Characterization Of Secondary Cell Wall Ultrastructure That Determines Lignocellulose Enzymatic Digestibility In Rice

Plant cell wall determines plant cell size and shape,and also affects plant mechanical property and stress resistance.Meanwhile,it represents the most abundant biomass resource convertible for biofuels and biochemical products.In general,plant secondary cell wall consists of an extremely complicated three-dimensional network via interactions of heterologous polymers including cellulose,hemicellulose and lignin.In comparison,cellulose microfibrils(CMFs)as the major loading-bear backbone,orientates a long stretch surrounding plant cells,hemicelluloses serve as soft linkers between cellulose and lignin,and lignin fills into the cell wall to maintain its strength.To address the network recalcitrance,it thus becomes scientifically important to understand plant cell wall biosynthesis and to explore its ultrastructure structure.Because the fragile culms of rice have been examined as excellent experimental materials for cell wall characterization,this study first selected a new rice fragile culm 24(Osfc24/Os UGE2)mutant,and identified its involvements in two metabolic pathways to co-modulate cellulose biosynthesis by providing UDP-Gal substrates.Combined with another previously-identified fragile culm 16(Osfc16/OsCESA9),this study explored CMFs formation and assembly in two mutants,and raised the mechanism models about cellulose biosynthesis and lignocellulose enzymatic hydrolysis in rice.Here were major results achieved in this study.Chapterl.Function analysis of a novel rice fragile culm mutant Osfc24 with its cell wall structure identification1.The rice mutant Osfc24 was of genetic-stable fragile culm phenotypes including brittleness of leaves and internodes.Based on TEM observation and chemical analysis,the Osfc24 mutant exhibited significantly reduced cell wall thickness and altered cell wall composition.Meanwhile,the Osfc24 mutant showed paler leaves and stunned growth,with a significant reduction of plant height,tiller number and second internode length.In addition,the Osfc24 mutant showed significantly reduced cell widths measured in the transverse direction and decreased cell number accounted in the longitude direction.2.Based on the integrated analyses including map-based gene cloning,genetic complementary and co-segregation survey,the Osfc24 mutant was identified with the null mutation on the 9th exon of LOC_Os08g28730,and it was then designed as OsUGE2 subjective to phylogenetic tree analysis and protein sequence alignment.3.The OsUGE2 protein was further observed to localize in the cytoplasm,and its transcript was much expressed in the green and mature tissues of rice.By comparison,the OsUGE1,OsUGE2 and OsUGE3 expression levels were significantly reduced in the Osfc24 mutant,whereas the Os UGE4 and OsPHD1 were increased.Enzyme assays in vitro indicated that the OsUGE2 was of bidirectional epimerase activities,and the catalytic activities in vivo of the entire UGEs were significantly decreased in the Osfc24 mutant,compared to the wild type.4.Biochemical analyses revealed that the Osfc24 mutant was of significant reduction of galactolipids,chlorophyllin and soluble sugars,photosynthesis parameters,and biomass yield,as compared to the wild type.Accordingly,two related family genes were all downregulated such as OsSUSs(sucrose synthases)and OsCESAs(cellulose synthases)in the Osfc24 mutant,suggesting that the reduced photosynthetic capacity may cause less carbon supply for cellulose synthesis.Meanwhile,total galactose and glucose levels of cell walls were significantly reduced in the Osfc24 mutant,and arabinogalactan protein(AGP)was less accumulated into the cell walls,based on the histological staining and immunolabeling assay with several anti-AG mAbs,which may lead to defective cellulose biosynthesis in the mutant.Furthermore,this study detected significantly reduced cellulose DP and CrI in the Osfc24 mutant,which should account for much enhanced biomass enzymatic saccharification after alkali and acid pretreatments with biomass residues,compared to the wild type.Using AFM observation,the Osfc24 mutant showed its CMFs of disrupted orientation and more defects,compared to the wild type.5.In conclusion,this study has identified the OsUGE2 activity for UDP-Gal biosynthesis de novo by interconverting UDP-Glc,leading