Functional Characterization Of OsCPK24 And OsCPK12 Genes In Rice

As one of the second messengers,Ca2+mediates the response to external stimuli in plants,and participates in the regulation of a variety of physiological and biochemical processes in cells.Calcium-dependent protein kinases(CPKs or CDPKs)are a specific class of Ca2+binding proteins in plants and pivotal components of intarcellular calcium signaling cascade.CPKs is a multi-gene family.Genome-wide analysis showed that there are 31 members in rice,but the function of most genes is still unknown.Since CPKs play important roles in plant growth and development and stresses response,isolating OsCPK genes and identifying their biological functions is beneficial to improving the agronomic traits of rice,and providing potential genetic resources for developing green super rice.Based on the expression profiles of OsCPKs family genes under stresses and hormone treatment,seven candidate genes,which were up-regulated by hormone or stresses treatment,were chosen for functional investigation via the overexpression,RNA interference and mutant materials.By screening the phenotypes of the over-expression plants under nomal or stresses conditions,two genes OsCPK24 and OsCPK12 were chosen for further functional characterization We analyze the two genes from many aspects such as gene expression,protein localization,biochemical characteristics,and substrate screening and identification.The main results are as follows:1.OsCPK24 is a functional protein kinase.CPKs encode typical Ser/Thr protein kinases.Kinase assay in vitro showed that OsCPK24 kinase activity was induced by Mg2+but inhibited by Ca2+,which was contrary to previous results.Under normal conditions,the concentration of Ca2+ in cytoplasm is low in rice,but concentrations of Mg2+is high,suggesting that OsCPK24 is present in rice in functional form,and Ca2+flow induced by environment plays an inhibitory role in its function.2.Expression pattern and subcellular localization of OsCPK24 in rice.Via RT-PCR analysis,OsCPK24 mRNA was detected in roots,leaves,leaf sheaths,stems and panicles.The 1949-bp promoter fragment of OsCPK24 was fused with a GUS reporter gene and then transferred to Nipponbare.GUS staining of transgenic plants showed that except for the anther,the root,stem,leaf blade,leaf sheath,glume and seed were stained blue.Although the promoter region of OsCPK24 contains a variety of cis elements predicted to respond to hormones and abiotic stresses,its expression is only induced by cold stress.Transient expression of OsCPK24-EGFP fusion proteins in rice protoplasts and tobacco leaf epidermal cells showed that OsCPK24-EGFP was mainly distributed in cytoplasm.3.Biological function of OsCPK24.We overexpressed or suppressed OsCPK24 in Nipponbare.After the detection of copy number and expression,some transgenic lines were selected for further analysis.Compared with wild type plants,the growth of overexpression plants were inhibited but its tolerance to cold were increased.The growth of OsCPK24-knockdown plants were not affected significantly,but the,tolerance to cold was decreased.In OsCPK24-overexpressing plants the total glutathione and proline content were increased.In OsCPK24-knockdown plants,the total glutathione and proline content were decreased,and ROS content was reduced under cold stress.In addition,OsCPK24 positively regulates the expression of glutathione reductases(OsGR10-1 and OsGR10-2),and proline synthase(OsPC5S-1 and OsPC5S-2).These results suggest that OsCPK24 participates in cold tolerance via controlling redox reaction and permeation.4.Interaction between OsCPK24 and OsGrx10.The truncated fragments of OsCPK24 were fused with the GAL4 DNA-Binding domain.By yeast two-hybrid screening,a glutaredoxin OsGrx10 was identified to interact with OsCPK24 kinase region.BiFC showed that they interacted with each other in vivo.Biochemistry study shows that OsGrx10 is a phosphorylated substrate of OsCPK24.OsGrx10 is a glutathione-dependent reductase,its activity was inhibited due to phosphorylation.The expression of OsGrx10 neither affected by cold nor by OsCPK24,and overexpression of OsGrx10 could not effectively improve cold resistance of rice.OsGrx10 may be a downstream signaling receptor or transporter of OsCPK24.5.The characteristics of OsCPK12.OsCPK12 is a functional protein kinase,its kinase activity is also induced by Mg2+and competitively inhibited by Ca2+.OsCPK12 is a tissue-specificly expressed gene.Promoter analysis and RNA in situ hybridization revealed that OsCPK12 was mainly expressed in the meristem and microtubule organization.Its expression level was induced by GA and BR treatment.Expressed CPK12-EGFP fusion protein in rice protoplast,BY2 cell line and tobacco epidermal cells showed that OsCPK12 protein was mainly localized on the plasma membrane.6.Biological function of OsCPK12.Plant height and the length of each node were increased in OsCPK12-overexpvessing plants,but reduced in mutant and knockdown plants.The expression of cell cycle related genes CYCB-2,CYC1A2M and MCMB-1 were increased in OSCPK12-overexpressing materials.7.HADS is a phosphorylated substrate of OsCPK12.Via yeast two-hybrid experiments,three candidate genes were identified,and one candidate gene HADS was analyzed in depth.HADS interacted with full-length protein of OsCPK12,but not interacted with the kinase region The Thr208 site plays a key role in the interaction between OsCPK12 and HADS.In vitro phosphorylation experiments demonstrated that HADS could be phosphorylated by OsCPK12.These results indicate that HADS is a potential phosphorylation substrate for OsCPK12.