Study On The Mechanisms Of OsCaM1-1 And OsCML16 To Regulate Stress Tolerance In Rice

Ca²⁺,as a mineral element,is necessary for plant growth and development,and it is also one of the most important second messengers in plant cells.Studies have shown that the concentration of intracellular Ca²⁺ is increased instantaneously,following by the special changes occurred in spatiotemporal,amplitude,and frequency which produces calcium signature,when plants are subjected to abiotic stresses.The Ca²⁺ signal is recognized,decoded and transmitted by intracellular Ca²⁺ sensor proteins?CaMs/CMLs,CDPKs,CBLs?to induce the expressions of downstream responsive genes,thereby contributing to a series of physiological and biochemical changes in plant,finally enabling plants to adapt to external stress.To date,the CaMs/CMLs family has been identified to be involved in the regulation of plant growth and abiotic stress?cold,heat,drought and salt?resistances.Therefore,it is necessary to elucidate the regulation mechanism of abiotic stress signal transduction and provide a theoretical basis for enhancing stress tolerances in plants.Rice is one of three major food crops in China,and it is also a thermophilic grass crop that originates from the tropical-subtropical zone and is sensitive to moderate salt damage and cold stress.Salt damage and low temperature have been the main abiotic stress factors affecting rice growth and development,yield and quality.Herein,we attempt to carry out excavation and functional identification of genes related to salt tolerance and cold tolerance signaling pathways in rice,and to explore the molecular physiological and genetic mechanisms of salt stress tolerance and cold stress tolerance,which has important theoretical and application significance for improving salt stress tolerance and cold stress tolerance in physiological,cultivation and molecular breeding pathways.This paper is mainly divided into the following two parts:In the first part,we developed the studies of the function and regulatory mechanism of OsCaM1?rice calmodulin 1?in rice salt tolerance,proposing a salt stress signal transduction pathway OsCaM1-OsCCaMK-OsMKK1/6 in rice.Four conserved phosphorylation sites T80,S82,S102,and T118 were found by alignment of CaM amino acid sequences in eukaryotes.EMSA and protein interaction experiments demonstrated that S102 and T118 affect the binding of OsCaM1 to Ca²⁺ in vitro,but don't affect the interaction between OsCaM1 and the target protein OsCCaMK in vitro and in vivo;Yeast two-hybrid and BiFC assay confirmed that OsCCaMK interacts specifically with OsMKK1 and OsMKK6,respectively;Phosphorylation experiments in vitro clarified that OsMKK1 phosphorylates OsCCaMK,whereas OsMKK6 is phosphorylated by OsCCaMK.Therefore,there may be an OsCaM1-OsCCaMK-OsMKK1/6 pathway in rice.qRT-PCR analysis showed that there is a"cross-talk"between Ca²⁺ signaling and MAPK signaling pathway in response to ABA signaling,and Ca²⁺/CaM mediates the effects of ABA on the transcriptional regulation of ABA on OsCCaMK,OsMKK1,OsMKK6,and its downstream target genes OsMPK3,OsMPK4 and OsMPK6.OsCaM1-1 overexpression and antisense expression transgenic rice plants exposure to salt stress showed that the OsCaM1-1 overexpressing transgenic lines showed stronger salt tolerance than the antisense transgenic lines;Simultaneously,exogenous calcium promoted the growth of lateral roots in rice germination,enhanced salt stress tolerance and induced different expression levels of genes related to lateral root development.In the second part,genetic and biochemical methods were used to identify OsCML16?Calmodulin-like protein 16? interacting proteins and its expression level induced by cold stress.The results showed that OsCML16 was significantly induced by 4? and 12?;In vitro protein experiments showed that OsCML16 protein has the characteristics of binding to Ca²⁺ and Mg²⁺.However,Ca²⁺ and Mg²⁺ induce different degrees of conformational changes in OsCML16;GUS tissue staining showed that OsCML16 is mainly expressed in the young leaves and seedling roots,the hull at the booting stage,and the stem segments at maturity in rice.At the same time,we constructed OsCML16 overexpression and RNAi transgenic rice plants and obtained homozygous lines.The subcellular localization indicated that OsCML16 is mainly located in the cytoplasm and nucleus in rice protoplasts;OsCML16 was used as bait protein to screen the yeast two-hybrid cDNA library of rice seedlings treated with low temperature,and nine candidate proteins interacting with each other were obtained,including 2 kinase proteins?Y2,Y12?,2 transcription factors?Y26,Y336? and 5 functional proteins?Y1,Y14,Y64,Y304,Y419?,and then point-to-point yeast two-hybrid and BiFC assay identified the authenticity interaction between OsCML16 and these candidate proteins.qRT-PCR analysis showed that expression levels of 6 candidate interaction genes were induced by 4? and 12?.