The Functional Analysis Of Plant Cytokinin Oxidases/dehydrogenases Gene AtCKX3 And Fe(?)-and 2-oxoglutarate Dependent Dioxygenase Gene SAG203 Regulating Leaf Senescence

Plants senescence is an important age-dependent stage of development.The obvious phenomenon of senescence is color-change of leaf,during this process the loss of chlorophyll could directly affect vegetative and reproductive growth of plant.Impaired photosynthesis result from leaf senescence will lead to the product limitation of crop.Discovering the molecular mechanism of leaf senescence will provide more evidence for improving crop's phenotypic trait and quality.Phytohormones play a vital role in plant growth and development.It is reported that cytokinin(CKs)is a negative regulator of leaf senescence,whereas salicylic acid(SA)is a positive regulator.AtNAP is a NAC family transcription factor gene,which could physically bind to the promoter region of target genes and regulate the process of leaf senescence.In this study,we perform two genes,cytokinin oxidases/dehydrogenase 3 AtCKX3 and salicylic acid synthesis related gene SAG203,which involve in AtNAP regulating pathway,and playing crucial role in leaf senescence.The primary results of this research are listed as followed:1?The cytokinin content is decreased during leaf senescence.Cytokinin oxidases/dehydrogenases(CKXs)are the major enzymes which catalyze the irreversible degradation of cytokinins.qPCR and promoter-GUS analysis showed that the transcription of cytokinin oxidases/dehydrogenase 3 AtCKX3,which is one of cytokinin oxidases/dehydrogenases family members of Arabidopsis(AtCKX1-AtCKX6),was induced in the senescent leaves.Yeast one hybrid assay demonstrated the AtNAP TF physically binded to the 7-bp core sequence of the AtCKX3 promoter,and the transcriptional level of AtCKX3 was positively regulated by the AtNAP.The atckx3 knockout plants exhibited significantly delayed leaf senescence,and the null phenotype can be rescued by the wild-type AtCKX3.In addition,inducible overexpression of AtCKX3 accelerating leaf senescence,all these result showed AtCKX3 was a positive regulator of leaf senescence.Cytokinin content(IPA)analysis also proved transcriptional level of AtCKX3 was negative correlated to cytokinin content in leaf,and this result further suggesting that the transcriptional change of AtCKX3 was the major factor leading to leaf senescence by degenerating CK in leaves.These work uncovered a regulatory chain,AtNAP-AtCKX3,which controlled cytokinin content during leaf senescence.2?Two knockout lines of leaf senescence related gene SAG203 exhibited significantly delayed leaf senescence compared with that of the wild type,and inducible overexpression of SAG203 led to precocious leaf senescence.The yeast one hybrid assay confirmed that SAG202 TF,the direct downstream gene of AtNAP,was able to physically bind to the promoter region of SAG203,and positively regulated SAG203 transcription.qPCR analysis showed the transcript level of SAG203 was significantly induced by SA treatment,but the induction of SAG203 in sag202 null mutant was severely weakened comparing with that of wild type.Moreover,the free SA level in the senescing leaves of sag203 null mutant was obviously lower than that of wild type in the same development stage.So we hypothesized SAG203 may be involved in salicylic acid synthesis.These results indicated that there was a unique regulatory pathway,AtNAP-SAG202-SAG203,which controled SA biosynthesis during leaf senescence.The necrotrophic fungus Botrytis cinerea is one of an important postharvest pathogenic fungus.In this study,we worked on the functional analysis of a putative phytochrome-like histidine kinase(HK)gene Bcphy3,which involved in light response and pathogenicity of Botrytis cinerea.The transcriptional level of the Bcphy3 gene was both induced by light intensity(0,50,100,and 150 ?umo1·m-2·s-1)and time of light treatment(0,0.5,1.0,and 2.0 hours after light treatment).A Bcphy3 null mutant ?Bcphy3-P21 was obtained by homologous recombination.ABcphy3-P21 grew more slowly than B05.10,and was unable to produce sclerotia.Moreover,?Bcphy3-P21 displayed significantly reduced pathogenicity on different tested plant tissues.In addition,?Bcphy3-P21 was more susceptible to cell wall stress caused by Congo red.The TEM images showed that there was variable thinness of the?Bcphy3-P21 mutant cell wall,and the edge of the cell wall was obscure.The results of gene transcription analysis and the chitin content assay demonstrated that chitin synthesis was inhibited in ?Bcphy3-P21 mutant cells.