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ENVIRONMENTAL STRESS AND ADAPTATION TO STRESS

Characterization of Stress-Responsive CIPK Genes in Rice for Stress Tolerance Improvement^1,[W]

National Center of Plant Gene Research (Wuhan), National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan 430070, China

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Plants respond to adverse environments by initiating a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein-interacting protein kinases (CIPKs). In this study, putative CIPK genes (OsCIPK01–OsCIPK30) in the rice (Oryza sativa) genome were surveyed for their transcriptional responses to various abiotic stresses. The results showed that 20 OsCIPK genes were differentially induced by at least one of the stresses, including drought, salinity, cold, polyethylene glycol, and abscisic acid treatment. Most of the genes induced by drought or salt stress were also induced by abscisic acid treatment but not by cold. A few CIPK genes containing none of the reported stress-responsive cis-elements in their promoter regions were also induced by multiple stresses. To prove that some of these stress-responsive OsCIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) were overexpressed in japonica rice ‘Zhonghua 11’. Transgenic plants overexpressing the transgenes OsCIPK03, OsCIPK12, and OsCIPK15 showed significantly improved tolerance to cold, drought, and salt stress, respectively. Under cold and drought stresses, OsCIPK03- and OsCIPK12-overexpressing transgenic plants accumulated significantly higher contents of proline and soluble sugars than the wild type. Putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants than in the wild type. The differentially induced expression of OsCIPK genes by different stresses and the examples of improved stress tolerance of the OsCIPK transgenic rice suggest that rice CIPK genes have diverse roles in different stress responses and some of them may possess potential usefulness in stress-tolerance improvement of rice.

Most abiotic stresses can elicit an increase of cytosolic free Ca²⁺ concentration in almost all eukaryotic cells, and the change of Ca²⁺ concentration has been generally accepted as a secondary messenger to transduce the cellular responses to extracellular stimuli (Sanders et al., 1999; Berridge et al., 2000, 2003; Hofer and Brown, 2003; Kolukisaoglu et al., 2004). In plants, calcium acts as a universal messenger in various signal transduction pathways, including responses to a diverse array of biotic (Chung et al., 2004; Ludwig et al., 2005) and abiotic stresses (Frohnmeyer et al., 1999; Knight and Knight, 2001; Kim et al., 2003a) and regulation of various cellular and developmental processes (Trewavas and Malho, 1998; Tran et al., 1999).

In plants, many Ca²⁺-sensing protein kinases have been reported for their involvement in the stress responses. These protein kinases include calcium-dependent protein kinases (Sheen, 1996; Saijo et al., 2000; Romeis et al., 2001; Pagnussat et al., 2002; Asano et al., 2005; Zhang et al., 2005; Chandran et al., 2006; Mori et al., 2006) and Suc non-fermentation-related kinases (SnRKs; Nozawa et al., 2001; Guo et al., 2002). In Arabidopsis, there are 38 SnRKs (Hrabak et al., 2003), including the SnRK3 subfamily that has been called CIPK (calcineurin B-like [CBL] protein interaction protein kinase; Shi et al., 1999; Albrecht et al., 2001; Batistic and Kudla, 2004; Mahajan et al., 2006) or SOS2 (SALT OVERLY SENSITIVE2)-like protein kinase (PKS) associated with the AtCBL/SOS3-like calcium-binding proteins (SCaBPs; Kolukisaoglu et al., 2004). The plant CIPK contains a specific Ser/Thr protein kinase domain that is activated through interaction with CBL containing four EF hands for Ca²⁺ binding (Ishitani et al., 2000; Nagae et al., 2003). Activated CIPKs can subsequently transduce calcium signals by phosphorylating downstream signaling components (Liu et al., 2000). Sequence analysis of Arabidopsis genome revealed 10 AtCBL/SCaBP and 25 CIPK/PKS genes (Luan et al., 2002; Kolukisaoglu et al., 2004). Expression patterns of these CBL/SCaBP and CIPK/PKS genes suggest diverse functions in different signaling processes, such as light, hormone, sugar, and stress responses (Kudla et al., 1999; Guo et al., 2002; Gong et al., 2004; Pandey et al., 2004). In Arabidopsis, a SOS pathway containing SOS3 (a Ca²⁺ sensor of the CBL family; Ishitani et al., 2000; Sanchez-Barrena et al., 2005), SOS2 (a CIPK protein; Ishitani et al., 2000; Gong et al., 2004; Sanchez-Barrena et al., 2005), and SOS1 (a Na⁺/H⁺ antiporter activated by SOS2; Shi et al., 2000; Qiu et al., 2002, 2004) has been well elucidated for its role in transducing Ca²⁺ signal, thereby maintaining ion homeostasis during salt stress (Liu et al., 2000; Shi et al., 2000; Xiong et al., 2002; Zhu, 2002; Sanchez-Barrena et al., 2005). SOS2 encodes a Ser/Thr protein kinase with an N-terminal catalytic domain (sharing sequence homology with SNF), C-terminal regulatory domain that is unique to CIPK family kinases, and a FISL/NAF motif containing 21 amino acids. Both the N-terminal and C-terminal domains are essential for salt tolerance (Albrecht et al., 2001), and the FISL motif acts as an autoinhibitory domain by interacting with the kinase domain to keep the kinase activity inactive under normal conditions. SOS3 can interact with SOS2 via the FISL motif thus to release SOS2 from an autoinhibition state to a kinase-active state in a calcium-dependent manner (Halfter et al., 2000). Through the SOS pathway, excess Na⁺ ions can be transported out of the cell via the plasma membrane Na⁺/H⁺ antiporter SOS1 that is phosphorylated and activated by the SOS3-SOS2 complex, thus reinstating cellular ion homeostasis (Qiu et al., 2002). Constitutively overexpressing SOS2 under the control of cauliflower mosaic virus 35S promoter can rescue the salt-hypersensitive phenotypes of the sos3 and sos2 mutants (Zhu, 2002; Cheng et al., 2004; Guo et al., 2004). Coexpression of all three functional components of the SOS pathway in a salt-sensitive yeast mutant led to a great increase in Na⁺ tolerance (Quintero et al., 2002).

