Study Of Endogenous Gibberellin Regulation On Tomato Fruit Ripening

Fruit ripening process involves a series of physiological and biochemical changes,such as pigment accumulation,sugar formation,fruit softening,and so on.Ethylene is a necessary phytohormone for tomato in ripening process.prevent ethylene biosynthesis and signal transduction will inhibition of tomato fruit ripening process,exogenous ethylene can promote the process.Other hormones,Exogenous GAs can delay fruit ripening,But the chemical nature of exogenous and endogenous hormones may vary.The effect of exogenous hormone treatment is not completely equal to endogenous hormone in plant growth and development.Therefore,it is worthwhile to investigate the roles of GAs,particularly endogenous GAs in the fruit ripening process.In this study.We used the wild type and the transgenic tomato materials,which saved in our lab.Preliminary revealed how the endogenous gibberellins influence DNA methylation and ethylene biosynthesis and signal transduction in tomato,then regulate its ripening process further more.The main results were as follows:1.Our result showed that,the expression of GA biosynthetic gene GA3ox1 decreased significantly during mature green to breaker stage,while the main GA deactivation genes GA2oxs increased significantly.The expression of SIGAST1 had a significant reduction from the mature green stage to the breaker stage.In ethylene biosynthetic gene families,ACS2,ACS4,and ACO1 are all highly expressed at the breaker stage or subsequent ripening stages,and the expression of ethylene response gene E8,had a significant increasing by a dozen fold in this period,and reached a very high level at the breaker stage.Consistent with the result,the concentration of endogenous GAs decreased significantly,but ethylene production started to increase from the mature green stage to the breaker stage of tomato fruits.Therefore,we speculated that there may have a relationship between the decreasing of endogenous GAs and the activation of ethylene biosynthesis in tomato fruit ripening.2.To demonstrate our speculation mentioned above,we firstly accelerated endogenous GA degradation in tomato fruits by transferring the fruit-specific promoter TFM7 controlling the GA catabolic gene SlGA2ox1 into the Micro-Tom tomato cultivar,to study its effects on fruit ripening.Real time RT-PCR(qPCR)result showed that expression of the SlGA2ox1 gene in transgenic fruits was increased compared to the wild type,,as they displayed an accelerated ripening rate.These results demonstrate that reducing endogenous GA concentrations accelerates total carotenoid and lycopene accumulation and chlorophyll degradation.Expression of carotenoid and lycopene biosynthetic genes in transgenic fruits was significantly increased.The expression of polygalacturonase gene PG2A was significantly increased in GA-deficient transgenic tomatoes.In addition,Exogenous GA3 was applied to transgenic tomato can reverse the accelerated ripening process of transgenic fruits.We also studied the effects of exogenous GAs on tomato fruit ripening.Results showed that the fruit ripening process of GA3-treated fruits were significantly delayed compared to that of control fruits.The expression of above relate genes were decreased.These results suggest that the decreasing of endogenous GAs accelerated tomato ripening process.Exogenous GAs delayed the process.3.Ethylene is the major hormone regulating most aspects of fruit ripening Compared with the WT fruits,qPCR analysis showed that transcript levels of ACS2,ACS4,ACO1 increased early in GA-deficient transgenic tomatoes,but decreased and delay in GA3-treated fruits.The ethylene production consistent with the result.Transcript levels of the genes ETR3?ETR4?EIN2?EIL2?EIL3 and CTR1 were increased in transgenic tomatoes.but decreased in GA3-treated fruits The expression of several key ripening regulators assessed at the transcript level was strongly increased in the transgenic lines,but decreased in in GA3-treated fruits,which include NOR,CNR,RIN,and so on.These results suggest that during tomato ripening process,the decreasing of endogenous GAs activation of ethylene biosynthesis and signaling in tomato through regulate the expression of ripening regulator genes.Exogenous GA3 showed the opposite effect.4.The DNA demethylase gene SlDML2 is an important regulator of the transcriptional levels of ripening regulator genes through methylation of these gene promoters during the tomato ripening process,so we studied the effects of GAs on SIDML2 gene expression.The results showed that SlDML2 transcript levels upregulated in transgenic fruits.By contrast,the expression of SlDML2 was downregulated in GA3-treated fruits after injection compared to control fruits Therefore,we used bisulfite sequencing to analyze the methylation levels of these promoters sequences in GA-deficient transgenic and GA3-treated tomatoes.The results revealed that the methylation levels of NOR and CNR gene promoters were decreased transgenic fruits,Conversely,when mature-green fruits were treated with exogenous GA3.The NOR and CNR gene promoters all displayed increased methylation levels.These results suggest that endogenous GAs regulates the methylation levels of some ripening regulator gene promoters,and then influence ethylene production and signaling in tomato ripening process.Taken together,in this study,we propose an approach regulation during the fruit ripening process.GAs act as negative regulators of ethylene biosynthesis and the subsequent ripening process.During tomato fruit early development,GAs are at high levels to promote fruit enlargement before the mature green stage,and then concentrations of endogenous GAs gradually decrease.When these concentrations fall below a certain threshold,the DNA demethylase gene SIDML2 expression will be activated,the methylation levels of some ripening regulator promoters such as CNR and NOR will decrease,then the expression of these genes will increase,triggering the downstream ethylene biosynthetic pathway,and resulting in ethylene production and activation of the ethylene signaling pathway,thereby causing fruit ripening in tomatoes.Our research provides new sight in how endogenous gibberellin regulates SlDML2 expression and then regulates the ripening mechanism of tomato.It may also provide a basis for the improvement of tomato quality and fruit preservation breeding.