Functional Characterization Of AXS And UXS Genes In Arabidopsis

UDP-GlcA is an important intermediate in nucleotide sugar metabolism which could be used for the synthesis of UDP-apiose(UDP-Api)and UDP-xylose(UDP-Xyl)by UDP-apiose synthase(also called UDP-apiose/UDP-xylose synthase,AXS)and UDP-xylose synthase(UXS).UDP-Api participates in the synthesis of rhamnogalacturonan II(RG-II).RG-II could form borate cross-linking with the two apiosyl residues of side chain A of two monomeric RG-II which though to result in a stable cell wall pectin network.Boron-RG-II complex is thought to be important for the mechanical property of cell wall.UDP-Xyl could be synthesized in cytosol and Golgi under the role of UXS.UDP-Xyl synthesized in cytosol is transported to Golgi by UDP-xylose transporter(UXT)and will participate in the synthesis of xylan and xyloglucan.All of the AXSs found so far,are bifunctional enzyme,which could form two kinds of nucleotide sugar: UDP-Api and UDP-Xyl.Moreover,the AXS and UXS genes are homologous in evolution.Therefore,this research characterized the AXS,UXS and UXT genes in this paper.The expression pattern,subcellular location and enzyme activity of AXS genes were analyzed.Then this research got the corresponding mutants of AXS,UXS and UXT and characterized their growth phenotypes.The effects of multi gene mutations on the plant development and cell wall composition were analyzed.Then main research results are as follows.1.There are two AXS genes in Arabidopsis genome.AXS2 had a higher expression level in all of the tested tissues.AXS1 and AXS2 proteins were located in cytosol and could form home or hetero dimer.The catalytic activities of AXS proteins were very low.2.The axs1 and axs2 single mutants showed no obvious phenotype which indicated the redundant function of the two AXS genes.After the cross of axs1 and axs2,no homozygous mutant was found which indicated that the plant without AXS gene maybe lethal.There were two types of mutants containing one functional copy of AXS gene and they showed different phenotypes.The axs1/+ axs2 mutant could not grow up and dead after a month.axs1 axs2/+ mutant was smaller than Col-0 wild type and could form seeds.From the dosage effect of the two types of mutant,we could draw a conclusion that AXS2 gene played a more important role in Arabidopsis than AXS1 gene.In consideration of the similar catalytic activity of the two AXS protein,the reason of the function difference of the two AXS genes was maybe the different expression level of the two AXSs.3.The axs1 axs2/+ mutant could form multi shoot apical dominance growth points without the main stem.The axs1 axs2/+ mutant seedlings contained shorter main root and more lateral roots.All of the above phenotypes indicated that the apical dominance of axs1 axs2/+ mutant was influenced which maybe follow the same reason with boron deficiency.With less Api and boron in the cell wall,there will be not enough boron-RG-II complexes in the cell wall which influence the localization of auxin in the growth point.The changed auxin distribution finally weakens or vanishes the apical dominance.4.Pollen grains without AXS gene were collapsed and could not germinate which indicated that RG-II is important for the pollen development and germination.With the axs1-1 single mutant background,AXS2 gene was RNAi with DEX which could kill the transgenic seedlings.The microarray analysis results of DEX:RNAi-axs2 transgenic plants showed that numbers of stress related genes were affected which accompanied with cell death.These results indicated that RG-II is indispensable for plant growth.5.RG-II structure of the axs mutants was uninfluenced.However,the UDP-Api contents of the four axs single mutants and two axs1 axs2/+ mutant were reduced.The Api,boron and RG-II contents of the two axs1 axs2/+ mutant were reduced.Monosaccharide composition analysis of axs1 axs2/+ and axs1/+ axs2 mutants showed that they had a lower pectin sugar content than Col-0 wild type.This was indicated that RG-II content had influenced cell wall pectin content.The microarray analysis results of axs1-1 axs2-1/+ mutant showed that the expression levels of lots of cell wall related genes were affected by the cell wall RG-II content.6.Ultrasonic assisted mucilage extraction method was developed which could accomplish the extraction in 20 s.Compared with the traditional methods,ultrasonic extraction could obtain the most amount of Arabidopsis inner mucilage in the least time.The outer mucilage had a more relationship with the growth conditions than the inner mucilage which indicated that this part of mucilage may contribute not so much to the Arabidopsis seeds survival.The germination rate of Arabidopsis seeds after 2 min ultrasonic treatment was not reduced which could be used for other experiments.7.The inner mucilage of axs1 axs2/+ mutant could not expand normally because of the broken obstacle of the seed coat outer cell wall.The mucilage of axs1 axs2/+ mutant contained more free carboxyl and less methyl ester which indicated that RG-II may have a role in the regulation of the HG methylation degree.Besides,the outer mucilage content of axs1 axs2/+ mutant was reduced which indicated that RG-II content had influenced the outer mucilage content.8.The analysis of cytosol located uxs3uxs5uxs6 and Golgi located uxs1uxs2uxs4 triple mutants found that the xylan content was reduced;the methylation degree of xylan and the saccharification efficiency of cell wall was increased in the uxs3uxs5uxs6 triple mutant.As such,we considered that cytosol located UXSs were more important for the secondary growth in Arabidopsis.AXS2 gene could not complement the uxs3uxs5uxs6 triple mutant which indicated that AXS has a very low catalytic activity for UDP-Xyl.The principal role of AXS is supplying UDP-Api.9.The triple mutants of UXT and UXS genes were used to comparative study.In contrast to the uxs mutants,the loss of UXT genes had no impact on the nucleotide sugar contents.The uxt1uxt2uxt3 triple mutant had a similar phenotype that collapsed vessel and reduced xylan content which indicated that UXTs were important for the translocation of UDP-Xyl.The saccharification efficiencies of uxs3uxs5uxs6 and uxt1uxt2uxt3 triple mutant were increased.The uxt1uxt2uxt3 triple mutant could bear very less seeds which indicated the abortion of this mutant.One reason is that the anthers of uxt1uxt2uxt3 triple mutant could not contact with the stigma entirely.Another reason is that there was less lignin in the anther which leaded the incomplete open of the anther and the failed release of pollen grains.