The CBL/CIPK or SCaBP/PKS signaling pathway has also been evidenced in rice (Oryza sativa; Ohba et al., 2000; Kim et al., 2003b). Sequence analysis suggested 30 putative CIPK genes in the rice genome (Kolukisaoglu et al., 2004), and the predicted rice CIPK proteins have the same domain compositions as Arabidopsis CIPK proteins. However, the functions of almost all rice CIPK proteins remain to be revealed except very few reports on the expression of rice CIPK genes, including OsCK1, which is induced by diverse stimuli such as cold, light, salt, sugar, calcium, and cytokinin (Kim et al., 2003b). Toward uncovering the roles of rice CIPK genes in responses to abiotic stresses, a complete survey of the expression levels of all the putative rice CIPK genes in response to various abiotic stress and abscisic acid (ABA) treatments was conducted in this study. Based on the gene expression patterns, three stress-responsive CIPK genes were selected and overexpressed in rice to evaluate the potential usefulness of the stress-responsive CIPK genes in genetic improvement of stress tolerance.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification and Sequence Analysis of OsCIPK Genes

To identify all putative genes of the CIPK family in the rice genome, the reported CIPK protein sequences from Arabidopsis (Kolukisaoglu et al., 2004) were used as queries to search against the rice genome annotation database release 5.0 (http://www.tigr.org/tdb/e2k1/osa1/) and the full-length cDNA database (http://cdna01.dna.affrc.go.jp/cDNA). Through this approach, all the 30 putative CIPKs that had been designated as OsCIPK01 to OsCIPK30 by Kolukisaoglu et al. (2004) were identified. Although no additional putative CIPK was identified, some information of the OsCIPK genes was updated, including accession numbers of full-length cDNAs (locus ID of the predicted genes in the updated The Institute for Genomic Research [TIGR] database if cDNA not available), accession numbers of corresponding BAC/PAC clones, and physical locations of OsCIPK genes on chromosomes (Table I ).

View larger version (17K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Intron-exon organization of the 30 OsCIPK genes. Rectangles and lines represent exons and introns, respectively. White rectangles represent UTR regions. The // and line bar represent 2 kb and 1 kb, respectively. Genes in Groups I and II have many (>10) and few (<5) exons, respectively.

To reveal the responses of OsCIPK genes to different stresses, RNA gel-blot analyses were performed using total RNA from the leaves of upland rice IRAT109 treated by drought, salt, cold, polyethylene glycol (PEG), and ABA. Reverse transcription (RT)-PCR and real-time PCR were also used because the hybridization signals of 11 OsCIPK genes in RNA gel-blot analyses were too weak to distinguish the difference between samples. Three genes (OsCIPK13, 14, and 27) could not be amplified in the leaves by RT-PCR (OsCIPK13 and OsCIPK27 had no cDNA support); the transcripts of all the other 27 OsCIPK genes could be detected by RNA gel-blot (Fig. 2 ) and /or PCR analyses (Fig. 3 ; Supplemental Fig. S2). Among them, 20 genes were induced by at least one of the stresses applied in this study. Among these 20 genes, 15 genes (OsCIPK01, 02, 05, 09, 11, 12, 15, 17, 20, 21, 22, 23, 24, 29, and 30) were induced by drought, 12 genes (OsCIPK07, 08, 09, 10, 11, 15, 16, 17, 21, 22, 29, and 30) were induced by salinity stress, 12 genes (OsCIPK01, 03, 09, 12, 15, 16, 17, 21, 22, 23, 24, and 29) were induced by PEG treatment, 16 genes (OsCIPK01, 02, 03, 05, 07, 09, 11, 12, 15, 16, 17, 20, 22, 24, 29, and 30) genes were induced by ABA treatment, and three genes (OsCIPK01, 03, and 09) were induced by cold (Figs. 2 and 3).

