Functional And Preliminary Mechanistic Study Of Ascorbate Peroxidase 1 On Resistance To Southern Corn Leaf Blight On Maize

Maize is one of the major food and feed crops in China,as well as an important industrial raw material and energy crop,occupying an important position in the national economy and agricultural production.Southern corn leaf blight is a foliar disease caused by dead nutritional fungal pathogen infestation,which is widespread and devastating worldwide.With the global warming,the restructuring of agricultural farming and the change of farming practices,the occurrence of maize diseases has shown an increasing trend year by year,which has become a major factor limiting the growth of maize production and poses a serious threat to global food security.Although southern corn leaf blight resistance genes have been cloned,however,SCLB resistance is a quantitative trait controlled by multiple genes,and the mechanism of SCLB resistance is more complex,so the identification of new SCLB resistance-related genes and the elucidation of their potential molecular mechanisms are of great theoretical and practical significance for maize mottle resistance breeding.Ascorbate peroxidase（APX）is an important antioxidant enzyme in reactive oxygen metabolism that uses ascorbate as an electron donor to catalyze the conversion of H₂O₂ to H₂ O.It has been shown that: APX expression shows differential expression under different stress conditions,and has played an important biological function in the response of dicotyledonous plants to stress.However,the function of APXs in plant response to dead trophic pathogens has not been reported.In this study,we first identified one of the differentially expressed proteins,ZmAPX1（B6U9S6）,within the previously reported QTL interval for SCLB resistance through comparative proteomic analysis before and after corn infestation with Bi-polaris.Further,ZmAPX1 overexpression transgenic maize plants were constructed,and the ZmAPX1 overexpression material was subjected to SCLB resistance identification and pathological observation,which demonstrated the positive regulatory function of APX on SCLB resistance.Finally,by studying the changes of H₂O₂ content,expression of immune defense-related genes and JA content of plant defense hormone in ZmAPX1 overexpression transgenic material,the mechanism of action of ZmAPX1 in regulating the resistance of maize to SCLB was preliminarily resolved.This study provides a theoretical basis for elucidating the function of APX in response to deadly nutritional pathogens,and also lays the foundation for further use of this gene for disease resistance breeding.The main studies are as follows:1.Comparative proteomic analysis of corn samples before and after infestation with Bi-polaris maydis using TMT marker technology identified 258 differentially expressed proteins,and KEGG and GO enrichment analysis indicated that these proteins were widely involved in various biological processes and cellular processes.One of the differentially expressed proteins associated with stimulus response,ZmAPX1（B6U9S6）,was also identified to be located within a previously reported QTL interval for SCLB resistance,further analysis through sequencing and expression profiling suggests that ZmAPX1 may be associated with resistance to SCLB.2.The overexpression transgenic material of ZmAPX1 was constructed,and the transgenic lines with stable inheritance in T₄ generations were obtained by consecutive self-crossing and screening.The expression of ZmAPX1 between transgenic positive and negative lines was examined by qRT-PCR and Western blot techniques at the transcriptional and translational levels,respectively,and the results indicate that ZmAPX1 can be stably expressed in transgenic positive lines and the expression level is significantly higher than that in negative lines.3.The transgenic positive strain and its negative control strain were inoculated with Bi-polaris maydis for Southern corn leaf blight resistance identification,and the results showed that overexpression of ZmAPX1 could improve the resistance of maize to SCLB.Pathological section observations of transgenic positive and negative control lines revealed that the transgenic positive lines had significantly lower amounts of invading hyphae in mesophyll cells compared to the negative control lines,and the leaf cells of the transgenic positive strain were more intact than those of the transgenic negative control strain,this research result further demonstrating the important role of ZmAPX1 in SCLB resistance.4.qRT-PCR analysis of JA biosynthesis and response-related genes（ZmAOS,ZmLOX2,ZmOPR8,ZmPDF1.2 and ZmVSP2）showed that the expression of JA biosynthesis and response-related genes in the transgenic positive strain was significantly higher than that in the negative control strain after inoculation with Bipolaris maydis.Further,we measured the content of JA and H₂O₂ in ZmAPX1 overexpression positive lines and showed that overexpression of ZmAPX1 reduced the accumulation of H₂O₂ and increased JA content.Exogenous JA treatment enhanced resistance to SCLB,and the resistance to SCLB has a positive correlation with JA concentration.In addition,we also found that resistance to SCLB had a negative dependent effect on H₂O₂.The above results indicate that ZmAPX1 may regulate resistance to SCLB through the JA and H₂O₂ pathways in the defense mechanism.In summary,this study identified 258 proteins responsive to SCLB using comparative proteomics techniques and identified a differential protein ZmAPX1（B6U9S6）located within a previously reported QTL interval associated with SCLB resistance.The resistance identification and pathological observations of ZmAPX1 overexpression transgenic materials have demonstrated the involvement of ZmAPX1 in the resistance of maize to SCLB.Further,physiological,biochemical and molecular biological studies clarified that ZmAPX1 positively regulates maize immune defense responses by reducing H₂O₂ accumulation and activating JA-mediated immune defense signaling pathways.The above results demonstrate the important role of ZmAPX1 in SCLB resistance and preliminarily elucidate the mechanism of ZmAPX1 regulation of SCLB resistance.This study provides a basis for further investigation of the role of APX family proteins in the resistance of dead plant nutritional fungal pathogens and provides potential genetic resources for maize disease resistance breeding.