., Chen, H.C. and Sheen, J. (2002) Calcium signaling through protein kinases. The Arabidopsis calcium-dependent protein kinase gene family. Plant Physiol. 129, 469–485. Claes, B., Dekeyser, R., Villarroel, R., Van den Bulcke, M., Bauw, G., Van Montagu, M. and Caplan, A. (1990) Characterization of a rice gene showing

organ-specific expression in response to salt stress and drought. Plant Cell, 2, 19–27. Cutler, S.R., Rodriguez, P.L., Finkelstein, R.R. and Abrams, S.R. (2010) Abscisic acid: emergence of a core signaling network. Annu. Rev. Plant Biol. 61, 651–679. Fan, J., Hill, L., Crooks, C., Doerner, P. and Lamb, C. (2009) Abscisic acid has a key role in modulating diverse plant–pathogen interactions. Plant Physiol. 150, 1750–1761. Finkelstein, R.R., Gampala, S.S.L. and Rock, C.D. (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell, 14, S15–S45. Fitzgerald, H.A., Chern, M.S., Navarre, R. and Ronald, P.C. (2004) Overexpression of (At)NPR1 in rice leads to a BTH- and environment-induced lesion-mimic/cell death phenotype. Mol. Plant Microbe Interact. 17, 140– 151. Foyer, C.H. and Noctor, G. (2005) Redox homeostasis and antioxidant signaling: a metabolic interface between stress perception and physiological responses. Plant Cell, 17, 1866–1875. Fukuda, A., Nakamura, A., Tagiri, A., Tanaka, H., Miyao, A., Hirochika, H. and Tanaka, Y. (2004) Function, intracellular localization and the importance in salt tolerance of a vacuolar Na+/H+ antiporter from rice. Plant Cell Physiol. 45, 146–159. Harmon, A.C., Gribskov, M. and Harper, J.F. (2000) CDPKs – a kinase for every Ca2+ signal? Trends Plant Sci. 5, 154–159. Harper, J.F. and Harmon, A. (2005) Plants, symbiosis and parasites: a calcium signalling connection. Nat. Rev. Mol. Cell Biol. 6, 555–566. Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H. and Harmon, A.C. (1991) A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science, 252, 951–954. Harper, J.F., Breton, G. and Harmon, A. (2004) Decoding Ca2+ signals through plant protein kinases. Annu. Rev. Plant Biol. 55, 263–288. Hayashi, N., Ando, I. and Imbe, T. (1998) Identification of a new resistance gene to a chinese blast fungus isolate in the Japanese rice cultivar aichi asahi. Phytopathology, 88, 822–827. Hirayama, T.and Shinozaki,K.(2010)Research onplantabiotic stress responses in the post-genome era: past, present and future. Plant J. 61, 1041–1052. Hong, C.Y., Hsu, Y.T., Tsai, Y.C. and Kao, C.H. (2007) Expression of ASCORBATE PEROXIDASE 8 in roots of rice (Oryza sativa L.) seedlings in response to NaCl. J. Exp. Bot. 58, 3273–3283. Hood, E.E., Gelvin, S.B., Melchers, L.S. and Hoekema, A. (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res. 2, 208–218. Hrabak, E.M., Chan, C.W.M., Gribskov, M. et al. (2003) The Arabidopsis CDPKSnRK superfamily of protein kinases. Plant Physiol. 132, 666–680. Igarashi, Y., Yoshiba, Y., Sanada, Y., Yamaguchi-Shinozaki, K., Wada, K. and Shinozaki, K. (1997) Characterization of the gene for D1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L. Plant Mol. Biol. 33, 857–865. Imbe, T. and Matsumoto, S. (1985) Inheritance of resistance of rice varieties to the blast fungus strains virulent to the variety’’Reiho’’. Jpn. J. Breed, 35, 332–339. Jain, M., Nijhawan, A., Tyagi, A.K. and Khurana, J.P. (2006) Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR. Biochem. Biophys. Res. Commun. 345, 646–651. Jia, W., Wang, Y., Zhang, S. and Zhang, J. (2002) Salt-stress-induced ABA accumulation is more sensitively triggered in roots than in shoots. J. Exp. Bot. 53, 2201–2206. Jiang, C.J., Shimono, M., Sugano, S., Kojima, M., Yazawa, K., Yoshida, R., Inoue, H., Hayashi, N., Sakakibara, H. and Takatsuji, H. (2010) Abscisic acid interacts antagonistically with salicylic acid signaling pathway in riceMagnaporthe grisea interaction. Mol. Plant Microbe Interact. 23, 791–798. Kobayashi, M., Ohura, I., Kawakita, K., Yokota, N., Fujiwara, M., Shimamoto, K., Doke, N. and Yoshioka, H. (2007) Calcium-dependent protein kinases regulate the production of reactive oxygen species by potato NADPH oxidase. Plant Cell, 19, 1065–1080. Kotchoni, S.O., Kuhns, C., Ditzer, A., Kirch, H.H. and Bartels, D. (2006) Overexpression of different aldehyde dehydrogenase genes in Arabidopsis thaliana confers tolerance to abiotic stress and protects plants against lipid peroxidation and oxidative stress. Plant Cell Environ. 29, 1033–1048. Kwak, J.M., Mori, I.C., Pei, Z.M., Leonhardt, N., Torres, M.A., Dangl, J.L., Bloom, R.E., Bodde, S., Jones, J.D. and Schroeder, J.I. (2003) NADPH


