KNAT7 Positively Regulates Xylan Biosynthesis In Arabidopsis

The plant secondary wall is primarily composed of cellulose,hemicellulose and lignin.The secondary wall hemicellulose in dicots and most monocotsis mainly xylan.Xylan is the second most abundant polysaccharide after cellulose in nature and accounts for almost one third of all renewable organic carbon on earth.A transcriptional regulatory network mainly composed of NAC and MYB transcription factors,and someother transcription factors regulate the secondary wall biosynthesis,including MYB46,MYB20,MYB52,MYB69,MYB85,MYB103,KNAT7,IFL1 and ERF72.However,the transcriptional regulation of xylan biosynthesis is still not very clear.In this study,we screened out some transcription factors in Arabidopsis thaliana which activatedxylan biosynthetic key genepromoters by transcriptional activation assay.Then a transcription factors with relativelystrong transcriptional activation activity was selected from them for further stud ies.The effect of the transcription factor geneexpression level on xylan biosynthesis,secondary wall biosynthesis and xylan biosynthetickey gene expression levels was detected.Moreover,whether the transcription factor is able to directly bind to the promoter of a xylan biosynthetickey gene was tested.The main results are as follows:(1)The transcriptional activation assay showed that MYB46,MYB20,MYB85,MYB103,KNAT7 and ERF72 are able to activate one or more xylan biosynthesis key gene promoters.KNAT7 can activate the promotersof IRX9,IRX10,IRX14-L and FRA8.(2)Non-cellulosic monosaccharidecompositionof wild type plants and knat7 mutants was analyzed by ion chromatography.The results indicated that the main compotents of xylan,xylose and glucuronic acid,inknat7 mutantssignificantly decreased.The xylan content of wild type plants and knat7 mutants was determined by enzyme linked immunosorbent assay.There was a significant reduction of xylan in the knat7 mutants.(3)The stained sections of wild type,knat7,KNAT7over-expession and 35S:KNAT7/knat7 plantinflorescence stems were observedand the thicknesses of vessel element,xylary fiber and interfascicular fiber cell walls were measured utilizing light microscopy.knat7 mutants exhibitedinward collapsed vessel elements,butthevessel elements of 35S:KNAT7/knat7 plantswere similar to those of wild type plants.knat7 mutants had thinner vessel element and xylary fiber cell walls but thicker interfascicular fiber cell walls,and KNAT7 over-expession plants showed the opposite phenotype.(4)The expression levels of IRX9,IRX9-L,IRX10,IRX10-L,IRX14,IRX14-L,FRA8 and F8 H in wild type,knat7,and KNAT7 over-expession plantsweremeasured by quantitative real-time PCR.The results showed that the expression of IRX9,IRX10 and FRA8 decreased in knat7 mutants and the the expression of IRX9,IRX10,IRX14-L and FRA8 increasedin KNAT7 over-expession plants.(5)The possible binding regionin IRX9 promoter of KNAT7 was determinedby transcriptional activation assay with deletions of the IRX9 promoter.Then KNAT7 was demonstrated to be able to directly bind to the IRX9 promoter by electrophoretic mobility shift assay with biotin labeled about 50 bp IRX9 promoter fragments.These results indicate that MYB20,MYB85,MYB103 and ERF72 probably positively regulate xylanbiosynthesis,and KNAT7 positively regulates xylanbiosynthesis by activating IRX9,IRX10 and FRA8 expression at least and directly inducesIRX9 expression.