View larger version (137K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. RNA gel-blot analysis of transcript levels for OsCIPK genes under drought, salt, cold, PEG, and ABA treatments. Multiple RNA blots were prepared from the same set of RNA samples (RNA loading of one set was shown as an example) and hybridized with gene-specific probes for different genes. Each stress treatment was repeated twice, and result from one repeat is shown.

View larger version (28K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. Real-time PCR analysis of expression levels of the OsCIPK genes that were undetectable by RNA gel blot. RNA samples were the same as those used in the RNA gel-blot analysis. The x axes are time courses of stress treatments and the y axes are scales of relative expression level. d, drought; s, salt; a, ABA; p, PEG; c, cold. For the gene OsCIPK09, the bars for d3 (>50-fold) and s1 (>70-fold) indicated by asterisks were intentionally reduced (fold of actual induction minus 30) so that the induction folds for other stresses could be seen clearly. A threshold of 2-fold was used to determine the stress responsiveness of genes.

View larger version (29K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Putative ABRE, DRE, and LTRE core sequences in the 1-kb promoter regions of the stress-inducible genes identified by RNA gel-blot and RT-PCR analyses. The lines represent promoter sequences. The elements located in the forward strand (sense strand of the gene) and the reverse strand are indicated above and below the lines, respectively. Induction of the genes by stress treatments is indicated to the right of each gene with checks (D, drought; S, salt; A, ABA; P, PEG; C, cold).

To further confirm that some of these stress-responsive genes are potentially useful in improving stress resistance of crop plants, three genes (OsCIPK03, OsCIPK12, and OsCIPK15 strongly induced by cold, drought, and salt stress, respectively) were overexpressed in rice ‘Zhonghua 11’ and the transgenic plants were tested for stress tolerance.

Overexpression of OsCIPK03 in Rice Results in Enhanced Cold Tolerance

A total of 23 independent transgenic plants were generated for the OsCIPK03 construct under the control of the cauliflower mosaic virus 35S promoter. RNA gel-blot analysis suggested that the gene was overexpressed in 15 independent plants (Fig. 5A ). Transgenic seeds of four OsCIPK03-overexpressed T₁ families (T10, T24, T29, and T30) were germinated on hygromycin-containing Murashige and Skoog (MS) medium and at least 20 positive (hygromycin-resistant) seedlings for each family were selected for cold tolerance at the five-leaf stage. Overexpression plants and wild type with similar vigor under normal growth conditions (Fig. 5B) were used for treatment. After cold treatment (4°C for 5 d) and recovery (25°C for 5 d), almost all wild-type plants were completely dead, whereas most of the transgenic plants remained alive (Fig. 5C). In another cold treatment (4°C for 4 d and 25°C for 7 d for recovery) experiment, the survival rates of all the four transgenic families (68.2%–90.4%) were significantly (t test, P < 0.01) higher than the survival rate of the wild type (18.5%; Fig. 5D). These results suggested that overexpression of OsCIPK03 could enhance the tolerance of transgenic plants to cold shock. In addition, no obvious difference in growth or development was observed between transgenic and wild-type plants under normal growth conditions (Supplemental Fig. S3). Drought and salt tolerance of OsCIPK12 transgenic plants were also tested, but no significant effect was detected for these two stresses (data not shown).

View larger version (56K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Identification and cold-tolerance testing of OsCIPK03-overexpressing rice. A, RNA gel-blot analysis of part of the transgenic plants and the wild type. B, Transgenic and wild-type plants right before cold treatment. C, Performance of cold tolerance of two transgenic families and wild-type plants after cold treatment (4°C for 5 d) and recovery (25°C for 5 d). D, Survival rate of four transgenic lines and the wild type after cold treatment (4°C for 4 d and 25°C for 7 d for recovery). Error bars are based on three replicates.

A total of 27 independent transgenic plants were generated for the OsCIPK12 overexpression construct. Among them, 15 plants showed overexpression of the gene, as determined by RNA gel-blot analysis (Fig. 6A ). At least 20 hygromycin-resistant transgenic seedlings with similar plant height and vigor as the wild type (Fig. 6B) for each of six OsCIPK12-overexpressed T₁ families were selected for drought-resistance testing at the vegetative stage. With water withheld for 1 week, only a few plants of the transgenic families showed slight leaf-rolling, while almost all leaves of the wild type became rolled or withered. After recovery (rewatering when all leaves of the wild type were completely rolled) for 3 d, only about 30% of wild-type plants survived, whereas most of the transgenic plants (62.7% to 87.4%) survived quite well (Fig. 6C). The survival rates of the six transgenic families were significantly (t test, P < 0.01) higher than the survival rate of the wild type (Fig. 6D). This result suggested that overexpression of OsCIPK12 could increase the drought tolerance of rice at the vegetative stage. We also tested the salt and cold tolerance of OsCIPK12 transgenic plants, but no significant effect on improving tolerance to these two stresses was detected (data not shown).

