Physiological Function Study In Plant Calcium Dependent Growth Of Arabidopsis CPK6

Calcium plays an important role in plant growth and development as an essential mineral element.It can maintain the structure and function of cell membrane and enhance the stability of cell wall structure.It also regulates biological enzyme activity and stabilizes the intracellular environment.The cytoplasmic calcium ion acts as a second messenger in plants.It responds to various stress stimuli and plays an important role in cell signal transduction.In plants,calcium-dependent protein kinases?CPKs?bind to Ca²⁺and participate in the Ca²⁺signal transduction.In Arabidopsis thaliana,there are 34 proteins encoded by AtCPK gene.In this subject we focuse on the physiological function of AtCPK6.In order to study the function of AtCPK6 in Arabidopsis,we performed homozygous identification of its two gene deficient mutants,atcpk6-2?Salk₀33392?and atcpk6-3?Salk₀25460?and obtained two homozygous mutants.We analyzed the growth phenotype of wild-type Arabidopsis Col-0 and atcpk6 mutants on the medium of different Ca²⁺concentrations.And we found that the mutants showed a growth-inhibiting phenotype relative to wild-type Col-0 in the deficiency of Ca²⁺.There was no significant growth difference between wild-type Col-0 and atcpk6 in the deficiency of Fe²⁺,Mg²⁺,Zn²⁺and Mn²⁺.We obtained the Arabidopsis transgenic material expressing At CPK6 promoter-driven?-glucuronidase.Using GUS histochemical staining we found that At CPK6 promoter had activity in the root and the young leaf primordium.Since the AtCPK6-encoded calcium-dependent protein kinase was a Ca²⁺binding protein,we further studied the effect of Ca²⁺deprivation on its promoter activity and found that the activity of AtCPK6 promoter in leaves was enhanced under Ca²⁺deficient condition.In order to further study whether AtCPK6 gene influences the absorption and accumulation of Ca in plants,the total Ca content of the plants was determined by atomic absorption spectrometry?AAS?.The results showed that there was no significant difference in Ca content between Col-0 and atcpk6 under control and Ca²⁺deficiency conditions,indicating that AtCPK6 gene does not participate in the absorption and accumulation of Ca in plants.A variety of external stimuli induce the production and accumulation of H₂O₂ in plants.Using 3,3'-diaminobenzidine?DAB?histochemical staining methods,it was found that the accumulation of H₂O₂ in the leaves of atcpk6 was significantly higher than that in Col-0 under Ca²⁺deficient condition.Using Dihydrorhodamine 123?DHR?fluorescent probes,we found that the accumulation of H₂O₂ in the root of atcpk6 was significantly higher than that in Col-0in the deficiency of Ca²⁺.It demonstrated that AtCPK6 was a negative regulator of the accumulation of H₂O₂ induced by Ca²⁺deficiency in plants.The study is helpful to reveal the mechanism of plants to adapt to Ca²⁺deficient environment.