36 Takayuki Asano et al. oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J. 22, 2623–2633. Leshem, Y., Seri, L. and Levine, A. (2007) Induction of phosphatidylinositol 3-kinase-mediated endocytosis by salt stress leads to intracellular production of reactive oxygen species and salt tolerance. Plant J. 51, 185–197. Lu, Z., Liu, D. and Liu, S. (2007) Two rice cytosolic ascorbate peroxidases differentially improve salt tolerance in transgenic Arabidopsis. Plant Cell Rep. 26, 1909–1917. Ludwig, A.A., Romeis, T. and Jones, J.D.G. (2004) CDPK-mediated signalling pathways: specificity and cross-talk. J. Exp. Bot. 55, 181–188. Ma, S.Y. and Wu, W.H. (2007) AtCPK23 functions in Arabidopsis responses to drought and salt stresses. Plant Mol. Biol. 65, 511–518. Mauch-Mani, B. and Mauch, F. (2005) The role of abscisic acid in plant– pathogen interactions. Curr. Opin. Plant Biol. 8, 409–414. Mehlmer, N., Wurzinger, B., Stael, S., Hofmann-Rodrigues, D., Csaszar, E., Pfister, B., Bayer, R. and Teige, M. (2010) The Ca2+-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis. Plant J. 63, 484–498. Midoh, N. and Iwata, M. (1996) Cloning and characterization of a probenazoleinducible gene for an intracellular pathogenesis-related protein in rice. Plant Cell Physiol. 37, 9–18. Miki, D., Itoh, R. and Shimamoto, K. (2005) RNA silencing of single and multiple members in a gene family of rice. Plant Physiol. 138, 1903–1913. Miller, G., Suzuki, N., Rizhsky, L., Hegie, A., Koussevitzky, S. and Mittler, R. (2007) Double mutants deficient in cytosolic and thylakoid ascorbate peroxidase reveal a complex mode of interaction between reactive oxygen species, plant development, and response to abiotic stresses. Plant Physiol. 144, 1777–1785. Miller, G., Shulaev, V. and Mittler, R. (2008) Reactive oxygen signaling and abiotic stress. Physiol. Plant. 133, 481–489. Miller, G., Suzuki, N., Ciftci-Yilmaz, S. and Mittler, R. (2010) Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant Cell Environ. 33, 453–467. Miyao, A., Tanaka, K., Murata, K., Sawaki, H., Takeda, S., Abe, K., Shinozuka, Y., Onosato, K. and Hirochika, H. (2003) Target site specificity of the Tos17 retrotransposon shows a preference for insertion within genes and against insertion in retrotransposon-rich regions of the genome. Plant Cell, 15, 1771–1780. Moons, A., De Keyser, A. and Van Montagu, M. (1997) A group 3 LEA cDNA of rice, responsive to abscisic acid, but not to jasmonic acid, shows varietyspecific differences in salt stress response. Gene, 191, 197–204. Mori, I.C., Murata, Y., Yang, Y. et al. (2006) CDPKs CPK6 and CPK3 function in ABA regulation of guard cell S-type anion- and Ca2+-permeable channels and stomatal closure. PLoS Biol. 4, 1749–1762. Mundy, J. and Chua, N.H. (1988) Abscisic acid and water-stress induce the expression of a novel rice gene. EMBO J. 7, 2279–2286. Ogasawara, Y., Kaya, H., Hiraoka, G. et al. (2008) Synergistic activation of the Arabidopsis NADPH oxidase AtrbohD by Ca2+ and phosphorylation. J. Biol. Chem. 283, 8885–8892. Raghavendra, A.S., Gonugunta, V.K., Christmann, A. and Grill, E. (2010) ABA perception and signalling. Trends Plant Sci. 15, 395–401. Rossel, J.B., Walter, P.B., Hendrickson, L., Chow, W.S., Poole, A., Mullineaux, P.M. and Pogson, B.J. (2006) A mutation affecting ASCORBATE PEROXIDASE 2 gene expression reveals a link between responses to high light and drought tolerance. Plant Cell Environ. 29, 269–281. Saijo, Y., Hata, S., Kyozuka, J., Shimamoto, K. and Izui, K. (2000) Overexpression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants. Plant J. 23, 319–327. Takahashi, F., Mizoguchi, T., Yoshida, R., Ichimura, K. and Shinozaki, K. (2011) Calmodulin-dependent activation of MAP kinase for ROS homeostasis in Arabidopsis. Mol. Cell, 41, 649–660.