View larger version (63K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. Identification and drought-tolerance testing of OsCIPK12-overexpressing rice. A, RNA gel-blot analysis of part of the transgenic plants and the wild type. B, Transgenic (two independent families are shown as examples) and wild-type plants right before treatment. C, Phenotypes of transgenic and wild-type plants after water withholding for 1 week and recovery for 3 d. D, Survival rate of transgenic lines and the wild type. Error bars are based on three replicates.

A total of 27 independent transgenic plants were also generated for the OsCIPK15 overexpression construct. RNA gel-blot analysis suggested that the gene was overexpressed in 17 independent plants (Fig. 7A ). Transgenic seeds of four OsCIPK15-overexpressed T₁ families (T4, T14, T19, and T22) were germinated on MS medium containing hygromycin and then at least 20 seedlings for each family were transferred to MS medium containing 100 mM NaCl for salt-tolerance testing. Transgenic shoots with similar length and vigor as the wild type were used as control. At 12 d after transplanting, all the transgenic families on salt-containing medium had significantly (t test, P < 0.01) longer shoot and root length (Fig. 7, B and C) and higher fresh weight per plant (Fig. 7D) than the wild type. No significant difference in shoot and root length (data not shown) or fresh weight (white bars in Fig. 7D) was observed between transgenic families and the wild type growing in the normal MS medium. These results suggested that overexpression of OsCIPK15 could enhance salt tolerance of rice. We also tested the drought and cold tolerance of OsCIPK15 transgenic plants, but no significant effect on improving tolerance to these two stresses was detected (data not shown).

View larger version (36K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Identification and salt-tolerance testing of OsCIPK15-overexpressing rice. A, RNA gel-blot analysis of part of the transgenic plants and the wild type. B, Growth performance of transgenic (one family is shown as an example) and the wild type in medium containing 100 mM NaCl for 12 d. C and D, Shoot and root length (C) and fresh weight (D) of transgenic lines and wild type growing in the NaCl-containing medium for 12 d. The average values were calculated from measures of at least 20 plants.

Plant adaptation to environmental stresses is often associated with metabolic adjustment, such as accumulation of Pro and soluble sugars (Abraham et al., 2003). To inquire the physiological basis for the improved stress tolerance of transgenic rice, we measured the Pro and soluble sugar contents in the overexpression plants of OsCIPK genes and the wild type under normal growth and stress conditions. No significant difference of Pro and soluble sugar contents in the leaves was detected between the overexpression plants of the three genes (OsCIPK 03, 12, and 15) and the wild type grown under normal growth conditions. After cold stress for 3 d, OsCIPK03-overexpressing plants (three independent lines were tested) accumulated approximately 8-fold higher content of Pro (Fig. 8A ) and 5-fold higher content of soluble sugars (Fig. 8B) than the plants did before cold stress. The cold stress-induced increases of Pro and soluble sugar contents in the transgenic plants were significantly higher (t test, P < 0.01) than those in wild-type plants, in which only about 2- to 3-fold increases of Pro and the soluble sugars after cold stress were detected. Under drought stress, OsCIPK12-overexpressing plants (three independent lines were tested) also accumulated Pro and soluble sugars much quicker than wild-type plants. After water withholding for 6 d, transgenic plants accumulated approximately 7-fold higher content of Pro (Fig. 8C) and 5-fold higher content of soluble sugars (Fig. 8D) compared to the contents of Pro and sugars before drought stress. Such increases were significantly higher than that in wild-type plants, in which Pro and soluble sugars were increased less than 3-fold after drought stress. In OsCIPK15-overexpressing plants, however, the Pro and soluble sugar contents had no significant difference compared to the wild type under both normal and salt-stressed conditions (data not shown).

View larger version (34K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Contents of soluble sugars and free Pro in the transgenic rice plants overexpressing OsCIPK03 and OsCIPK12 genes compared to the wild-type plants. A and B, Free Pro (A) and soluble sugar (B) contents in the OsCIPK03-overexpressing plants under cold stress (4°C) for 0, 1, 3, and 6 d. C and D, Free Pro (C) and soluble sugar (D) contents in the OsCIPK12-overexpressing plants under drought stress for 0, 4, 6, and 10 d. Values are means ± SD (n = 4). FW, Fresh weight. E and F, Expression levels of putative Pro synthetase genes (J033099M14 and J033031H21) and Pro transporter genes (03g44230 and 07g01090) in cold-stressed (4°C for 1 d) OsCIPK03 (E) and drought-stressed (drought for 6 d) OsCIPK12 (F) transgenic plants by real-time PCR. The bars represent three repeats. Accession numbers of the genes are as follows: J033099M14, AK102633; J033031H21, AK101230; 03g44230, AK067118; and 07g01090, AK0666298.

