| As the largest crop planted in China,maize plays an important role in ensuring food security.In recent years,the corn seed stock has been overstocked seriously.The anti-aging ability directly affects the storage life of the seed.Therefore,mining the key genes and analyzing its regulatory mechanism of anti-aging ability,and cultivating new varieties maize are effective ways to solve the above problem.In this paper,zmmyb59 loss-of-function mutants created in the laboratory were used to study the function of Zm MYB59 transcription factor regulating anti-aging ability of maize seeds.The molecular mechanism of Zm MYB59 regulating anti-aging ability at the transcriptional level was explored through omics and molecular biology experiments such as RNA-Seq,CUT & Tag,dual fluorescence reporter system,EMSA,and so on.The main results of the study are shown as follows:(1)Zm MYB59 transcription factor positively regulated the anti-aging function of maize seeds.In this paper,the zmmyb59 mutants,which were obtained by genome editing technology in our previous studies,were used to germination experiments after artificial aging treatment for 4 days.It was found that there was no difference in germination rate between zmmyb59 mutant and WT without aging treatment,but the root and shoot lengths of mutant are significantly longer than those of the wild type.However,aging treatment reduced the germination rate,root length and shoot length of zmmyb59 mutant were lower than those of wild type.Compared with wild type,germination rate,root length and shoot length of zmmyb59-1 was decrease by 20.0%,24.0%,32.0%,and the germination rate,root length and shoot length of zmmyb59-2 was decrease by 23.0%,27.2%,34.3%.The results,indicated that Zm MYB59 transcription factor may positively regulate anti-aging ability of maize seed.(2)The Zm MYB59 transcription factor directly binds to the AC element on the Zm ICDH1 promoter to regulate its expression.By joint analysis of RNA-Seq and CUT & Tag data from Zm MYB59 overexpressing protoplasts,and 3 candidate genes were further screened according to the literature review and annotation of gene functional region characteristics.In maize protoplasts,the promoter activity of the candidate gene was identified by dual-luciferase reporter system,and it was found that the promoter activity of Zm ICDH1 was upregulated by 29.3 times in Zm MYB59 overexpressing protoplasts.The cis-acting element prediction of the Zm ICDH1 promoter shows that there are 2 AC elements on the promoter.In order to clarify the function of the predicted element,we constructed dual fluorescent reporter vectors which drived by different lengths of Zm ICDH1 promoter,and then co-transformed with Zm MYB59 or GFP overexpression vectors into maize protoplasts,respectively.Then,we found that the second AC element located-175 bp upstream of the starting codon was functioned.After further mutation of this element,it was found that Zm MYB59 had a significant decrease in its regulatory fold,indicating that Zm MYB59 could bind to the second AC element located in the Zm ICDH1 promoter region to regulate the expression of this gene.EMSA experiments were performed using the purified Zm MYB59 protein and a biotin-labeled ACcontaining element probe,and we found that Zm MYB59 could bind to identified AC elements in the Zm ICDH1 promoter region.(3)Functional study of Zm ICDH1 in seed aging tolerance.By detecting the expression of Zm MYB59 and Zm ICDH1 in the maize developing seeds,it was found that Zm MYB59 was expressed earlier than Zm ICDH1.This result indicates that the accumulation of Zm MYB59 expression may be promoted the expression of Zm ICDH1,and Zm MYB59 may be mediated the anti-aging ability of seeds by regulating the expression of Zm ICDH1.Subcellular localization of Zm ICDH1 was found to function in the cytoplasm.By measuring the enzymatic activity of the Zm ICDH1 prokaryotic expression protein,it was found that the protein has isocitrate dehydrogenase activity.Since NADPH catalyzed by isocitrate dehydrogenase in the cytoplasm is an important coenzyme for reduced glutathione,which plays an important role in the removal of reactive oxygen species produced due to aging stress,we speculate that Zm ICDH1 may play an important role in seed anti-aging by upregulating the activity of isocitrate dehydrogenase.In summary,we found that the transcription factor Zm MYB59 regulated seed aging tolerance by using the maize zmmyb59 mutant,and further found that Zm MYB59 could directly bind to AC element,which was located at the promoter region of Zm ICDH1,to regulate its expression by omics and molecular techniques.The results of this study lay a foundation for revealing the regulatory mechanism of seed anti-aging ability,and provide key genes for the cultivation of new maize varieties with strong aging tolerance. |
