The Molecular Mechanism Of CBL1 And CIPK1 Proteins Involved In Low Phosphate Stress In Arabidopsis Thaliana

Phosphorus is an essential element for plant growth and development.Insufficient supply of phosphorus in soil will cause physiological stress to lead to in huge yield loss.The availability of soil phosphorus resources is the main factor affecting the absorption and utilization of phosphorus by plants.However,soil phosphorus resources are generally scarce in China.Meanwhile,inorganic phosphorus which can be directly absorbed and utilized by plants accounts for a relatively small proportion.This part of inorganic phosphorus is easy to form insoluble complex with calcium,magnesium,iron and aluminum.In order to pursue the increase of crop yield,excessive application of phosphate fertilizer,people cause the huge deterioration of soil physical and chemical properties and water pollution.Therefore,Phosphate deficiency has become one of the main factors limiting crop yield.It is of great significance to study how plants coordinate the molecular mechanism of phosphate uptake,transport,migration and storage.As a ubiquitous second messenger,Ca²⁺plays an important role in the response of eukaryotic cells to intracellular and extracellular stimuli.CBL-CIPK network can regulate the responses of drought,high salt,low temperature,pathogenic microorganisms and the dynamic balance of various intracellular ions.However,CBL-CIPK response to phosphate deficiency signaling pathway is still rarely reported.In view of the above reasons,The involvement of Ca²⁺in the response to low phosphorus was further investigated,and explore new regulatory factors of phosphorus starvation response,so as to provide technical support for improving plants phosphate utilization efficiency and tolerant breeding crops to low phosphorus stress.In this study,we used T-DNA insertion mutants of Arabidopsis thaliana as materials to screen two regulatory factors in response to phosphate starvation by reverse genetics approach,and studied the biological function of CBL-CIPK in response to low phosphate stress.The results show that:1.The existing cbls mutants in the laboratory were used as the breakthrough point to screen the phenotype of the mutants under phosphate deficiency.Fortunately,we found that cbl1 mutant had significant tolerance to low phosphorus stress.Compared with wild type,cbl1 mutant had longer primary root,shorter root hair and less anthocyanin accumulation,Meanwhile,the cbl1 mutant was sensitive to arsenic stress.The q RT-PCR experiment showed that the expression of phosphate starvation response genes in cbl1 mutant was increased.The content of total phosphorus increased and the content of acid phosphatase and reactive oxygen species decreased in plants,which confirmed that CBL1 mutation could enhance Pi uptake in plants.2.In order to explore the CBL1 interacting with CIPK protein kinase,relieve the self inhibition of the NAF domain of CIPK,activate the protein kinase activity of CIPK protein,and further analyze the physiological process involved in the coordinated regulation of root architecture changes under phosphate deficiency.The homozygous cipks T-DNA insertion mutants preserved in the laboratory were used to screen cipks mutants with low phosphate phenotype.Unfortunately,no phenotype similar to cbl1 mutant was found.Fortunately,Under low phosphate stress,the primary roots of cipk1 mutant were significantly shortened,and the number and density of lateral roots were significantly increased compared with the wild type.Physiological changes of cipk1 mutant in response to low phosphorus stress were determined by acid phosphatase assay and H₂O₂assay.Subsequently,CIPK1complementation lines could compensate for low phosphate stress phenotype.Further arsenic stress experiments showed that there was no significant difference between cipk1 mutant and wild-type mutants,suggesting that CIPK1 may not act on the PHT1.These results suggested that CIPK1 responded to the signal transduction pathway of low phosphate stress.3.In order to elucidate the molecular mechanism of cipk1 mutant under low phosphate stress,Bimolecular fluorescence complementation and yeast two hybrid system experiments were used to verified the PHT5;1 interacting with CIPK1.The results show that CIPK1 interacted with PHT5;1 located in the vacuole membrane.In conclusion,we found two new components of phosphate pathway in Arabidopsis thaliana,and elucidated the mechanism of CBL-CIPK in response to low phosphate stress,which can improve the regulation mechanism of phosphate absorption,transportation and redistribution in plants,and provide a theoretical basis for the creation of germplasm resources with high phosphorus absorption efficiency.