For probing the mechanism of enhanced salt tolerance of OsCIPK15-overexpressing plants, concentrations of Na⁺ and K⁺ in the root, stem, and shoot were measured for transgenic and wild-type plants growing in the Hoagland solution containing 100 mM NaCl. Interestingly, no significant difference in the concentration of either ion was detected between transgenic plants and the wild type in the examined tissues, including root, leaf, and stem (Supplemental Table S1), despite the fact that the transgenic plants still showed improved salt tolerance similar to the result shown in Figure 7B.

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Differential Responses of OsCIPK Genes to Abiotic Stresses

Rice is one of the most important crops and the model plant for monocot species. In this study, as a first step toward systematic functional characterization of CIPKs in rice, all the putative rice CIPK genes were checked for their expression changes upon various stress treatments. Our results clearly suggest that different OsCIPK genes have different responses to stresses. Among the 27 genes detected with transcripts by the techniques used in this study, 20 genes were found to be induced by single or multiple stresses. The OsCIPK genes induced by different stresses may provide new starting points to uncover the unique molecular basis of rice response to different stresses considering the nature of CIPKs as putative signaling components. Although the transcript levels of most OsCIPK genes were up-regulated by at least one stress, a few OsCIPK genes did not exhibit obvious change of transcript level upon the stress treatments. Since the CIPK family has been largely diversified and different members may be involved in responses to different stresses, it would be necessary to include more stress treatments in further studies to check the expression of OsCIPK genes, especially for those genes showing no response to the stresses in this study.

Besides the differential responses of OsCIPK genes to various stresses, responses of specific OsCIPK genes to multiple stresses were also detected. Noticeably, most ABA- or PEG-inducible genes were also induced by drought or salt stress, and most salt-inducible genes were also induced by drought (Fig. 4). These results indicate that OsCIPK genes may be involved in the substantial common regulatory systems or cross talks triggered by different stresses. As many abiotic stresses ultimately result in dehydration and osmotic imbalance of plant cells, there is a large overlap of genes induced by drought and salt stresses (Xiong et al., 2002; Ludwig et al., 2005; Nambara and Marion-Poll, 2005). Our results based on the stress-induced expression patterns of OsCIPK genes also support the notion of cross talks between drought and salt stresses (Seki et al., 2002a, 2002b). Stress-induced changes of gene expression in turn may affect the generation of hormones such as ABA, which has been considered an important "stress hormone" and plays a critical role in response to various stresses. The application of ABA to plant partially mimics the effect of stress conditions (Nambara and Marion-Poll, 2005). In this study, most ABA-inducible genes were also induced by drought or salt stress, suggesting that these OsCIPK genes may participate in the cross talks of signaling pathways for drought and salt stresses and ABA treatment. Studies have suggested that osmotic stress imposed by high salt or drought can be transmitted through ABA-dependent and ABA-independent signaling pathways (Uno et al., 2000; Chinnusamy et al., 2004). We also found that quite a few drought- or salt-inducible OsCIPK genes were not induced by ABA treatment. However, further experiments are required to clarify which OsCIPK genes function in ABA-dependent or ABA-independent signaling pathways. In this study, most genes induced by drought, salt, ABA, or PEG were not induced by cold stress, which may imply that some OsCIPK genes might be specifically involved in the signaling pathways triggered by cold stress.

Complex Relationships of Predicted Stress-Related cis-Elements and Actual Stress Induction Patterns of OsCIPK Genes

cis-Elements and trans-acting factors are two basic types of molecular modulators for transcriptional regulation of genes controlling various biological processes, including abiotic stress responses. Several types of cis-elements have been identified and analyzed for their activation by different stresses. Among these stress-responsive cis-elements, ABRE, DRE, and LTRE have been extensively characterized for their important roles in activation of gene expression under stress conditions (Yamaguchi-Shinozaki and Shinozaki, 1994; Shinwari et al., 1998; Narusaka et al., 2003). In this study, 20 genes were induced by different stresses, including drought, salt, cold, PEG, and ABA treatments, which prompted us to investigate the relationship of induction pattern and distribution of putative stress-responsive elements of these genes. Most of the stress-inducible genes (16 of 20) contain at least one of the three cis-elements (DRE, ABRE, or LTRE) within the 1,000-bp promoter regions of these genes (Fig. 4). Generally, the genes containing putative stress-responsive cis-elements are actually induced by the corresponding stress, which is especially true for ABRE (all ABRE-containing genes are induced by ABA, drought, or salt stress). However, the predicted cis-elements do not support the actual expression patterns for some genes. For example, there are four OsCIPK genes (OsCIPK06, 19, 26, and 28) that are not induced by any of the stresses applied in this study but actually contain at least one of the three cis-elements in their promoter regions. On the other hand, there are four genes (OsCIPK07, 08, 12, and 20) that contain none of the three cis-elements mentioned above in their promoter regions, but these four genes are actually induced by at least one stress. These discrepancies could be due to the sequence variation in the promoter regions since we used variety IRA109 in this experiment while the promoter sequences analyzed were from ‘Nipponbare’. The discrepancies may also suggest that the activation of the cis-elements largely depends on the cis-background (sequences flanking the cis-element) and the trans-background (activity of the corresponding transcription factors). The discrepancies are not very surprising since only three major cis-elements were included in this analysis and increasing number of stress-responsive cis-elements have been identified and deposited in the PLACE database. It cannot be excluded that novel stress-responsive cis-elements yet to be identified may exist in the promoters of these stress-inducible genes containing none of the reported cis-elements.

