Functional Analysis Of SiCBL5 In Response To Salt Stress In Foxtail Millet

Salinity is one of the main abiotic stresses of foxtail millet,which can adversely affects the growth of foxtail millet and severely limits the yield.Previous studies have shown that the calcineurin B-like proteins（CBL）played vital roles in regulating salt response.The earliest discovered in plant is the CBL4/SOS3-CIPK24/SOS2-SOS1 pathway,also known as the SOS pathway,which is the most critical pathway in the CBL-CIPK signaling network.At CBL4/SOS3 interacts with At CIPK24/SOS2 to form a complex,thereby activating the Na⁺/H⁺transport function of At SOS1 and pumping excess Na⁺out of plant cells.Foxtail millet genome encodes seven CBLs,and only SiCBL4 has been shown to be involved in salt response.In this paper,we found that SiCBL5 was invoved in response to salt tolerance using the complementation test in Arabidopsis thaliana sos3-1 mutant.Further,we investigated the role of SiCBL5 in enhancing the tolerance of foxtail millet to salt stress.The main results are as follows:Overexpression of SiCBL4 or SiCBL5 in Arabidopsis thaliana sos3-1 mutant could rescue its salt hypersensitivity phenotype,but the other SiCBLs（SiCBL1,SiCBL2,SiCBL3,SiCBL6 and SiCBL7）could not rescue salt hypersensitivity of Atsos3-1 mutant.Secondly,subcellular localization experiments indicated that SiCBL5 with a N-myristoylation motif is located in the plasma membrane,which is consistent with the localization of At CBL4.Next,with"Yugu 1"as experimental material,different treatments are performed on transformed receptors by Agrobacterium-mediated transformation.We used an PCR-based assay to screen transformants in third-leaf stage seedlings.We determined an optimal lethal concentration of glufosinate（Basta）when sprayed to millet seedlings,and tested the different PCR-based genotyping methods with or without Basta spary.On the basis of the above,the infection conditions were further optimized.Finally,the optimal genetic transformation system was determined:After dark treatment,millet about 1 cm in length was placed directly into the infection solution with OD₆₀₀ concentration of 1.4,then 800μM acetylsyringone was added and treated at 0.05MPa for 40 minutes.In addition,a more convenient detection method of"Combination of spraying Basta and PCR"was established.In order to study whether SiCBL5 enhances the salt tolerance of foxtail millet,the transformation system was used to construct OE-SiCBL5（overexpression,OE）and RNAi-SiCBL5（RNA interference,RNAi）transgenic foxtail millet materials.Then,the fresh weight,chlorophyll content and Na⁺contents in shoots of wild type plants,SiCBL5 overexpressors and RNAi-SiCBL5 transgenic lines were investigated.In the presence of Na Cl,SiCBL5overexpressors exhibited less severe wilting and chlorosis than wild type plants,while RNAi-SiCBL5 lines exhibited more severe wilting and chlorosis,the shoot fresh weight and chlorophyll content of SiCBL5 overexpressors were greater than those of the wild type,while the shoot fresh weight and chlorophyll content of RNAi-SiCBL5 lines were lesser than those of the wild type.Also,the Na⁺contents were significantly higher in SiCBL5-RNAi lines but markedly lower in SiCBL5 overexpressors.However,no significant difference in the growth and Na⁺content between the wild type millet and all transgenic plants in the absence of Na Cl.Shoots of wild type and transgenic lines had approximately equal K⁺contents both in the absence and presence of Na Cl.Na⁺/K⁺ratio in shoots were also calculated.Under Na Cl treatments,Na⁺/K⁺ratio were obviously higher in SiCBL5-RNAi lines compared to those in wild type,while the Na⁺/K⁺ratio of SiCBL5 overexpressors were the lowest among them.Together,these results clearly showed that SiCBL5 positively regulates the Na⁺homeostasis in shoots of foxtail millet.To explore the mechanism of SiCBL5 in response to salt stress,we used yeast two-hybrid assay and firefly luciferase complementation imaging assay to confirm the interaction between SiCBL5 and SiCIPK24.Then,the yeast mutant strain AXT3K,which lacks plasma membrane Na⁺transporters,was used to perform yeast functional complementation experiments.The expression of SiSOS1 or the co-expression of SiSOS1 and SiCIPK24 could not alleviate the salt sensitivity of the AXT3K mutant strain.The yeast cells co-expressing SiSOS1,SiCIPK24 and SiCBL5 was more tolerant to Na Cl compared with the others.The results showed that SiCBL5-SiCIPK24 regulates the function of SiSOS1 in yeast cells.These results clearly showed that SiCBL5 positively regulates the salt tolerance of foxtail millet by adjusting the Na⁺steady state.This study provides the foundation for further research on the mechanism of SiCBLs under salt stressconditions.