Functional Analysis Of Two Maize NAC Transcription Factors,ZmNST3 And ZmNST4,in Secondary Wall Biosynthesis

Maize(Zea mays L.)is a staple food crop worldwide with tremendous economic benefit and biological values,and its straw is a major source of biomass in our country.Secondary walls are the most abundant biomass produced by plants,which consist mainly of lignin,cellulose and hemicellulose.As a component of plant cell wall,secondary wall is important for plant development.For example,secondary wall in anther is required for its automatic opening and that in stem is required for straight growth of plant.Currently,little works have focused on secondary wall biosynthesis in maize.NAC transcription factor(TF)is a plant-specific family which widely involved in plant growth and development processes.In this study,two N AC TFs,ZmNST3(Zea mays NAC secondary wall thickening promoting factor 3)and ZmNST4,were selected to function analysis after systematically analyzing of Secondary Wall NACs in maize(ZmSWNs).In situ hybridization showed that ZmNST3 and ZmNST4 were expressed specifically in secondary wall-forming cells.ZmNST3 and ZmNST4 were localized in the nucleus,showed transcriptional activation activity in yeast and could specifically bind to SNBE in vitro.All these findings indicated that ZmNST3 and ZmNST4 were typical NAC TFs.ZmNST3 and ZmNST4 were homologous to Arabidopsis NST3.The Arabidopsis nst1 nst3 mutants exhibited pendent inflorescence stems because of the lack of lignified secondary walls in interfascicular fibers.When expressed in nstlnst3 mutant background,ZmNST3 and ZmNST4 could restore the phenotype of the mutant.And when overexpressed in Arabidopsis WT background,ZmNST3 and ZmNST4 could promote secondary wall formation in interfascicular fibers.To research the functions of ZmNST3 and ZmNST4 in maize,the transgenic maize with down-regulated expression of ZmNST3(ZmNST3kd)and ZmNST4(A4)and up-regulated expression of ZmNST3(ZmNST3ox)were obtained.Because the transgenic maize plants overexpressing ZmNST4 were lethal,no ZmNST4ox plant was obtained.The phloroglucinol-HCI stained and SEM images of sections of transgenic internodes showed that the thickness of secondary walls in bundle sheath,tracheary element and vessel in ZmNST3ox maize were significantly increased,and in A4 or ZmNST3kd maize they were significantly decreased.The expression level of ZmCESA4/9,which are key genes for cellulose biosynthesis of secondary wall,and ZmHCT,which is a key gene for lignin biosynthesis,were up-regulated in the internode of ZmNST3ox maize,and were down-regulated in that of A4 and ZmNST3kd maize.Besides,the content of cellulose and lignin in cell wall of ZmNST3ox maize internode were both increased and that in A4 and ZmNST3kd maize internode were both decreased.Further,the function mechanism and gene expression regulation of ZmNST3 and ZmSNT4 were analyzed.MYB46 is a direct target of NST3 in Arabidopsis.Using homologous alignment,homologues of MYB46,ZmMYB109,ZmMYB 128 and ZmMYB149,were identified in maize.The results of gene expression quantification and transactivation assay in tobacco system showed that ZmNST3 and ZmNST4 could regulate the expression of these MYBs.Y1H was performed to screen the maize seedling cDNA library to identify the upstream regulatory factors of ZmNST3 and ZmNST4 and as results,ZmWRKY82 and ZmARF2 were identified.ChIP assay and transactivation assay in tobacco system revealed that ZmWRKY82 can bind to the promoter region of ZmNST3 and ZmSNT4 and activate their promoter activities.ZmARF2 can bind to the promoters but repress their activities.In conclusion,ZmNST3 and ZmNST4 are master switches for secondary wall deposition in maize.Through regulating the expression level of ZmMYB109,ZmMYB128 and ZmMYB149,ZmNST3 and ZmNST4 can regulate the biosynthesis of cellulose and lignin,and thus regulate secondary wall biosynthesis.Besides,our work also demonstrated that ZmWRKY82 and ZmARF2 were regulator of ZmNST3 and ZmNST4 in maize seedlings.