Functional Study Of Rice UAP1 And UAP2 Genes Involved In Early Leaf Senescence And Defense Response

Inactivation of the UDP-N-acetylglucosamine pyrophosphorylase 1(UAP1)mutation leads to the appearance of premature leaf senescence and defense response phenotype in rice.The UAP1 gene in rice has a homologous gene,UAP2.Similar to UAP1,UAP2 also has the catalytic function of UDP-N-acetylglucosamine pyrophosphorylase.The UAP1 gene is critical,but the mutant uap1 leaves can grow normally for a period of time before they die prematurely.The mechanism for maintaining the transient survival of the uap1 mutant leaves remains to be further explored.In addition,it remains to be systematically verified whether the UAP1 gene participates in the two biological processes of premature leaf senescence and defense response with positive or negative regulatory effects.This study investigated the phenotypic effects of rice UAP2 and UAP1 genes on uap1 mutants from the phenotypic,physiological,and molecular levels,and explored the positive and negative regulatory modes of UAP1 gene for early leaf senescence and defense response by utilizing RNA interference technology and over-expression technology.The main findings are as follows:1.The UAP2 gene is the same as UAP1,showing function on restoring the leaf mutation phenotype of the lesion-mimic and early leaf senescence mutant uap1.The function of the UAP2 gene is the reason that the uap1 mutant can grow normally for a period of time without the normal UAP1 enzymatic function.Firstly,the phenotypic observation of UAP2 gene overexpression in uap1 plants revealed that the UAP2 gene could restore the mutant phenotype of leaves of the uap1 mutant.Transgenic lines of UAP1 gene complementation in uap1 and UAP2 gene overexpression in uap1 were analysed.It was found that the chlorophyll content,the expression levels of aging-induced SGR gene,senescence-related transcription factors and senescence-related genes in transgenic lines of UAP1 gene complementation in uap1 and UAP2 gene overexpression in uap1 have recovered to be without significant differences when compared with the wild type.These evidences from the phenotypic,physiological,and molecular levels confirmed that the UAP2 gene can restore the early leaf senescence phenotype of the uap1 mutant as the UAP1 gene.Secondly,when inoculated with the bacterial blight strain PXO99,it was found that the resistance to the bacterial blight strain of the transgenic lines of UAP1 gene complementation in uap1 and UAP2 gene overexpression in uap1 can restore to have no significant difference compared with the wild type.There was no significant difference for the expression of defense signal-related genes PR1 a and PBZ1 in leaves of the transgenic lines in comparison with the wild type.The UAP1 gene plays a key role in the synthesis of UDP-N-acetylglucosamine(UDP-Glc NAc),while UDP-Glc NAc is related to protein glycosylation and may affect endoplasmic reticulum stress to trigger unfolded protein responses.The expression levels of endoplasmic reticulum stress-related transcription factors and marker genes were tested in the transgenic lines of UAP1 gene complementation in uap1 and UAP2 gene overexpression in uap1.The induced expression levels of these genes in uap1 mutant were found to recover to have no significant difference when compared with the wild type.In addition,the UAP1 complementation in uap1 and UAP2 gene overexpression in uap1 could not completely restore the agronomic traits of the uap1 mutant,such as plant height,effective panicle number,panicle length,filled grain number per plant,total grain number per plant,seed-setting rate,and thousand-grain weight.2.Knocking down the expression of UAP1 gene by using the RNA interference technology leads to leaf premature senescence and defense response in rice.Detection of leaves at the tillering stage of RNA interference transgenic lines of rice UAP1 gene,it showed that the the positive transgenic lines showed a phenotype of spotted leaves,premature senescence and decreased chlorophyll content;and the expression of transcription factor and genes related to senescence,like SGR,was up-regulated.All of these results confirmed that the feature of premature senility of rice leaves occuerd in positive RNAi strain of UAP1 gene at the phenotypic,physiological and molecular levels.Through the inoculation experiment of bacterial blight,it was found that the RNAi transgenic lines with down regulated expression of UAP1 gene were more resistant to bacterial blight,the expression of defense signaling genes PR1 a and PBZ1,endoplasmic reticulum stress related transcription factors and genes were significantly increased,suggesting that the defense response and endoplasmic reticulum stress response were activated.In terms of agronomic characters,RNAi strain with down-regulated expression of UAP1 gene affected the plant height,effective panicle number,panicle length,filled grain number per plant,total grain number per plant,seed-setting rate and thousand-grain weight,and had a certain impact on the decline of rice yield.3.The overexpression of UAP1 gene did not lead to the delay of leaf senescence and the change of defense response.Phenotypic observation and chlorophyll content analysis of UAP1 gene overexpressing plants at the tillering stage and mature stage,and found that UAP1 gene overexpression plants and leaves developed normally,showing normal green phenotype,and no significant change in chlorophyll content in leaves.Detecting the the expression of SGR genes,transcription factor and genes related to senescence,there was no obvious difference from its UAP1 overexpression lines,it revealed that the overexpression of UAP1 can not delay the leaf senescence and change leaf development process.It was also found that the resistance of UAP1 overexpression lines to bacterial blight had not changed,and the expression levels of PR1 a and PBZ1,which indicated that the overexpression of UAP1 gene had no effect on the process of defense response.Except this,the results showed that there was no significant difference in the expression level of endoplasmic reticulum stress related genes,which indicated that endoplasmic reticulum stress did not occur at the molecular level.Through the investigation of agronomic characters,the overexpression of UAP1 has no significant positive effect on the agronomic characters of plant height,effective panicle number,panicle length,filled grain number per plant,total grain number per plant,seed-setting rate and thousand-grain weight.