Cell Wall Polysaccharide Changes Of Miscanthus Druing Cell Wall Degradation And Function Explorations Of NcPMO-2 In Plants

Plant cell walls are highly complex composites.The majority of lignocellulose stored in plant cell walls can be converted to various biofuels such as bioethanol,via physi-chemical pretreatment followed by enzymatic hydrolysis,and cellulose hydrolyzation is the key point during the degragation of biomass.Cell wall recalcitrance is the major barrier for biomass degradation and therefore bioenergy industrialization.Research on cell wall construction and components of biomass contributes to improve the procedure of biomass utilization and also breeding of low-degradability crop species.There are two parts in this research:First part focuses on the changes of cellulosic,hemicellulosic and pectic polysaccharides during direct enzymatic saccharification,or during alkali/acid pretreatment followed with enzymatic saccharification.We used polysaccharide-specific antibodies,combined with pentose and hexose determination by spectrometry and GC-MS,to reveal the dynamic change and interactions of cell wall components.The result would be useful for deeper understanding of Miscanthus cell wall and breeding purpose.The main results are as following:1.The ground tissue and parenchyma cells were degraded in the early stage during pretreatment and enzymatic saccharification,followed by phloem of vascular bundles.Protoxylem vessels were the hardest tissue to be removed.2.Alkali pretreatment had almost no effect on cellulose,while acid pretreatment could remove little amout of cellulose.3.The release of hexose,pentose and aldonic acid by acid pretreatment-enzymatic saccharification was less than those in alkali pretreatment-enzymatic saccharification.The releases of hexose,pentose and aldonic acid by pretreatment-enzymatic saccharification were much higher than with direct enzymatic saccharification.4.The main components of primary cell wall Xyloglucan(XyG)counldn't be removed by alkali or acid pretreatment,but could be degraded immediately with enzymatic saccharification.Pretreatments had evident impact on homogalacturonan(HG),but little effect on hemicellulosic polysaccharide.5.The releases of the hextose,pentose and aldonic acid from Miscanthus sinensis material were always higher than those from Miscanthus sacchariflorus.6.Xylan,as the major components of hemicellulose,was the hardest polysaccharide to remove during degradation,although there was an even increase of the Xylan release as determined by spectrometry,and an increase of xylose by GC-MS during both pretreatments and enzymatic saccharification.Second part about reseach on fungi polysaccharide monooxygenases is blank in higher plants Cellulose hydrolyzation is the key point during the degragation of biomass.The classical enzymes are hydrolases,they cleave glycosidic bonds by addition of a water molecule and dehydrogenation,generating oligosaccharides which are converted to monosugars such as glucose in the end.A recently discovered new type of enzymes called“PMO” enzymes,could catalyze oxidative cleavage of crystalline cellulose at both the reducing end and non-reducing end.PMOs have a flat substrate binding surface with a tightly bound Cu ion within the catalytic center,that catalyzes the region-specific hydroxylation of crystalline cellulose,the reaction also requires electron donor and oxygen molecule.PMO enzymes are widely distributed in bacteria and fungi,with diverse substrates such as hemicellulose and starch,PMOs play a crucial part in the degradation of cell wall.Most studies about PMOs are from microorganisms,none in plants.In this study,we used a PMO gene from Neurosopora crassa,Nc PMO-2,created transgenic plants with overexpression of Nc PMO-2,to illustrate its possible function in plants.The main results are as following:1.The cDNA sequece of NcPMO-2 was synthesized,and overexpression vectors PMO-p D1301 S and PMO-p UBI were constructed and insertedtransformed into Arabidopsis thaliana and Nipponbare rice,respectively.Positive transgenic lines with high expression of Nc PMO-2 were selected.2.The Nc PMO-2ox transgenic Arabidopsis had a lower branching phenotype,whereas Nc PMO-2ox transgenic rice showed an increase in tiller number and biomass of stem,but a decrease in fruiting.3.For cell wall components measured from mature stem,cellulose abundance was significantly increased in Nc PMO-2ox transgenic Arabidopsis,but crystalline cellulose was reduced.For Nc PMO-2ox transgenic rice,both cellulose and crystalline cellulose were reduced.4.Both NcPMO-2ox transgenic Arabidopsis and rice showed a much higher enzymatic saccharification rate revealed by higher hexose release during enzymatic hydrolysis,the improvement over control was 12%-25%.