Enhanced Stress Tolerance Mediated by OsCIPK Genes

Response of a gene to a specific stress at the transcriptional level does not necessarily mean that the gene must have significant effect in conferring tolerance to the stress. However, a survey of stress-induced expression patterns can often provide clues for speculating the putative functions of the genes, which may be especially true for a functionally diversified gene family such as the CIPK family. To prove that some of these stress-responsive OsCIPK genes may be potentially useful for enhancing stress tolerance in rice, three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15, responsive to cold, drought, and salt stress, respectively) were chosen as examples and overexpressed in japonica rice ‘Zhonghua 11’. Our results indicate that transgenic rice plants overexpressing OsCIPK03, OsCIPK12, and OsCIPK15 can indeed significantly improve rice tolerance to cold, drought, and salt stress, respectively.

Plants have evolved an array of mechanisms in response to abiotic stresses, such as high salt, drought, cold, heat, and excessive osmotic pressure. In response to these stresses, many plants can accumulate compatible osmolytes, such as free Pro (Liu and Zhu, 1997; Armengaud et al., 2004; Ito et al., 2006) and soluble sugars (Gilmour et al., 2000; Garg et al., 2002; Tabuchi et al., 2004; Gupta and Kaur, 2005; Chyzhykova and Palladina, 2006), to protect their subcellular structures from damage by adjusting the intracellular osmotic potentials. Overproduction of Pro in tobacco (Nicotiana tabacum) could lead to an increase in tolerance of osmotic stress (Kishor et al., 1995). To address the possible mechanisms of the enhanced stress tolerance of the OsCIPK transgenic rice, we first checked the contents of Pro and soluble sugars in the transgenic plants under normal growth and stress conditions. The data showed that the OsCIPK03- and OsCIPK12-overexpressing plants indeed accumulated significantly more Pro and soluble sugars than the wild type under cold (for OsCIPK03 overexpressor) and drought (for OsCIPK13 overexpressor) stress conditions. Real-time PCR results also showed that Pro synthetase and Pro transport genes had significantly higher expression level in the OsCIPK03 and OsCIPK12 overexpression lines than in the wild type. The significantly improved Pro and sugar contents of OsCIPK03- and OsCIPK12-overexpressing plants under the stress conditions may partially explain the improved tolerance of OsCIPK03- and OsCIPK12-overexpressing plants to cold and drought stresses, respectively. No significant difference in Pro and sugar contents was detected between the transgenic and wild-type plants under normal growth conditions, despite the fact that the constitutive promoter was used to drive the transgenes. This might suggest that these CIPKs are not active under normal growth conditions. As for the OsCIPK15-overexpressing plants, we did not detect significant difference either in Pro and sugar contents or Na⁺ and K⁺ concentrations in root, stem, and leaf between the OsCIPK15-overexpressing plants and the wild type under both normal and salt stress conditions, which contrasts with the result of the homologous CIPK gene SOS2 in Arabidopsis (Zhu et al., 1998; Zhu, 2002). Nevertheless, this observation has provided a starting point to identify a possible new mechanism of salt tolerance in addition to the SOS2-mediated pathway.

Although the molecular basis of the improved stress tolerance of the transgenic rice has not been completely resolved in this report, our data clearly suggest a functional diversification of the CIPK family in response to different stresses. Further characterization of the OsCIPK genes involved in different stress responses will largely expand our understanding the functions of the CIPK family. Even though only three OsCIPK genes were tested for their effectiveness in improving stress tolerance in rice, some other stress-responsive OsCIPK genes (most of them are under transgenic testing) may also be potentially useful for stress-tolerance improvement in rice.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Identification and Sequence Analysis of CIPK Genes in Rice

The protein sequences of Arabidopsis (Arabidopsis thaliana) CIPK proteins available from The Arabidopsis Information Resource (http://www.arabidopsis.org; Garcia-Hernandez et al., 2002) and a previous report (Kolukisaoglu et al., 2004) were used to search for their homologous genes in the databases of rice, including the genome annotation database at TIGR (http://www.tigr.org/tdb/e2k1/osa1/) and the KOME (http://cdna01.dna.affrc.go.jp/cDNA). The resulting homologous protein sequences (with E-value less than 1.0E10^–5) were manually checked for existence of the feature motifs and domains of CIPK proteins. The exon-intron organizations of the rice CIPK genes were determined by comparing the full-length cDNA or predicted CDSs with their corresponding genomic sequences. The position of each gene in the rice genome was determined by BLASTN search against the genomic sequences of rice chromosomes available at the GRAMENE database (http://www.gramene.org).

