Research On The Mechanism Of SlPP2C In Regulating Tomato Senescence

Senescence is an important process of biological programmed death,which is regulated by both genetic and environmental factors.Plant organ senescence not only affects growth and development but also affects plant yield and product quality.Plant organs senescence is a complex physiological process,and it is regulated by complex physiological metabolism and multiple genes,including members of the family of PP2 C which is thought to be important regulatory factors to animal and plant cell in death and senescence.Because PP2 C family is huge,its members behave in different ways in different plant species.Therefore,in order to understand the PP2 C regulation mechanism in tomato organ senescence,this paper carried out a systematic study.The main research results are as follows:1.We clarified that a senescence related gene Sl PP2C(Solyc07g062970)was found in tomato,and its gene expression,protein accumulation and GUS staining all confirmed that it was highly expressed in the senescence and abscission.During the development of tomato leaves,flowers,fruits and pedicel,the gene expression and protein accumulation of SlPP2C increased with the process of maturation,senescence and abscission.GUS staining in Pro Sl PP2C::GUS plants showed deep staining in the senescence leaves,flowers and pedicel as well as in the mature fruits and the staining was deep in the senescent tissues,such as petals,stamens,tapetum,seeds,fruit vascular bundles,distal end of flower pedicel,etc.Under the treatment inducing leaf senescence in vitro,SlPP2C expression was also significantly up-regulated,especially in the dark treatment.Subcellular localization has found that SlPP2C is located in the cell membrane,nucleus and cytoplasm,and its extensive localization indicates that SlPP2C may have diverse functions.2.It was clarified that tomato SlPP2C belongs to the F subfamily of PP2 C phosphatase and it has nine homologous genes.SlPP2C RNAi plants significantly delayed the maturation,senescence and abscission of tomato leaves,flowers and fruits,respectively.We obtained SlPP2C specific silencing plants and observed their phenotypes of senescence and abscission.After limited root cultivation for 60 days,the first and second true leaf of SlPP2C RNAi plants still remains green,while the leaves of wild type plants has turned yellow.The determination in chlorophyll fluorescence,chlorophyll content and senescence relative gene expression all obtained consistent results.Detached leaves cultivated under dark observed similar results.In addition,SlPP2C RNAi plants exhibited significantly delayed senescence of petals and stamens and prolonged flower period for up to 15 days.Under cultivation for 15 days,detached fruits from control plants reached red ripening stage,while SlPP2C RNAi plants still remained at green stage or turning stage.Pedicel abscission rate of SlPP2C RNAi plants was significantly lower than that of control plants.3.Two important factors,Sn RK1 and PIN1,interacting with SlPP2C were found in yeast base of senescent leaf and abscisic pedicel.SlPP2C bait vector and yeast library were used for hybridization to select eight interacting proteins.The four proteins with the most frequent occurrences were selected for co-transformation verification,and the results showed that the four proteins did interact with SlPP2C.Subsequently,the genes encoding these four proteins were silenced respectively in SlPP2C RNAi lines.When investigating the leaf senescence phenotype,only silencing Sn RK1 could reverse the delayed-senescence of leaf phenotype of SlPP2C RNAi lines,which can be regarded as a key candidate protein in the pathway of SlPP2C regulating leaf senescence.When investigating the abscission,we found that silencing PIN1 and Sn RK1 both could reverse the phenotype of delayed pedicel abscission in SlPP2C RNAi plants,while silencing PIN1 showed a more significant effect.We further confirmed the interaction between Sn RK1 and SlPP2C through Pull down and BIFC experiments.Then BIFC experiment was used to verify the interaction between SlPP2C and PIN1.4.Clarified that SlPP2C regulates Sn RK1 through dephosphorylation and silenced Sn RK1 can accelerate leaf senescence.In vitro dephosphorylation experiment showed weakened bands of Sn RK1 after added SlPP2C,which demonstrated SlPP2C can dephosphorylate Sn RK1.After 37 days of root-limited cultivation,it significantly promoted the chlorsis of the first and second true leaves of Sn RK1 RNAi lines,while the control plants were not affected.The results of the corresponding physiological and molecular indexes were consistent with the phenotype.The detached leaves cultured under dark conditions obtained consistent results.5.Clarified that SlPP2C inhibits Sn RK1 signal.Transcriptome analysis of the leaves of SlPP2C RNAi and Sn RK1 RNAi silencing lines as well as wild type plants showed that senescence mark genes were down-regulated in SlPP2C RNAi lines and up-regulated in Sn RK1 RNAi lines.51% of the genes down-regulated in SlPP2C RNAi lines were up-regulated in Sn RK1 RNAi lines,while 54% of the genes up-regulated in SlPP2C RNAi lines were down-regulated in Sn RK1 RNAi lines.There are 2902 genes regulated both by SlPP2C and Sn RK1.KEGG analysis of co-regulated genes revealed that the top 10 pathways are mainly related to senescence,such as photosynthesis,chlorophyll degradation,macromolecular degradation and transport,and sugar signaling pathways(sucrose starch decomposition and carbon and nitrogen metabolism),etc.This indicated that the downstream genes regulated by SlPP2C and Sn RK1 had opposite regulatory mechanisms during the senescence of tomato leaves,and more than half of the genes regulated by SlPP2C are also regulated by Sn RK1.6.We make it clear that SlPP2C inhibits the response of Sn RK1 to high sugar content,in addition,SlPP2C regulates Sn RK1 in an ABA receptor-independent way.It was found that Sn RK1 RNAi plants were less sensitive to hyper-glucose than control plants.The enzyme activity of Sn RK1 kinase was significantly up-regulated in SlPP2C RNAi plants compared with control plants.These demonstrate that SlPP2C RNAi lines enhanced the response to sugar signal by increasing Sn RK1 activity,thus delaying leaf senescence.In addition,no interaction was found between SlPP2C and tomato ABA receptor(Sl PYLs),indicating ABA receptors don't participate in the regulation of SlPP2C to Sn RK1.