Functional Analysis Of TRNA Nucleoside Methylation Gene And Study Of Genes Involved In Arabinoxylan Biosynthesis In Rice

Methylated nucleosides are ubiquitous modification occuring at a variety of tRNAs from different kingdoms.Among more than 100 identified RNA modifications,two thirds of them involve methylation.MostRNA nucleoside modifications occur post-transcriptionally,and tRNA is the most heavily modified RNA species.Sacharomyces cerevisiea has thirteen Trm proteins for tRNA nucleosides methylation,Trm1,Trm2,Trm3,Trm4,Trm5,Trm6,Trm7,Trm8,Trm9,Trm10,Trm11,Trm13 and Trm140.The other three Trm proteins Trm61,Trm82 and Trm112 are the auxiliary proteins for Trm6,Trm82 and Trm112,respectively.These are not methyltransferase themselves,but are required for methylation.In this study,Trm proteins in S.cerevisiae were used as query sequences to identify rice and Arabidopsis candidates.Three clusters of MTases were identified based on conserved residues within the MTase domains.Three pyrimidine MTases（Trm4p,Trm7p and Trm140p）which catalyze the methylation of m⁵C（5-methylcytidine）,Cm（2’-O-methylcytidine）and m³C（3-methylcytidine）exhibited a striking conservation of a Glycine（G）residue,and aconsensus‘VLD（L/M）CAA（P/N）’neighbor motif.A conserved‘Gx E/D’motif was found in purine MTases（Trm3p,Trm8p and Trm11p）,for Gm（2’-O-methylguanosine）,m⁷G（7-methylguanosine）,and m²G（2-methylguanosine）modifications,respectively.Finally,a conserved‘DLD’motif was found in Trm1p and Trm10p MTases,for m¹G（1-methylguanosine）and m₂²G（2,2-methylguanosine）methylations.Hierarchical clustering with nucleoside abundance and candidate MTase transcript levels indicate Am,Cm,m¹A and m⁷G being critical for stress response;whereas Gm,m⁵U and m⁵C for plant development.We identified LOC＿Os03g61750（OsTRM13）as rice MTase for Am modification.OsTRM13 transcript level increased significantly upon salt stress and ABA treatment,and the OsTrm13 protein was found to be located in the nucleus.OsTRM13 overexpression improved salt stress tolerance in transgenic rice.In vivo and in vitro study also support OsTRM13 for Am methylation activity.Therefore,OsTRM13 as a rice tRNA nucleoside methyltransferase plays important role for salt stress tolerance in rice.Xylans are major hemicelluloses of secondary cell walls in terrestrial plants.It has been reported that arabinose（Ara）substitution degree of xylan could largely affect biomass enzymatic saccharification（digestibility）under various physical and chemical pretreatments in grass plants examined,probably due to a reduction of cellulose crystallinity（Cr I）and degree of polymerization（DP）.However,much remains unknown about how Ara substitution degree significantly affects cellulose features（Cr I or DP）.In this study,we performed cell wall fractionation procedures,and then used cellulases（CBHI and EGII）and xylanases（Xyn10 and Xyn11）for distinct wall polymer digestions,providing evidences about the potential association between hemicellulose and cellulose.We also identified Os XAT4 gene and investigated its potential functions in xylan biosynthesis and lignocellulose enzymatic hydrolysis.The main results were described as follows:1.A potential covalent cross-linking may occur among xylan and cellulose via substituted Ara side chains,based on chemical composition analysis and interlinkage style detection using cellulase and xylanase digestions of specific wall polymer fractions.2.By selecting transgenic rice lines that either over-expressed or silenced Os XAT4 by RNAi,and identifying osxat4（T-DNA）mutant,this study found that the plant height,cell length of 2th stem and weight of 2th stem were much increased in the over-expressed transgenic rice lines,but reduced in the RNAi transgenic lines and osxat4 mutant.2)Overexpression of Os XAT4 could lead to increased Ara levels of hemicelluloses in both transgenic rice and Arabidopsis lines,suggesting that Os XAT4 may involve in branched Ara chain formation of xylan.3)Compared to the control cultivar,the Os XAT4 overexpressed transgenic rice plants showed significantly reduced Cr I by 7.18%-8.77%,with related reduced cellulose and lignin levels,leading to much enhanced biomass saccharification by 3.65%-8.17%,with raised ethanol yields by 7.43%-12.62%.