Preliminary Study On The Physiological And Molecular Mechanisms Of Ethylene Regulation In Watermelon Fruit Ripening

Fruit ripening is a complex but genetically programmed process.In climacteric fruits,ethylene has been identified as the key phytohormone.Recently,several physiological and genetic analyses uncovered its involvement in ripening progression of non-climacteric fruits as well,such as strawberry,grape,citrus etc.Watermelon is traditionally classified as non-climacteric.Different from non-ripening wide watermelon,dramatic changes occur in color,texture,flavor,and aroma of the fruit flesh when cultivated watermelon ripened.But now little is known about the regulation of these processes.In the present study,using tomato fruit as the reference,physiological and molecular mechanisms of ethylene regulation in two contrasting ripening type watermelon(one watermelon cultivar 97103 and one non-ripening wild germplasm PI296341-FR)was studied,and preliminary results were obtained as follows:1.The ethylene content was limited in watermelon flesh,but may involved in regulation of fruit ripening.Throughout development and ripening,very little ethylene evolved from watermelon fruit and was nearly undetectable.However,higher ethylene levels were discovered in watermelon flesh,which were found to represent the true level of ethylene synthesis.Ethylene content in flesh of watermelon cultivar 97103 was approximately ~10-fold lower on average than that of climacteric fruit tomato,and it increased considerably(~3-fold)and reached a peak at the onset of ripening(18DAP).In contrast,ethylene content was maintained at a continuously lower level in flesh of wild watermelon PI296341-FR,and did not show any increase over the course of fruit development.When treated with exogenous ethylene,accelerated softening and color development in 97103 flesh were observed,and synthesis of lycopene and ?-carotene increased.In PI296341-FR flesh,increased softening was also discovered but color development remained unchanged.The results above showed that,phytohormone ethylene may play an important role in modulating some metabolisms associated with ripening in watermelon flesh,such as cell wall modifications and carotenoid biosynthesis.2.During development and ripening,two ACS gens as well as two ACO gens in watermelon flesh may play key roles in ethylene biosynthesis,and act as important gene members in system II-like pathway of ethylene production.During development and ripening of 97103 and PI296341-FR,the activity of ACS and ACO was found to be in parallel with ethylene content in flesh.Eight ACS and eight ACO genes was identified in watermelon genome and their expressions were determined by qRT-PCR.Among of them,ACS genes Cla022653 and Cla011522 as well as ACO genes Cla014827 and Cla016287 in watermelon flesh of the two contrasting ripening type were discovered to be correlated with both of activity of ACS/ACO and ethylene content.Ethylene production in 97103 flesh increased dramatically when treated with exogenous ethylene,which showed its autocatalytic behavior of system II.And the system II-like pathway of ethylene synthesis was discovered not only existed in ripe flesh(18DAP)but also in unripe flesh(10DAP).Transcripts of ACS genes Cla022653 and Cla011522 as well as ACO genes Cla014827 and Cla016287 in 97103 flesh determined by qRT–PCR were all found to be induced by exogenous ethylene.The data above suggested that these four genes may play key roles in ethylene biosynthesis and act as important gene members in system II ethylene production in watermelon flesh during development and ripening.3.Several candidate genes of NAC,HD-Zip and ERF transcription factor family maybe involved in ethylene synthesis pathway were identified by transcriptome differential expression screening and co-expression network analysis.Combined with watermelon genome annotation and transcriptome profiling of fruit development and ripening of 97103 and PI296341-FR,differential expression screening and co-expression network analysis of genes in ethylene signal transduction pathway and transcriptional factors lying upstream of ethylene synthesis was performed.And thus several transcriptional factors were presumed to be involved in regulation of ethylene synthesis,including MADS-box gene Cla017291,NAC family gene Cla019099,HD-Zip family gene Cla017080 and so on.In addition,expressions of many ERF genes positively or negatively accompanying with ripening process were correlated with many other genes and formed three main clusters in co-expression network.And this suggested important roles of these ERF genes in watermelon ripening regulation mediated by ethylene.4.Over-expression of watermelon ERF gene ClERF069 in tomato resulted in inhibition of ethylene synthesis and ripening.One shared and major QTL on chromosome 2 was identified by high resolution genetic mapping of loci controlling SSC and carotenoids(lycopene and ?-carotene)content based on 103 watermelon RILs population from a cross between 97103 and PI296341-FR.Genes underlying this QTL were screened by combined genome annotation and comparative fruit transcriptome profiling analysis,and one candidate ERF gene was identified and named ClERF069.qRT-PCR analysis in flesh of RILs revealed that lycopene content and SSC were both negatively correlated with ClERF069 expressions.Based on these findings,the in vivo function of ClERF069 was analyzed by Agrobacterium-mediated transformation of tomato M82 with CaMV35 S :ClERF069.And over-expression of ClERF069 in tomato resulted in delayed ripening,accompanied by decreased ethylene production.Taken together,watermelon ERF gene ClERF069 may involved in ripening as a negative regulator mediated by or partly by exerting negative feedback control over ethylene synthesis.