Os03g0688300

This gene also know as OsCPK9 which is one of calcium-dependent protein kinase genes. It's a member of rice CDPK gene family.

Annotated Information

Function

OsCPK9 plays a positive role in drought stress tolerance and spikelet fertility. Physiological analyses revealed that OsCPK9 improves drought stress tolerance by enhancing stomatal closure and by improving the osmotic adjustment ability of the plant. It also improves pollen viability, thereby increasing spikelet fertility. And expression of OsCPK9 was elevated in seedlings infected by rice blast, indicating that this gene plays an important role in signaling in response to rice blast treatment.

OsCPK9 increases plants’ tolerance to drought, osmotic, and dehydration stresses. OsCPK9 functions in water retention by increasing proline and soluble sugars contents and improving stomatal closure under drought stress.

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

Mutation

OsCPK9 transgenic rice lines:

• OsCPK9-OX (OE)
• OsCPK9-RNAi (Ri)

To further study the function of OsCPK9 in planta, Wei et al. generated OsCPK9-OX (OE) and OsCPK9-RNAi (Ri) transgenic lines. The RT-PCR results showed that the transcript levels of OsCPK9 were markedly higher in OsCPK9-OX lines than in wild type (WT) with the highest transcriptional levels of OsCPK9 in OE28. In contrast, the transcript levels of OsCPK9 were reduced in OsCPK9-RNAi lines, with the lowest transcript levels of OsCPK9 in Ri2.

OsCPK9-OX lines are more sensitive to ABA than WT and VC.

Expression

OsCPK9 transcription was induced by abscisic acid (ABA), PEG6000, and NaCl treatments. Expression of OsCPK9 increased in response to rice blast treatment.

OsCPK9 expression was increased after 12–24 h of both avirulent and virulent rice blast treatment. These results suggest that OsCPK9 is involved in decoding the rice blast-induced Ca2+ signal pathway.

The OsCPK9 gene contains five exons and four introns,its protein is composed of 574 amino acid residues with a predicted relative molecular mass of 63.9 kDa.

OsCPK9 overexpression and interference analyses revealed that OsCPK9 positively regulates drought stress tolerance by enhancing stomatal closure and the osmotic adjustment ability of the plant. OsCPK9 also improves pollen viability, thereby increasing spikelet fertility. The OsCPK9-OX rice lines exhibited increased sensitivity to ABA. OsCPK9 expression also affects the transcription of ABA- and stress-associated genes.

Evolution

Figure 1.Phylogenetic relationships between CDPKs from rice and Arabidopsis.(from reference ).

'Figure 2.Phylogenetic relatedness among the rice, Arabidopsis and functionally characterized CDPKs from other plant species.(from reference ).

Figure 3.Phylogenetic relationships among CDPKs from rice (OsCPK1-OsCPK29) and Arabidopsis (AtCPK1-AtCPK34).(from reference ).

'Figure 4.Phylogenetic relationships among rice CCaMK, and rice and Arabidopsis CDPKs, CRKs and PEPRKs.(from reference ).

• OsCPK9 have four splicing sites, respectively , fewer than the number typical for CDPKs. This finding suggests that intron loss events have occurred within these rice CDPK genes during evolution.

• 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, OsCPK9 belongs to the Group III(Fig. 1).

• 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. 2).
• 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. 2).

• 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. 3). 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. 4, 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 5.Summary of the function of CDPKs in ABA and abiotic stress responses, as reported by several authors（from reference ).

• OsCPK9 gene had GC-rich sequences (66.8%) throughout their coding region.

• 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. 5).

• Overexpression of the AtbZIP60 gene in transgenic cell lines improved salt, drought, and cold stress tolerances by regulating expression of Ca(2+)-dependent protein kinase genes like OsCPK9 .