To analyze putative cis-acting regulatory DNA elements (cis-elements) in the promoters of the rice CIPK genes, 1,000-bp regions upstream the 5' end of the full-length cDNAs or predicted CDS were extracted from genomic sequences and subjected to cis-element search in PLACE 26.0, a frequently updated database of core DNA sequences of cis-elements identified in plants (http://www.dna.affrc.go.jp/PLACE/index.html; Higo et al., 1999).

Constructs of OsCIPK Genes and Rice Transformation

The full-length cDNAs of OsCIPK03, OsCIPK12, and OsCIPK15 were amplified from upland rice IRAT109 (Oryza sativa L. ssp. japonica) by RT-PCR with the following three pairs of primers, respectively: 5'-CACCATGTATAGGGCTAAGAGGGCT-3' and 5'-CTTGAAACTCACAAACTGTCA-3', 5'-CACCATGCTGATGGCGACCGTCTC-3' and 5'-GGAAGCTCCTGTCTCTAGCTC-3' (the four underlined nucleotides, CACC, were added in front of the gene-specific forward primer to facilitate the TOPO cloning of the PCR fragment into the TOPO-D entry vector), and 5'-TAAGGTACCGGCTAAAGAATTGCAGTCCA-3' and 5'-TAAGGATCCTTGCGACTGCTGCTATTC-3' (the underlined sequences are for restriction sites KpnI and BamHI, respectively). For OsCIPK03 and OsCIPK12, the PCR products were cloned into TOPO-D entry vector (Invitrogen), then introduced into destination vector pCB2004H (modified based on pCB2004) by LR reaction by following the manufacturer's instructions (Invitrogen). The Bar gene in pCB2004 (provided by Dr. Chengbin Xiang) was replaced by hygromycin-resistance gene Hpt (hygromycin phosphotransferase), resulting in the pCB2004H. For OsCIPK15, the sequencing-confirmed PCR fragments were double digested by KpnI and BamHI and ligated into the binary expression vector pCAMBIA1301U digested with KpnI and BamHI, thus allowing the genes to be driven by a maize (Zea mays) ubiquitin promoter. The constructs were introduced into japonica ‘Zhonghua 11’ by Agrobacterium-mediated transformation (Hiei et al., 1994).

Plant Growth and Treatments

For transcript level measurement of OsCIPK genes, rice plants of IRAT109 were grown in the green house with a 14-h-light/10-h-dark cycle. Two-week-old seedlings were treated with chemical and abiotic stresses. Chemical treatments were conducted by spraying leaves with 0.1 mM ABA followed by sampling at 0, 3, 6, 12, and 24 h, or irrigating the plants with 20% PEG 6000 followed by sampling at 0, 1, 5, and 12 h. Abiotic treatments were conducted essentially according to Saijo et al. (2000). For cold stress, seedlings were transferred to a growth chamber at 4°C and sampled at 0, 3, 6, 12, and 24 h after treatment. Drought stress was applied by exposing intact plants in the air without water supply and plant leaves were sampled at 0, 3, 6, 12, and 24 h after treatment. The 2-week-old seedlings were submerged with 200 mM NaCl solution for salt stress and sampled at 0, 5, 14, and 24 h after treatment.

For drought-resistance testing of transgenic rice at the seedling stage, the plantlets that germinated on plates with 50 mg/L hygromycin and positive seedlings and wild-type control (both with shoot height of 4–5 mm) were transferred into barrels filled with a mixture of soil and sand (1:1). To minimize the experimental error, each barrel was filled with 2 kg of soil/sand mixture and supplied with the same volume of water. When the plants grew up to five-leaf stage, water supply was cut off to allow drought stress to develop. About 10 d later, the survival rates of plants and the dry weight per plant were investigated. For salt-tolerance testing, transgenic seeds were germinated on MS medium with 50 mg/L hygromycin and wild-type seeds were germinated on MS medium without hygromycin at 26°C. The plantlets (shoot length 4–5 mm) were transferred onto MS medium containing 100 mM NaCl and allowed to grow in a growth chamber at 26°C with a 14-h-light/10-h-dark cycle. Plants were measured for fresh weight, shoot length, root length, and number of roots at 12 d after transplanting. For cold-tolerance testing, positive transgenic and wild-type plantlets (with shoot height 2–3 cm) were transferred into pots filled with soil. When the plants grew up to the five-leaf stage, the plants were transferred to growth chamber at 4°C and subsequently kept under normal growth conditions for 5 d. The survival rate as well as the area of green leaves after cold stress were investigated.

