Cell Wall Polysaccharide Changes Of Rice In Cell Wall Degradation And Genetic Analysis Of Cellulose Synthase Like Family F6 In Rice

One of the key barriers for low cost biological conversion of cellulosic biomass into renewable fuels and chemicals is recalcitrance,which refers to the resistance of cell wall to deconstruction by chemical,enzymes and/or microbial degradation.Antibodies specific for different cell wall polymers is very useful for studying plant cell wall recalcitrance,since they can be used to detect epitope-specific polysaccharide.In this study,we used different glycan-specific antibodies to monitor the structural changes during the chemical pretreatment and enzymatic hydrolysis.Meanwhile,we examined the amount of hexose,pentose and aldonic acid released during pretreatment and enzymatic hydrolysis,as well as hemicellulose monosaccharide by GC-MS,to illustrate the changes of cell wall components.The results are summarized below:1.The effect of alkali pretreatment on cellulose is not obvious,while acid pretreatment could remove some cellulose.Compared to directly enzymatic hydrolysis,the pretreatment followed with enzymatic hydrolysis has more significant effect on cellulose removal.The polysaccharides from the hemicellulose and pectin could be completely removed by alkali and acid pretreatment followed with enzymatic hydrolysis.2.The enzymatic hydrolysis only removes the xyloglucan epitope,revealing the masked xylan epitope on ground tissue cell wall.There is no obvious impact on pectin epitope.3.The ground tissue and parenchyma cells were firstly degraded during pretreatment and enzymatic hydrolysis,the phloem cell walls were degraded gradually,the protoxylem vessels except the phloem twere the last type of tissues that were removed.4.In the process of cell wall degradation,we found that pectin is the first wall component for remove,followed with xyloglucan and xylan.In other words,xylan show remarkable resistance than pectin and xyloglucan.5.Compared with the wild type Nipponbare(NPB),the rice mutant Osfc16 had higher enzymatic digestibility.The difference of the hemicellulose monosaccharide,hexose,pentose and aldonic acid releases between NPB and the rice mutant Osfc16 remain consistent,no matter of which pretreatment method used.Mix-linkage-glucan is an important component of hemicellulose in grass cell wall.Since MLG has an important application on dietary value,biomass conversion and the processes of malting and brewing,recently increasing interest was brought for study of the biosynthesis of MLG.Most of the genes responsible for MLG synthesis belongs to the glycosyltransferase(GT)family,in particular the cellulose synthase-like(CSL)superfamily.Among them,two grass-specific subfamilies,CslF and CslH families,were shown to mediate the biosynthesis of MLG.CslF and CslH are small gene families,with eight and three members in rice,respectively.This study focus on the genetic transformation of CslF6 in biosynthesis MLG in rice.The results are as following bellowed:1.The overexpression(35S promoter)and the complementation vector(with CslF6 own promoter)with GFP or m Cherry protein tag were transformed into Arabidopsis or NPB rice,providing materials for subsequent analysis.2.By membrane protein extraction from leaves of T0 genneration OsCslF6 overexpression rice,we can detect a weak band in the western-blotting when using the GFP-bingding antibody.It show that the OsCslF6 regulate the MLG synthase on the plasma membrane.3.The overexpression or complementation vector were also ectopically transformed into Arabidopsis thaliana.Positive transgenic lines were verified by qRT-PCR for OsCslF6 gene expression,T2 transgenic plant of OsCslF6 showed reduced plant height and prolonged generation time compared to wild type.