Os02g0126400

The OsCPK4 gene is a member of the complex gene family of calcium-dependent protein kinases in rice (Oryza sativa), and its overexpression confers salt and drought tolerance by preventing membrane lipid peroxidation.

Annotated Information

Function

• The OsCPK4 gene (Os02g0126400) encodes a CDPK containing the four-domain structure typical of CDPKs: an N-terminal variable domain, a Ser/Thr kinase domain, a junction autoinhibitory domain, and a C-terminal calmodulin domain (Fig. 1A).The protective effects of OsCPK4 overexpression in conferring salt and drought tolerance might rely more on the protection against oxidative damage of membranes and the activation of genes involved in lipid metabolismrather than on the activation of the typical salt/drought stressassociated transcriptional networks in root tissues.
• OsCPK4 functions as a positive regulator of the salt and drought stress responses in rice via the protection of cellular membranes from stress-induced oxidative damage. The protective effects of OsCPK4 overexpression in conferring salt and drought tolerance might rely more on the protection against oxidative damage of membranes and the activation of genes involved in lipid metabolismrather than on the activation of the typical salt/drought stressassociated transcriptional networks in root tissues

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

Mutation

• loss-of-function mutants: two transfer DNA (T-DNA) insertion mutants of OsCPK4:
  

To further investigate the function of OsCPK4, Campo et al. searched for loss-of-function mutants. Two transfer DNA (T-DNA) insertion mutants of OsCPK4 were identified in the POSTECH collection: 2D-00040 (cv Dongjin background) and 1D-03351 (cv Hwayoung background). Both of them contained the T-DNA insertion in the kinase domain of the OsCPK4 gene.

• In each experiment:
  • wild-type (WT)
  • vector control(pC; three independent lines)
  • OsCPK4 overexpressor (OsCPK4-OX; five independent lines)

Expression

Figure 1. Expression of OsCPK4 in response to salt and drought stress.(from reference ).

• OsCPK4 gene expression is induced by salt and drought stress. OsCPK4 and OsLEA23/OsDip1 share similar expression patterns in response to salt stress, these two genes representing early salt stressresponsive genes:
  

OsCPK4 was rapidly and transiently activated upon exposure to salt stress in rice roots (Fig. 1C, left). Under drought stress imposed by either air-drying treatment or polyethylene glycol (PEG) treatment, induction of OsCPK4 expression was also observed, its activation being maintained during the entire period of stress treatment (Fig. 1C, middle and right). To assess the effectiveness of treatment, Campo et al. analyzed the expression of the marker gene OsLEA23, a dehydrin gene also known as OsDip1 (Fig. 1D). The expression of OsLEA23/OsDip1 is known to be up-regulated by salt and drought stress (earlyresponsive gene). OsLEA23/OsDip1 transcripts increased in response to high-salt conditions, with the highest levels recorded at 1 h of salt treatment and then decreasing at subsequent times of salt treatment (Fig. 1D, left). Air-drying and PEG treatment also induced OsLEA23/OsDip1 expression (Fig. 1D, middle and right).

• OsCPK4 expression is induced in response to treatment with ABA, the transcriptional activation of OsCPK4 expression in response to salt and drought stress or ABA treatment is accompanied by the accumulation of the OsCPK4 protein in rice roots.

OsCPK4 overexpression confers tolerance to salt and drought stress in rice plants. OsCPK4 knockout results in severe growth inhibition, which suggesting OsCPK4 disruption has a strong impact on plant growth, OsCPK4being required for the normal growth and development of rice plants.

• Overexpression of OsCPK4 results in few transcriptional changes in either root or leaf tissues. In root tissues, OsCPK4 positively regulates the expression of genes involved in metabolic processes, mainly lipid metabolism, as well as genes involved in

protection against oxidative stress. No significant alterations in the expression of salt-associated genes appear to occur in roots of OsCPK4 rice plants, whereas a reduction in the expression of certain salt-associated genes occurs in leaves of OsCPK4 plants.

• OsCPK4 overexpression prevents salt stress-induced lipid peroxidation and electrolyte leakage in cellular membranes under salt stress conditions. Under salt stress conditions, the OsCPK4 transgenic plants accumulate less Na+ in their roots than control plants.

Subcellular localization

To localize OsCPK4 in the plant cell, Campo et al. transiently expressed an OsCPK4-GFP fusion gene in onion epidermal cells: A plasma membrane localization of OsCPK4 was observed by transient expression assays of green fluorescent protein-tagged OsCPK4 in onion (Allium cepa) epidermal cells.

Evolution

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

'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, OsCPK4 belongs to the Group IV (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

• Centre for Research in Agricultural Genomics, Consejo Superior de Investigaciones Científicas-Institut de Recerca i Tecnologia Agroalimentàries-Universitat Autònoma de Barcelona-Universitat de Barcelona, Campus UAB, Bellaterra, Cerdanyola del Valles, 08193 Barcelona, Spain (S.C., P.B., J.M., M.C., B.S.S.)
• Oryzon Genomics, Cornella de Llobregat, 08940 Barcelona, Spain (E.L.)