Molecular Mechanism Of SiPLATZ12 In Regulating Foxtail Millet Growth And Salt Tolerance

Yield and plant resistance to stresses are critical for crop production.Foxtail millet（Setaria italica）plays important roles in crop industry diversification and sustainable agriculture development.There are few studies on critical genes that regulate foxtail millet development and abiotic stress resistance.It has been reported that the plant-specific zinc-dependent DNA-binding proteins（PLATZs）play crucial roles in regulating plant development and stress resistance.The biological function and molecular mechanism of PLATZ have not been studied in foxtail millet.In this study,we explained the molecular mechanism of SiPLATZ12 in regulating development and salt tolerance in foxtail millet.The main results and conclusions are as following:（1）Tissue and induction expression patterns analysis of SiPLATZs in foxtail millet.To study the biological function of SiPLATZs,the homology alignment of PLATZ proteins was carried out,and a total of 16 members of SiPLATZs in foxtail millet was identified.The tissue expression patterns showed that all SiPLATZs were highly expressed in panicles and 9SiPLATZs were highly expressed in roots.These results indicate that SiPLATZs might regulate root growth,nutrient absorption,and panicle and seed development.All SiPLATZs were significantly induced by PEG while most SiPLATZs were induced by 4℃.Moreover,about 8,4,1,and 7 SiPLATZs were induced by Na Cl,Na HCO₃,ABA,and 42℃,respectively.These results suggest that SiPLATZs may respond to various abiotic stresses in foxtail millet.（2）SiPLATZ12 regulates growth and development of foxtail millet and Arabidopsis.SiPLATZ12 overexpression lines were generated in Ci846 foxtail millet and WT Arabidopsis.In foxtail millet,the three SiPLATZ12 overexpression lines showed decreased plant height,increased stem width,prolonged heading date,larger panicles with increased length,and width and significantly longer and wider seeds,indicating that SiPLATZ12 positively regulates multiple elite yield traits and positively regulates seed development in foxtail millet.In Arabidopsis,the three SiPLATZ12 overexpression lines showed bigger seeds compared with WT,indicating that SiPLATZ12 positively regulates seed development in Arabidopsis.（3）SiPLATZ12 reduces salt tolerance in foxtail millet and Arabidopsis.The salt tolerance of transgenic foxtail millet was much lower than Ci846,as well as the relative root length and relative fresh weight were significantly lower than Ci846 under salt stress.SiPLATZ12overexpression Arabidopsis showed lower fresh weight and survival rate under salt stress.These data indicate that SiPLATZ12 negatively regulate salt tolerance in foxtail millet and Arabidopsis.（4）SiPLATZ12 targets SiNHX2,SiCBL4,and SiCBL7.The expression levels of NHXs,CBLs and SOSs in SiPLATZ12 overexpression lines were lower than control in both foxtail millet and Arabidopsis.The increased Na⁺content,decreased K⁺content,increased Na⁺/K⁺ratio,and decreased vacuolar p H were showed in transgenic foxtail millet.Furthermore,it was verified that SiPLATZ12 directly targets SiNHX2,SiCBL4,and SiCBL7 by yeast one-hybrid essays,micro-scale thermophoresis essays,chromatin immunoprecipitation-q PCR,and electrophoretic mobility shift assays.Overexpression of SiNHX2 and SiCBL4 can rescue the salt-sensitive phenotype of SiPLATZ12 overexpression line#28,indicating that SiPLATZ12functions upstream of SiNHX2 and SiCBL4.（5）Hap A and Hap A9 haplotype are closely related to multiple agronomic traits and salt tolerance.Haplotype analysis of SiPLATZ12 revealed that there were 18 natural variants between gene body,3 kb upstream and 2 kb downstream of SiPLATZ12,resulting in five dominant haplotypes including Hap1,Hap2,Hap3,Ha14,and Hap18.The haplotype distribution frequency showed that Hap1 was the predominant haplotype,with 1099 foxtail millet accessions（cultivated varieties）.Hap2 was represented by 77 accessions,with only 1wild and 76 cultivated accessions.Hap3 was assigned to 44 accessions including 11 cultivated and 33 wild accessions.Hap14 was represented by 64 accessions with 1 wild and 63 cultivated accessions.Hap18 was the second major haplotype with 264 green foxtail accessions（wild varieties）.Further analysis showed that 9 bp insertion at V6 site in SiPLATZ12 promoter produced Hap A and Hap A9（9 bp insertion）haplotypes.Hap A9 includes all modern cultivated accessions and a few of wild accessions while all Hap A accessions belong to wild accessions,indicating that Hap A9 might be naturally mutated from green foxtail millet and artificially selected.The expression level of SiPLATZ12,TGW,stem diameter width,and panicle length in Hap A9 varieties were significantly higher than Hap A varieties,while the survival rate under salt stress was significantly lower in Hap A9 varieties.These results indicate that the 9 bp insertion in SiPLATZ12 promoter improves the expression level of SiPLATZ12 and further influences multiple agronomic traits associated with development and salt tolerance in foxtail millet.（6）The expression levels of SiNHX1,SiNHX2,SiNHX3,and SiNHX7 were correlated with seed size and salt tolerance in foxtail millet.SiNHX1,SiNHX2,SiNHX3,and SiNHX7 were all induced by Na Cl,although the expression levels were lower in Hap A9 varieties than Hap A varieties.The expression levels of the four SiNHXs were negatively correlated with TGW and positively correlated with SUR in foxtail millet,indicating that SiNHX1,SiNHX2,SiNHX3,and SiNHX7 negatively regulate growth and development,and positively regulate salt tolerance in foxtail millet.（7）SiPLATZ12 interacts with SiDi19-3.To further reveal the regulatory mechanism of SiPLATZ12,the interaction protein SiDi19-3（Drought induced protein）of SiPLATZ12 was screened by yeast two-hybrid assays.The interaction between SiDi19-3 and SiPLATZ12 was confirmed by pull-down and bimolecular fluorescence complementation assays.To verify the biological function of SiDi19-3,we generated the SiDi19-3 overexpression lines in foxtail millet and Arabidopsis,respectively.The growth of SiDi19-3 overexpression foxtail millet was better than control under salt stress with longer root length and heavier fresh weight.SiDi19-3overexpression Arabidopsis also showed higher salt tolerance with faster germination rate,longer root length,and heavier fresh weight than WT under salt stress.Moreover,SiDi19-3promotes the expression of most NHXs,CBLs and SOSs genes in both foxtail millet and Arabidopsis.These data indicate that SiDi19-3 positively regulates the salt tolerance in foxtail millet and Arabidopsis.SiDi19-3 and SiPLATZ12 have opposite effects on salt response in foxtail millet and Arabidopsis,indicating that they might antagonize each other in this process.However,the specific molecular mechanism needs further investigation.