Functional Analysis Of Transcription Factor ZmNAC84 In Response To Salt Stress In Maize

Salinity is a major worldwide environmental problem,which severely limits the growth,development and yield of crops,such as maize（Zea mays L.）.Plants have evolved various strategies to adapt the salinity.In general,plants sense the stimulus and transmit signals that regulate the downstream genes responsible for the adaptive response to salt stress.Transcription factors（TFs）play important roles in affecting various physiological and biochemical processes via regulating the expression of stress-related genes.NAC（NAM,ATAF1/2 and CUC2）transcription factors are plant specific transcription factors,which are widely found in different plant species.Although the roles of few NAC proteins in response to salt stress have been identified,the mechanism of the other important members in this process are still unclear.In our previous study,we found that NaCl could significantly induce ZmNAC84 expression,and Ser-113,a phosphorylation site,of ZmNAC84 is an important site enhancing the activity of antioxidant enzyme.However,the role of ZmNAC84 in tolerance of maize to salt stress is not clear.In this study,we analyzed the mechanism of ZmNAC84 participating in salt stress response and the role of Ser-113 of ZmNAC84 in this process.The results are as follows:1.To investigate whether ZmNAC84 functions in the tolerance of maize to salt stress,the ZmNAC84 overexpression and ZmNAC84 interference plants were generated and used to perform the salt sensitivity assays.The results showed that overexpression of ZmNAC84increased the tolerance of maize seedlings to salt stress,while knock-down of ZmNAC84 in maize by RNA interferencing decreased the salt tolerance.In the presence of NaCl,overexpression of ZmNAC84 significantly reduced the malondialdehyde（MDA）content and the electrolyte leakage rate,and increased the chlorophyll content.Knock-down of ZmNAC84 in maize by RNA interferencing significantly increased the MDA content and electrolyte leakage rate,and obviously decreased the chlorophyll content.To explore the effect of ZmNAC84 in H₂O₂accumulation exposed to salt stress,3,3’-diaminobenzidine（DAB）in situ staining and xylenol orange（XO）method were used to determine the H₂O₂content in WT plants and ZmNAC84 transgenic plants.The results showed that overexpression of ZmNAC84 obviously decreased the salt-induced H₂O₂accumulation,while knock-down of ZmNAC84 in maize by RNA interferencing significantly increased the salt-induced H₂O₂accumulation.Further,the activity of CAT,which is the main antioxidant enzyme involved in H₂O₂scavenging was measured.In the presence of NaCl,overexpression of ZmNAC84 significantly increased the CAT activity,while knock-down of ZmNAC84 in maize by RNA interferencing significantly decreased the CAT activity.However,there were no significant differences in H₂O₂content and CAT activity between transgenic plants and WT plants under normal growth conditions.These results indicate that ZmNAC84 regulates CAT activity to eliminate the excess H₂O_2,thus positively regulates the tolerance to salt stress.2.To understand the mechanism of ZmNAC84 on regulating CAT activity in response to salt stress,the ZmCATs（ZmCAT1,ZmCAT2,ZmCAT3）expressions in ZmNAC84 transgenic plants and wild type exposed to salt stress were detected.The results showed that overexpression of ZmNAC84 only significantly increased the ZmCAT1 expression,while knock-down of ZmNAC84 in maize by RNA interferencing only significantly decreased the ZmCAT1 expression,which indicate that ZmNAC84 specifically regulates the ZmCAT1expression,but could not affect the expressions of ZmCAT2 and ZmCAT3 under salt stress.To further investigate whether ZmNAC84 directly regulates the ZmCAT1 expression,the dual luciferase reporter assay was used to analyze the activity of ZmCAT1 promoter.The results showed that overexpression of ZmNAC84 in tobacco obviously enhanced the promoter activity of ZmCAT1,implying that ZmCAT1 is the potential target gene of ZmNAC84.Also,the electrophoretic mobility shift assay（EMSA）and chromatin immunoprecipitation with quantitative real-time PCR（ChIP-qPCR）assay were used to confirm the interaction between ZmNAC84 and ZmCAT1 promoter.The results showed that ZmNAC84 could directly bind to the ZmCAT1 promoter in vitro and in vivo.Next,to further confirm the role of ZmCAT1 in response to salt stress,ZmCAT1 overexpression plants and ZmCAT1 interference plants were generated and perform the salt sensitivity assays.The results showed that overexpression of ZmCAT1 significantly increased the CAT activity,decreased the H₂O₂accumulation and improved the salt tolerance in the presence of NaCl.Knock-down of ZmNAC84 in maize by RNA interferencing significantly decreased the CAT activity,increased the H₂O₂accumulation and reduced the salt tolerance in the presence of NaCl.These results indicate that ZmNAC84 affects the salt tolerance by regulating ZmCAT1-mediated H₂O₂homeostasis.3.To explore whether the phosphorylation of Ser-113 functions in response to salt tolerance,ZmNAC84^S113A（substituted Ser-113 with Ala to mimic non-phosphorylation state）overexpression plants and ZmNAC84^S113D（substituted Ser-113 with Asp to mimic phosphorylation state）overexpression plants were generated and perform the salt sensitivity assays.Compared with ZmNAC84 overexpression plants,ZmNAC84^S113Aoverexpression plants and WT plants,ZmNAC84^S113Doverexpression plants had higher survival rate,lower degree of oxidative damage,higher CAT activity and lower H₂O₂content exposed to salt stress.Structural analysis showed that Ser-113 was located in the DNA binding domain of ZmNAC84.To explore the effect of phosphorylation of Ser-113 on DNA binding ability of ZmNAC84,the dual luciferase reporter assay,EMSA,and ChIP-qPCR assay were performed.The results showed that the phosphorylation of Ser-113 obviously affected the DNA binding ability of ZmNAC84 to ZmCAT1 promoter.These results suggest that the phosphorylation of Ser-113 of ZmNAC84 up-regulates the ZmCAT1 expression via enhancing the DNA binding ability,then modulates the CAT activity to maintain H₂O₂balance,thus enhances the tolerance to salt stress.4.To elucidate the regulatory network of ZmNAC84 response to salt stress,the transcriptome analysis of ZmNAC84 interference plants and WT plants was performed.The results showed that a total of 2945 genes were differentially expressed in ZmNAC84interference plants and WT plants in the presence of NaCl.Functional analysis of these genes revealed that they were involved in metabolic processes,cellular processes,response to stimulus,membrance part,catalytic activity,binding and antioxidant activity,respectively.After analysis,four HAK family genes-ZmHAK1,ZmHAK2,ZmHAK17 and ZmHAK22 with complete sequences were selected from the differentially expressed genes as possible target genes of ZmNAC84,and RT-qPCR proved that ZmNAC84 positively regulated the expression of these genes.The results of determination of Na⁺and K⁺in ZmNAC84 transgenic plants showed that ZmNAC84 could avoid the accumulation of Na⁺in the shoot.The conclusion is that ZmNAC84 directly up-regulates the ZmCAT1 expression,then modulates the CAT activity to maintain H₂O₂balance,thus enhances the tolerance to salt stress.In this process,the phosphorylation of Ser-113 of ZmNAC84 is very important for the DNA binding ability,ZmCAT1 expression,CAT activity,H₂O₂balance,and the tolerance of maize to salt stress.