Os08g0540400

OsCPK21, is a member of CDPKs (calcium-dependent protein kinases), which play important roles in regulating downstream components in calcium signaling pathways.

Annotated Information

Function

OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress. Calcium plays an important role as a messenger in various signal transduction pathways. Calcium-dependent protein kinases (CDPKs) are Ca²⁺-binding sensory kinases that regulate the downstream components of calcium signaling. CDPKs have been identified throughout the plant kingdom and in some protozoans but not in animals.
Overexpression of OsCPK21 in rice confers increased sensitivity to exogenous ABA, enhanced tolerance to salinity stress and elevated expression of ABA- and stress-inducible genes, suggesting a role for OsCPK21 as a positive factor in the ABA and salt stress signaling pathways,which could contribute to the elucidation of the CDPK-mediated ABA and salt stress signaling and to generate crops with improved tolerance to salt stress.

GO assignment(s): GO:0004672, GO:0004674, GO:0006468, GO:0005524, GO:0005509

Mutation

WT plants VS OsCPK21-FOX plants: Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, Asano et al. generated 250 independent transgenic rice plants overexpressing individual rice CDPKs(CDPK FOX-rice lines).These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type(WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants.

Expression

Figure 1.Tolerance of OsCPK21-FOX and OsCPK13-1-FOX plants to salt stress.(from reference ).

Figure 2.Phylogenetic relationships between CDPKs from rice and Arabidopsis.(from reference ).

• RT–PCR analysis confirmed that the OsCPK21 and OsCPK13-1 cDNAs were overexpressed in the OsCPK21- and the OsCPK13-1-FOX plants compared to WT(Fig. 1a). The OsCPK21-FOX plants also showed greater salt tolerance than WT plants (Fig. 1b). The survival rates of the OsCPK21-FOX plants under high salinity conditions reached 61–70%, whereas the survival of the WT plants was only 22% (Fig. 1c). No obvious differences in growth or development were observed between the transgenic and WT plants grown under normal growth conditions (data not shown). The survival rates of the OsCPK13-1-FOX plants under high salinity conditions were higher than those of

WT plants (Fig. 1c), and this result is consistent with that of Saijo et al..

• Asano et al. showed that OsCPK21 was strongly expressed in developing seeds. RT–PCR was used to monitor the expression of OsCPK21 in different tissues, and we detected high levels of expression in spikelets and developing seeds. RT–PCR analyses showed that the expression of OsCPK21 rapidly increased in the high salinity treatment, with the highest level recorded at 5 h. A relatively slow increase in OsCPK21 transcripts was also observed after ABA treatment. the abovementioned DRE, MYB and MYC recognition sequences may function as cis-acting elements responsive to abiotic stress in the OsCPK21 promoter.
• Asano et al. analysed the expression of ABA- and stressinducible genes in the OsCPK13-1-FOX plants compared to WT. When the plants were treated with exogenous ABA, the expression levels of the OsP5CS, OsLEA3 and OsNAC6 genes were more highly elevated in the OsCPK13-1-FOX plants than in WT plants. Expression of OsP5CS, OsLEA3, OsNAC6, OsbZIP23, OsNHX1 and OsSOS1 was also clearly enhanced by salt stress in the OsCPK13-1-FOX plants compared to WT.

Evolution

'Figure 3.Phylogenetic relatedness among the rice, Arabidopsis and functionally characterized CDPKs from other plant species.(from reference ).

Figure 4.Phylogenetic relationships among CDPKs from rice (OsCPK1-OsCPK29) and Arabidopsis (AtCPK1-AtCPK34).(from reference ).

'Figure 5.Phylogenetic relationships among rice CCaMK, and rice and Arabidopsis CDPKs, CRKs and PEPRKs.(from reference ).