RNA Gel-Blot and Real-Time PCR Analyses

Total RNA was isolated from rice leaves using TRIzol reagent (Invitrogen). Total RNA (15 µg from each sample) was separated on a 1.2% agarose gel containing 2% formaldehyde and then transferred onto a nylon membrane. RNA gel blots were hybridized with -³²P-dCTP-labeled gene-specific probes (primers for amplifying the OsCIPK gene probes are listed in Table I) overnight using Perfect-HYB Plus buffer (Sigma) at 65°C. Blots were washed three times (twice with 2x SSC/0.1% SDS for 10 min and once with 0.5x SSC/0.1% SDS for 5 min) at 65°C. The blots were briefly dried and then subjected to radiophotography. For real-time PCR analysis, first-strand cDNAs were synthesized from DNaseI-treated total RNA using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. Real-time PCR was performed in an optical 96-well plate with an ABI PRISM 7500 real-time PCR system (Applied Biosystems). Each reaction contained 10 µL of 2x SYBR Green Master Mix reagent (Applied Biosystems), 1.0 µL of cDNA samples, and 200 nM gene-specific primer in a final volume of 20 µL. The thermal cycle used was as follows: 95°C for 3 min; 40 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The primers for real-time PCR analysis of genes related to Pro synthesis and transport were as follows (in parentheses): J033099M14 (5'-CTCAAATCAAGGCGTCAACTAAGA-3' and 5'-TTTGTCAATATATACGTGGCATATACCA-3'), J033031H21 (5'-CGCCCCTCCCCGTATCT-3' and 5'-AGGAATGCGGCAACAAGTG-3'), 03g44230 (5'-AGGGACGATGGAGTTCTAAAGCT-3' and 5'-GGGATTCCAAAGGCAAAAAGA-3'), 07g01090 (5'-GAGGAGGCTACCTGACTGTCAAC-3' and 5'-GCTCATGAAGTCGCCAAGGA-3'). Rice Actin1 gene (accession no. X16280) was used as internal control with primers 5'-TGGCATCTCTCAGCACATTCC-3 and 5'-TGCACAATGGATGGGTCAGA-3'. The relative expression levels were determined as described previously (Livak and Schmittgen, 2001).

Measurement of Pro and Sugar Contents

Transgenic plants at the four-leaf stage were used for biochemical analysis. Free Pro content in leaves was determined by essentially following the reported methods (Troll and Lindsley, 1955). About 50 mg of leaves were homogenized in 1 mL of sulfosalicylic acid (3%) and the homogeneous mixture was centrifuged at 13,000 rpm for 15 min at 4°C. The extract (200 µL) was transformed to a new microcentrifuge tube and mixed with 200 µL of acid ninhydrin (0.1 g ninhydrin dissolved in 2.4 mL of glacial acetic acid and 1.6 mL of 6-mortho-phosphoric acid) and 200 µL of acetic acid. The reaction mixture was boiled in a water bath at 100°C for 30 min and cooled down at 4°C for 30 min. Then 400 µL of toluene was added to the leaf extract and thoroughly mixed. Finally, 120 µL of the toluene phase was removed for absorbance measurement at 520 nm in a DU640 spectrophotometer (Beckman).

Total soluble sugars in leaves were determined by the modified phenol-sulfuric acid method (Dubois, 1956). About 0.1 g of leaves were homogenized in 8 mL of double-distilled water, then boiled twice in a water bath at 100°C for 30 min. The extract (about 500 µL) was transferred to a new microcentrifuge tube, added with 1.5 mL of double-distilled water, 1 mL of 9% (v/v) phenol, and 5 mL of sulfuric acid added, and kept at room temperature for 30 min. The absorbance was measured at 485 nm in a DU640 spectrophotometer (Beckman).

Sequence data from this article can be found in the GenBank/EMBL data libraries under the accession numbers listed in Table I.

Supplemental Data

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

Supplemental Figure S1. Partial sequence alignment of part of OsCIPKs.

Supplemental Figure S2. RT-PCR analysis of nine OsCIPK genes that could not be detected with signals in northern analysis.

Supplemental Figure S3. Fresh weight of transgenic plants at four-leaf stage.

Supplemental Table S1. Content (mg/g) of Na⁺/K⁺ in root, stem, and leaf of OsCIPK15-overexpressing transgenic seedlings under normal and salt stress conditions.

	ACKNOWLEDGMENTS

 
We thank Dr. Chengbin Xiang for providing the pCB2004 vector and Yinglong Cao for providing the modified pCAMBIA1301U vector with maize ubiquitin promoter.

Received April 19, 2007; accepted May 22, 2007; published May 25, 2007.

	FOOTNOTES

 
¹ This work was supported by grants from the National Program on the Development of Basic Research (2006CB100103), the National Program on High Technology Development, the National Natural Science Foundation of China, and the Rockefeller Foundation.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Lizhong Xiong (lizhongx{at}mail.hzau.edu.cn).

^[W] The online version of this article contains Web-only data.

www.plantphysiol.org/cgi/doi/10.1104/pp.107.101295

^* Corresponding author; e-mail lizhongx{at}mail.hzau.edu.cn; fax 86–27–87287092.

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