?.Research On The OsXYN1,Which Encoding An Xylanase Is Critical For Plant Growth ?.Distinct And Cooperative Activities Of HESO1 And URT1 Nucleotidyl Transferases In MicroRNA Turnover In Arabidopsis

Secondary walls are mainly composed of cellulose,hemicelluloses and lignin,which is vital for maintaining cellular morphology and providing mechanical strength support and protection to plant body.Rice lodging-resistant is an important genetic base for high yield and yield stability.The degree of stem thickness,the number and structure of vascular bundle is significant for lodging-resistant.Thus,the search and characterization of genes encoding enzymes which are involved in synthesis and degradation of plant cell wall polysaccharides is vital for cultivation of rice varieties with high yield,good quality and resistance.As the second high abundant polysaccharides of the plant cell wall,Xylan is required for normal plant growth and development.In this study,we identified two mutants with thin stem,Osxyn1-1 and Osxyn1-2,from the Shuhui498 EMS mutant library,which is known as core parent of heavy panicle hybrid rice with elite lodging-resistant.The mutants show Pleiotropic defects,including dwarf plant height,shriveled leaf apex,lower fertility,lower tiller number,narrower vascular bundles in the stem.Genetic and allelic study indicated that the phenotypes of two mutants were controlled by one recessive gene,and two locus were allelic.The mapping result showed that the phenotypes of two mutants were caused by two different mutations in an unreported gene encoded a xylan hydrolases,named as OsXYN1.Based on these results,we further studied the cell and molecular biology,including temporal spatial expression,protein subcellular localization,internode monosaccharide composition analysis,cellular soluble oligosaccharide composition in leaves,gene regulation network and protein activity test in vivo or in vitro.The main experimental results are listed as follows:1.The agronomic traits comparison between the R498 and osxyn1-1 reveal that the mutation of Osxyn1 cause pleiotropic defects,including dwarf plant height,shriveled leaf apex,lower fertility,lower tiller number,narrower vascular bundles in the stem.2.Cytology analysis found that chloroplast grana laminar structure collapsed in the Osxyn1-1 mutant,the sclerenchyma cell and parenchyma cell of mutant vascular bundle show primary cell wall deficience.3.Genetic analysis indicate that the deficient phenotype of Osxyn1-1 was caused by recessive single gene mutation.Gene mapping found LOC_Os3g 47010 loci was the candidate gene.4.Real time PCR results indicated OsXYN1 preferentially highly expressed in leaves and low-level expression signals were also detected in reproductive stem and microspore by RNA insitu hybridization.5.Subcellular localization researches indicate that OsXYN1 is a membrane bound protein,the OsXYN1-GFP recombinant protein stick close with membrane during plasmolysis.6.Monosaccharide composition analysis found that most of monosaccharide tested were not significantly changed except for Arabinose.which implied that the mutation of Osxyn1-1 didn't affect the synthesis of xylan backbone,surprising,the AIR(alcohol insoluble remains)extracted from Osxyn1-1 was lower than the one from R498.7.The expression levels of the genes involved in the plant cell wall biosynthesis were down regulated.8.Cellular soluble oligosaccharide in young leaves tissue analyzed by GC-MS uncover that the amount of an oligosaccharide containing arabinose apparently decreased in Osxyn1-1.In brief,we can conclude that OsXYN1,encoded a Xylanase,maybe involve in the biogenesis of xylan during the plant cell wall deposition,and responsible for hydrolysis of xylan side chains containing arabinose.Our results will lay a foundation for unveiling the molecular regulatory network,and provide a theoretical basis for molecular design breeding related rice lodging-resistant.There are three major types of small RNAs in eukaryotes,microRNAs(miRNAs),small interfering RNAs(siRNAs)and piwi-interacting RNAs(piRNAs),which