Research On The Regulation Of Cucumber Ethylene-responsive Factors ERFs Participate In The Sex Determination Process

Cucumber is one of the most important vegetable crops cultivated and consumed over the world.Due to the diversity of floral sexual differentiation,cucumber has become a model crop for studying plant sexual determination.At present,there are five major genes related to sex types in cucumber,namely F(CSACS1G),M(CSACS2),A(CSACS11),CsACO2 and CsWIP1.Except for WIP1 as a transcription factor,the other four genes are all the members of the key enzyme family of ethylene synthesis pathway,and genetic tests have confirmed that there ara interactions between these five genes.Therefore,the research on the regulatory relationship between these key genes of sexual determination has become the starting point of this study.In this study,RNA-seq was performed using F and CsACS2(M)mutant materials.From the differentially expressed genes,four ERFs(ethylene response factor)probably involved in ethylene signaling pathway were identified as CsERF110,CsERF113,CsERF1b and CsERF1b-like.The possible regulatory functions of these four transcription factors in sexual determination were studied,and the main results were as follows:1.The expression of CsERF110,CsERF113,CsERF1b and CsERF1b-like were up-regulated after ethephon treatment,while the key B/E-type genes in cucumber male flower development were down-regulated.After treatment with ethylene synthesis inhibitor and ethylene response inhibitor,the expression of all four ERFs genes were significantly down-regulated,while these B/E-type genes were significantly up-regulated.This opposite expression trend between ERFs and B/E-type genes suggested that ethylene may regulate male flower development through these four ERFs transcription factors.Transgenic arabidopsis with the overexpression of CsERF110,CsERF113,CsERF1b or CsERF1b-like all showed typical “triple response” of ethylene,which confirmed their role as the components in ethylene response pathway.2.Both CsERF110 and CsERF113 can bind to the promoter of A gene and activate its transcription expression;CsERF113 also can bind to the CsERF110 promoter and activate its transcription.Protein interaction of CsERF110 and CsERF113 can enhance the transcriptional activation to A gene,which showed that these two ERFs co-regulate the cucumber sex determination gene A.In addition,it was found that CsERF110 can bind to the ERE element in the promoter of CsAP3 and inhibit its transcriptional expression.This finding provide evidence to the phenomenon that ethylene inhibits males,and help to link floral organ development to the process of sexual determining.3.Promoter fragment of CsPI was used to screen yeast library and CsERF was found can combine CsPI promoter sequence.Previous results have confirmed the transcription response of CsERF1b to ethylene and the correlation between the expression of CsERF1b and cucumber female performance.Further studies showed that CsERF1b inhibited the transcription of CsPI,which confirmed the opposite expression trend of these two genes in sexual mutants.CsERF1b also activated CsERF110 transcription by combining GCC-box element in its promoter.CsERF1b-like had previously been reported to directly bind ERE elements in the promoter of M gene and activate its transcription.In addition,it was found that CsERF1b-like could directly bind CsWIP1 promoter to inhibit its transcription,so CsERF1b-like may be involved in the process of sexual determination.These evidences provided further theoretical support for the regulation of key ERFs to sexual determining genes and the inhibition of stamen development mediated by ethylene.4.The amino acid sequence of ERF110 in watermelon,cucumber and melon were highly conserved,and we overexpressed CsERF110 in watermelon to analyze its functional conservatism in cucurbitaceae crops.Phenotypic analysis showed that the internodes of the transgenic plants were shortened,and most of the flowers failed to open and had faded prematurely.The results of tissue sections showed that pollen development was not affected at the early stage and pollen morphology was deformed at the later stage.The results of pollen staining showed that the pollen fertility was reduced and the morphology was deformed.The ethylene release of the top bud of transgenic plants was measured,and it was found that the ethylene release of plants was increased.Transcriptomic sequencing was performed on the top buds of transgenic plants,and it was found that ethylene synthesis related genes including Cl ACS11 were up-regulated,while B/E genes AP3,PI,SEP1,SEP3 and SEP4 were significantly down-regulated,which was consistent with the expression trend after ethephon in cucumber.In addition,genes related to pollen development were found to be significantly down-regulated in the transcriptome,such as MS1,AMS,DYT1,MS188,CYP704B1.Therefore,it is speculated that the increase of ethylene content after CsERF110 overexpression may be caused by the up-regulated expression of genes related to ethylene synthesis,which further affects plant height,development of pollen and flower organs.In conclusion,CsERF110,CsERF113,CsERF1b and CsERF1b-like mediate the regulation among cucumber sexual determining genes,and the link between cucumber floral organ development and ethylene-mediated sex determination could be established through these key ERFs.CsERF110 is not only involved in the regulation of sex determination,but also may play a regulator in the flower organ development after sex determination period,including pollen maturation and anther development.