to the supply either for AGP synthesis into the cell wall or galactolipids deposition in the chloroplasts.It is thus assumed that AGP accumulation may positively affect cellulose synthesis for altered plant mechanics,and that galactolipids as essential component of chloroplasts should play a role in plant photosynthesis to accumulate carbon source(sucrose)that provides the UDP-Glc substrate for cellulose biosynthesis.Hence,this study has proposed a mechanism model that OsUGE2 participates in two metabolic pathways to co-modulate cellulose biosynthesis by dynamically providing UDP-Gal substrates.Chapter 2.Characterization of the cellulose microfibril ultrastructure critical for lignocellulosic enzymatic hydrolysis1.Using our previously-identified rice mutant(Osfc16/Oscesa9)of fragile culms phenotypes,this study explored its CMFs ultrastructure.The innermost surface of secondary parenchyma cell wall was chosen as the model area for AFM observation.The gradient chlorite treatments were used to effectively remove lignin and partial hemicelluloses,leading to reduced cellulose DP.By comparison,the mild chlorite treatments had little impact on native cellulose structure,whereas the harsh ones largely extracted hemicelluloses with strongly disrupted cellulose CMFs.2.Based on the AFM observation,the CMFs exhibited well-preserved structures under mild chlorite treatment conditions,but they were broken down into short rods with harsh chlorite treatments.Particularly,under the 16%chlorite treatment,alternative groove breakpoints were observed in the axial direction of CMFs,whereas the CMFs retained native structure with the 8%chlorite treatment.Using AFM-based CBM3a single molecular technology,the interval regions of CMFs were observed with well-recognized by CMB3a,while the groove-like breakpoints had little recognition signals,indicating that the interval breakpoints generated by 16%chlorite treatment should be due to the destructed amorphous regions of CMFs.3.From AFM real-time observations,the endo-β-1,4-glucanase(EG)could cut CMFs transversely,while exo-β-1,4-glucnnase(CBHI)was able to peel off CMFs longitudinally.Furthermore,EG cut CMFs into even segments,and the segments of the Osfc16 mutant were much shorter than those of the wild type.CBHI hydrolysis also produced much shorter segments in the mutant,and the segments were rough and uneven.Using CBM3a single molecular technology,the cellulases attacking sites on CMFs were not recognized by CBM3a,whereas the interval segments were fully recognized,indicating that the cellulases may produce alternating amorphous sites along CMFs,and the Osfc16 mutant should possess higher frequency of amorphous sites.Hence,the results demonstrated that the amorphous sites may be the inherent property of CMFs in plant cell walls,and they could be modified by site mutation of cellulose synthases.4.Compared to the wild type,the Osfc16 mutant showed much more enhanced lignocellulose saccharification released from enzymatic hydrolysis of mixed-cellulases or single cellulase with biomass substrates,suggesting that more amorphous sites of CMFs may lead to increased accessibility of cellulases enzymes in the Osfc16 mutant.Notably,the 8%chlorite treatment caused the highest enzymatic saccharification in both mutant and wild type,but the 16%and 32%chlorite treatments had much reduced saccharification,suggesting that the destructed CMFs by harsh chlorite conditions may destroy the amorphous sites accessible for cellulases loading and digestion.5.Using several representative plant species with distinct lignocellulose compositions and features,this study detected their enzymatic saccharifications of two substrates(crystalline cellulose and holocellulose).Compared to the holocellulose substrates,the crystalline cellulose substrates were of extremely low enzymatic saccharification in all plant species,probably due to all amorphous sites of CMFs destroyed.Significantly,the substrates of both holocellulose and crystalline cellulose showed a negative correlation between the enzymatic saccharification and the cellulose DP,suggesting that cellulose DP should be a key parameter accounting for alternating crystalline regions of CMFs spaced by the amorphous sites.6.In conclusion,we raised a hypothesis model of groove-like CMFs structure in plant cell walls,providing insights into lignocellulose enzymatic hydrolysis in plants.