Os04g0584600

OsCPK13(also named OsCDPK7), is a member of CDPKs (calcium-dependent protein kinases),which confers both cold and salt/drought tolerance on rice plants].

Annotated Information

Function

Figure 1. A model for OsCDPK7-mediated cold-and salt/drought-stress signal transduction.（from reference ).

• Using the ZmCDPK7 cDNA as a probe, we isolated a highly homologous rice clone，designated OsCDPK7 (GenBank accession no. AB042550)' from a rice root cDNA library.The entire coding region of OsCDPK7 was subcloned into an expression vector, pGEX4T-1 (Amersham Pharmacia), and then transformed into Eschericia coli BL21(DE3).

• OsCDPK7(OsCPK13,Os04g0584600) is a positive regulator commonly involved in the adaptation to at least three distinct stress agents, cold, salt and drought. It belongs to a subclass of stress-inducible CDPKs, which is probably conserved from monocotyledo-nous to dicotyledonous plants.

• The induction of rab16A and salT by ABA was increased or decreased by OsCDPK7 over-expression or suppression, respectively, which indicates that OsCDPK7 is also involved in ABA-dependent pathways.

• Saijo et al. propose a model for the OsCDPK7 signalling pathway under the above stress conditions, in which the amount of activated OsCDPK7 determines the transduction current (Figure 1). The results also suggest that OsCDPK7 acts at one of the branch points of stress signal transduction in rice. Nevertheless, there seems little or no cross-talk downstream of these CDPK pathways even when each signal is amplifed by OsCDPK7 over-expression. There must be unknown mechanisms that maintain the signal-ling specifcity,Future analyses, especially on protein-protein interactions and protein localization, are awaited to verify this model.

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

Mutation

Figure 2. Cold and salt induction of the OsCDPK7 transcript.（from reference ).

Transgenic rice plants:

• S3
• S7
• S21

14 independent lines of transgenic plants(T0 generation) were generated. Among them, two lines over-expressing OsCDPK7, S3 and S1, and another co-suppressed line, S27, were chosen for further experiments.

Expression

• Expression of the OsCDPK7 mRNA, around 2.3 kb in size, was increased by cold and salt stresses in both shoots (Figure 2a) and roots (data not shown) of 10-day-old seedlings, but not by exogenous abscisic acid (ABA) application. In contrast, a rice stress-responsive gene，rab16A, was induced by the salt and ABA treatments (Figure 2a). This indicates that OsCDPK7 belongs to a subclass of stress-inducible CDPKs， which is probably conserved from monocotyledonous to dicotyledonous plants.

• Over-expression of OsCDPK7 conferred both cold and salt/drought tolerance on rice plants. In contrast, suppression of OsCDPK7 expression lowered the stress tolerance. Therefore, OsCDPK7 plays key roles in the tolerance to the two types of stress in rice.

• Expression of OsCDPK7 in rice roots:

In the elongation zone, intense signals were found predominantly in the central cylinder, which is composed of specialized conducting tissues, upon cold stress. A detailed view of a transverse section revealed that the protein is expressed highly in xylem parenchyma cells around metaxylem II and sclerenchyma cells. These cells are strengthened by lignified secondary walls, and thus may also serve as supporting tissues. Weak signals were also detected in endodermis cells. In contrast, signals were not apparently detected in the epidermis, exodermis or cortex parenchyma. In our most microtome sections, epidermal cells of soil-grown roots were so damaged that they are hard to be seen, although they remained intact in some sections like those of hydroponically grown roots.

• Localization of the OsCDPK7 pathway in stress-tolerant over-expressing transformants:

It seems likely that the OsCDPK7 pathway is amplified preferentially in the properly operating regions in the over-expressing transformants.

Evolution

Figure 3. Phylogenetic tree for the amino acid sequences of CDPKs.（from reference ).

Figure 4.Phylogenetic relationships between CDPKs from rice and Arabidopsis.(from reference ).

'Figure 5.Phylogenetic relatedness among the rice, Arabidopsis and functionally characterized CDPKs from other plant species.(from reference ).

Figure 6.Phylogenetic relationships among CDPKs from rice (OsCPK1-OsCPK29) and Arabidopsis (AtCPK1-AtCPK34).(from reference ).

• OsCDPK7 and ZmCDPK7 showed 88.6% identity at the amino acid level over the entire polypeptide. In addition, OsCDPK7 showed high amino acid identity to a mung bean CDPK, VrCDPK1 (85.4%), the transcript level of which was elevated by salt stress and mechanical strain. They were classifed into the same subclass of CDPKs in a phylogenetic tree for the amino acid sequences of the entire ORFs (Figure 3). OsCDPK7 and ATCDPK1 differ in substrate preferences.

• It seems likely that OsCDPK7, ZmCDPK7, ZmCDPK1, VrCDPK1 and the Arabidopsis protein may be orthologues that play identical roles undernstress conditions.

• 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. Phylogenetic analysis showed that rice CDPKs are divided into four distinct classes(Fig. 4).

OsCPK13 (OsCDPK7) belongs to Group I.

• 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. 5).

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. 5).

• 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. 6). 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.

Knowledge Extension

• 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.

• 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.

Labs working on this gene

• Division of Applied Biosciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan
• Laboratory of Plant Physiology, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, 606-8502 Japan
• Department of Applied Plant Science, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, 981-8555 Japan
• Laboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, 630-0101 Japan
• Department of Plant Production Sciences, Graduate School of Agricultural Science, Tohoku University, Aoba-ku, Sendai, 981-8555 Japan
• Plant Science Center, RIKEN (The Institute of Physical and Chemical Research), Hirosawa 2-1, Wako, Saitama, 351-0198 Japan