• The phylogenetic tree was created using the ClustalW program based on the alignment of the kinase catalytic domains of 29 rice (OsCPK1-OsCPK29) and 34 Arabidopsis (AtCPK1-AtCPK34) CDPKs. OsCPK21 is indicated by an arrow. Phylogenetic analysis showed that rice CDPKs are divided into four distinct classes(Fig. 2).
• To study the evolutionary relatedness of rice and Arabidopsis CDPKs with all the CDPK genes characterized so far from alfalfa, cucumber, ice plant, mung bean, potato, strawberry, tomato, Petunia, maize, tobacco and Medicago, an unrooted tree was constructed by using ClustalX 1.83. This exercise resulted in four distinct groups similar to that reported by Asano et al.(Fig. 3).
• The amplitude of difierential expression for these genes was not as significant as reported earlier, possibly due to use of difierent rice variety and/or experimental conditions. Most of the previously identiWed stress responsive CDPK genes cluster together in

subclades Ia and Ib(Fig. 3).

• Each calcium-dependent protein kinase (CDPK) consists of a variable N-terminal domain, a protein kinase domain, an autoinhibitory region and a calmodulin-like domain with EFhand Ca²⁺-binding sites. CDPKs are directly activated by the binding of Ca²⁺ to the calmodulin-like domain, and the activated CDPKs regulate downstream targets. CDPKs have been identified throughout the plant kingdom， and in some protozoans, but not in animals. CDPKs constitute a large multigene family in various plant species; CDPK genes have been identified in Arabidopsis thaliana, and CDPK genes have been found in Oryza sativa (rice) (Fig. 4). The expression and activities of CDPKs are upregulated by a variety of stimuli, such as hormones, abiotic stresses and biotic stresses. Red letters indicate CDPKs involved in abiotic stress signaling.
• Phylogenetic relationships among rice CCaMK, and rice and Arabidopsis CDPKs, CRKs and PEPRKs. A phylogenetic tree was created using the ClustalW program, based on the predicted amino acid sequences of the rice and Arabidopsis kinases, which are indicated by red and blue type, respectively.As shown in Fig. 5, the phylogenetic tree of these kinase sequences forms seven subgroups: CDPKs I–IV, CRKs, CCaMK and PEPRKs. Furthermore, the 29 rice CDPKs were divided into four distinct classes.

Knowledge Extension

'Figure 6.Summary of the function of CDPKs in ABA and abiotic stress responses, as reported by several authors（from reference ).

In rice, the CDPKs constitute a large family of 29 genes. CDPK genes (OsCPK1-29) contain multiple stress-responsive cis-elements in the promoter region (1 kb) upstream of genes. Analysis of the information extracted from the Rice Expression Database indicates that 11 of the CDPK genes are regulated by chilling temperature, dehydration, salt, rice blast infection and chitin treatment. RT-PCR and RNA gel blot hybridization were performed in this study to detect the expression 19 of the CDPK genes. Twelve CDPK genes exhibited cultivar- and tissue-specific expression; four CDPK genes (OsCPK6, OsCPK13, OsCPK17 and OsCPK25) were induced by chilling temperature, dehydration and salt stresses in the rice seedlings. While OsCPK13 (OsCDPK7) was already known to be inducible by chilling temperature and high salt, this is the first report that the other three genes are stress-regulated. OsCPK6 and OsCPK25 are up-regulated by dehydration and heat shock, respectively, while OsCPK17 is down-regulated by chilling temperature, dehydration and high salt stresses. Based on this evidence, rice CDPK genes may be important components in the signal transduction pathways for stress responses.

Three major classes of Ca²⁺-binding proteins have been characterized in higher plants: calciumdependent protein kinases (CDPKs), calmodulins (CaMs) and CaM-like proteins, and calcineurin B-like proteins.

The subcellular localization of each CDPK and the phenotypes of overexpression (OX) or knockout or knockdown (KO) lines are described. Red letters, Arabidopsis CDPKs; green letters, rice CDPKs; P, phosphorylation; ABF, ABA-responsive element binding factor; HSP1, a heat shock protein; OST1, open stomata 1 protein kinase(Fig. 6).

Labs working on this gene

• 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
• National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan
• National Agricultural Research Center, Joetsu, Niigata 943-0193, Japan
• Department of Applied Biological Chemistry, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
• National Institute of Crop Science, Tsukuba, Ibaraki 305-8518, Japan
• National Key Laboratory of Crop, Genetic Improvement and National Centre of Plant Gene Research, Huazhong Agricultural University, Wuhan 430070, PR China
• Interdisciplinary Centre for Plant Genomics and Department of Plant Molecular Biology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India