Molecular Mechanism Of UDP-Glucose Pyrophosphorylase Enhancing Vegetative Growth In Transgenic Plants

Dahurian larch,Larix gmelinii(Rupr.),deciduous tree of Pinaceae,is the major afforestation species in Great Khingan,which has high application value in the manufacture of glulam and composite wood because of its mechanical properties.Uridine diphosphate glucose pyrophosphate(UGPase)catalyzes glucose-1-phosphate(Glc-1-P)to produce uridine diphosphate glucose(UDPG),which is the precursor of cellulose and callose,the main components of plant cell wall.Therefore,UGPase plays a decisive role in plant growth and development,as well as in wood formation.Our previous study has confirmed that LgUGPase could promote the accumulation of cellulose and deposition of cell wall by regulating the metabolism of polysaccharides in plants,thereby enhancing the vegetative growth of transgenic plants.Therefore,it has great significance to further study the function,expression and regulation mechanism of the LgUGPase.The LgUGPase is encoded by two paralogous genes in L.gmelinii,the pLgUGPase1::GUS is highly expressed in seedlings of transgenic Arabidopsis thaliana and its expression level can be induced by the GA and MeJA.For analyzing the expression characteristics of LgUGPase1/2,the 1588 bp of LgUGPase2 was cloned by Genomic Walking.The PLANTCARE analysis showed that pLgUGPase1/2 contain not only the cis-acting elements responding to light,drought,mechanical damage and fungal elicitor but also the hormone-response elements such as auxin,methyl jasmonate,ethylene and gibberellin.GUS staining of transgenic Arabidopsis showed that pLgUGPase1/2::GUS showed different expression patterns,the pLgUGPase1::GUS was strong expressed in various tissues and organs of seedlings,its expression level decreased in mature leaves;the pLgUGPase2::GUS expressed in radicle,hypocotyl and cotyledon;the expression of pLgUGPase1/2::GUS were also detect in flowers and pods,which were induced by GA and MeJA.For investigating the subcellular localization of LgUGPase,the transient expression of LgUGPase-GFP fusion protein mediated by Agrobacterium in tobacco epidermal cells was performed,which showed that the LgUGPase-GFP fusion protein was localized in chloroplasts.To analyze the function of the LgUGPase,atugp1 mutant,atugp1 functional complement lines,LgUGPase-overexpressing lines were analyzed,which showed that LgUGPase can rescue the growth inhibition caused by the AtUGP1 mutation,and the expression of LgUGPase increased the contents of soluble sugar,cellulose and lignin,and promoted the vegetative growth of transgenic Arabidopsis.The transcriptome analysis of WT,atugp1 mutant and LgUGPase-overexpressing lines showed that there are 3214 of differentially expressed genes(DEG)between atugp1 mutant and wild type plants,and 2970 of DEG between WT and LgUGPase-overexpressing plants.The DEGs were annotated into phenylpropanoid biosynthesis,starch and sucrose metabolism,photosynthesis,carbon metabolism,plant hormone signal transduction,and plantpathogen interaction pathways.The yeast two-hybrid assay was performed to verify the interaction of LgUGPase with proteins predicted based on the transcriptome data,which showed that LgUGPase can not interact with these proteins.Therefore,it is speculated that UDPG,the catalytic product by UGPase,may act as a signal molecule to regulate growth,development and stress adaptation of plants by regulating metabolisms of saccharides,carbon,and phenylpropanoid,plant hormone signal transduction and plant-pathogen interaction pathway.These results lay a foundation for further understanding the regulation of sugar metabolism and analyzing the molecular regulation mechanism of LgUGPase,and provides theoretical and experimental basis for genetic improvement of crops and forest trees using LgUGPase.