Teixeira, F.K., Menezes-Benavente, L., Galvao, V.C., Margis, R. and MargisPinheiro, M. (2006) Rice ascorbate peroxidase gene family encodes functionally diverse isoforms localized in different subcellular compartments. Planta, 224, 300–314. Toki, S., Hara, N., Ono, K., Onodera, H., Tagiri, A., Oka, S. and Tanaka, H. (2006) Early infection of scutellum tissue with Agrobacterium allows highspeed transformation of rice. Plant J. 47, 969–976. Ton, J., Flors, V. and Mauch-Mani, B. (2009) The multifaceted role of ABA in disease resistance. Trends Plant Sci. 14, 310–317. Torres, M.A. and Dangl, J.L. (2005) Functions of the respiratory burst oxidase in biotic interactions, abiotic stress and development. Curr. Opin. Plant Biol. 8, 397–403. de Torres Zabala, M., Bennett, M.H., Truman, W.H. and Grant, M.R. (2009) Antagonism between salicylic and abscisic acid reflects early host-pathogen conflict and moulds plant defence responses. Plant J. 59, 375–386. Tseng, M.J., Liu, C.W. and Yiu, J.C. (2007) Enhanced tolerance to sulfur dioxide and salt stress of transgenic Chinese cabbage plants expressing both superoxide dismutase and catalase in chloroplasts. Plant Physiol. Biochem. 45, 822–833. Tsugane, K., Kobayashi, K., Niwa, Y., Ohba, Y., Wada, K. and Kobayashi, H. (1999) A recessive Arabidopsis mutant that grows photoautotrophically under salt stress shows enhanced active oxygen detoxification. Plant Cell, 11, 1195–1206. Tsugeki, R., Ditengou, F.A., Sumi, Y., Teale, W., Palme, K. and Okada, K. (2009) NO VEIN mediates auxin-dependent specification and patterning in the Arabidopsis embryo, shoot, and root. Plant Cell, 21, 3133–3151. Vinocur, B. and Altman, A. (2005) Recent advances in engineering plant tolerance to abiotic stress: achievements and limitations. Curr. Opin. Biotechnol. 16, 123–132. Ward, P., Equinet, L., Packer, J. and Doerig, C. (2004) Protein kinases of the human malaria parasite Plasmodium falciparum: the kinome of a divergent eukaryote. BMC Genomics, 5, 79. Wong, H.L., Pinontoan, R., Hayashi, K. et al. (2007) Regulation of rice NADPH oxidase by binding of Rac GTPase to its N-terminal extension. Plant Cell, 19, 4022–4034. Xiang, Y., Tang, N., Du, H., Ye, H. and Xiong, L. (2008) Characterization of OsbZIP23 as a key player of the basic leucine zipper transcription factor family for conferring abscisic acid sensitivity and salinity and drought tolerance in rice. Plant Physiol. 148, 1938–1952. Xiong, L. and Yang, Y. (2003) Disease resistance and abiotic stress tolerance in rice are inversely modulated by an abscisic acid-inducible mitogen-activated protein kinase. Plant Cell, 15, 745–759. Xiong, L., Schumaker, K.S. and Zhu, J.K. (2002) Cell signaling during cold, drought, and salt stress. Plant Cell, 14, S165–S183. Yoshida, S., Forno, D.A., Cock, J.H. and Gomez, K.A. (1976) Laboratory Manual for Physiological Studies of Rice. Philippines: The International Rice Research Institute, pp. 61–66. Yoshioka, H., Numata, N., Nakajima, K., Katou, S., Kawakita, K., Rowland, O., Jones, J.D. and Doke, N. (2003) Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell, 15, 706–718. Yoshioka, H., Asai, S., Yoshioka, M. and Kobayashi, M. (2009) Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity. Mol. Cells, 28, 321–329. Zhu, J.K. (2002) Salt and drought stress signal transduction in plants. Annu. Rev. Plant Biol. 53, 247–273. Zhu, S.Y., Yu, X.C., Wang, X.J. et al. (2007) Two calcium-dependent protein kinases, CPK4 and CPK11, regulate abscisic acid signal transduction in Arabidopsis. Plant Cell, 19, 3019–3036.