are involved in many biological processes such as development,disease resistance,genome stability and recognition,and environmental adaption.Considering the indispensible and widespread functions of small RNAs,it is important to research their biogenesis,turnover and balance in vivo.The biogenesis of siRNAs and miRNAs in plants and piRNAs and endogenous siRNAs in animals share a common step that is 2'-O-methylation on the 3'terminal of ribose carried out by the small RNA methyltransferase(HEN1 HUA ENHANCER1).In Arabidopsis hen1 mutants,miRNAs and siRNAs were 3'truncated and 3' uridylated,the abundance were reduced critically.Compared with the plant,in animal hen1 mutants,piRNAs and siRNAs become 3' truncated and 3' uridylated similarly.All of these indicate that 2'-O-methylation protect small RNAs against 3' truncation as well as 3' tailing carried out by a 3' to 5'exonuclease(s)and a nucleotidyl transferase(s)differently.According to previous study,we identified Arabidopsis HES01(HEN1 SUPPRESSOR1)as nucleotidyl transferase which is responsible for miRNA 3'tailing in vivo.Function mutation of HESO1 partially suppresses the developmental abnormalities of hen1 mutants,at the molecular level,the 3'tailing for most miRNAs were shorted and miRNA abundance were increased obviously.The analysis of small RNA profiling about hen1 and hen1 heso1 mutants reveals that 3' truncation is independent of 3' uridylation,as the reason that 3' uridylation occurs on both full-length and 3' truncated forms.Although the null heso1-1 mutation causes obvious reduction in 3' uridylation,but substantial levels of miRNA 3' tailing still remains in heso1 loss-of-function mutants,mainly mono-uridylation.This lead us to hypothesize that there is another nucleotidyl transferase also works on miRNAs in vivo.Several questions that were not well addressed in the previous study,whether uridylation is added to miRNAs when they are bound by AGO1 complex,the major miRNA effector in Arabidopsis,and how uridylation result in miRNA turn over in vivo.In this study,among ten potential nucleotidyl transferases in Arabidopsis,URT1 was identified as a nucleotidyl transferase is responsible for the uridylation of a small unmber of miRNAs,which have the second highest impact on miRNA uridylation.URT1 and HES01 show different preference on miRNA substrates with different 3' nucleotides in vitro.URT1 and HES01 act on different size variants of the same miRNAs in vivo.We found that URT1 and HESO1 act sequentially on some miRNAs,with URT1 mono-uridylating the miRNAs followed by their further uridylation by HES01.We showed that both HES01 and URT1 act on free miRNAs as well as AG01-bound miRNAs in vitro.Intriguingly,URT1,but not HES01,is highly effective in triggering the release of miRNAs from AGO1 upon their tailing beyond a certain length.Release from AG01 makes a miRNA susceptible to cellular nucleases.We also found that the tailing of AGO1-bound miR165/6 reduces its slicing activity while the monouridylation of miR171a endows an ability to trigger the biogenesis of phasiRNAs.Thus,miRNA tailing affects the activities of miRNAs in addition to causing miRNA degradation.This study sheds a light on distinct and cooperative activities of nucleotide transferases in miRNA modification.The main experimental results are listed as follows1.URT 1 is Responsible for the Uridylation of a Small Number of miRNAs2.URT and HESO1 act coordinately on miR158 by tailing different forms3.URT1 exhibits nucleotidyl transferase activity on unmethylated miRNA in vitro4.URT1 Initiate the Uridylation of miR173 and abolish the Biogenesis of tasiRNA in hen1 mutant5.URT1 and HES01 have different substrate specificities in vitro,HES01 prefer the miRNA ending with U and URT 1 prefer the miRNA ending with A6.Both of URT1 and HESO1 act on AG01-bound miRNA,the tailing miRNA still bound AGO1 complex7.The mutation Of URT1 blocks the generation of phased secondly siRNA triggered by 22 nucleotides miR171 In hen1-8.8.Tailing of miR165/6 by URT1 reduces its slicing 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