Functional Analysis And Fruit-ripening Molecular Mechanism Of SlFIE In Tomato

The control of fruit ripening is of great significance to the quality and shelf life for the harvested fruits.It is reported that fruit ripening is a complicated process and finely regulated by many factors,such as transcription factors,epigenetic modification,microRNA.Fleshy fruits are a large part of fruit crops,which can be classified into two groups,climacteric and non-climacteric fruits,by whether they show a rapid rise in respiration and a burst of ethylene production at the onset of the ripening process.As the second-largest fruit and vegetable crop in the world,the tomato is one of the model plants as well,which is a perfect research material for the development and quality of climacteric fruit and molecular breeding.According to our previous work,we have found that SlDDB1(UV-damaged DNA binding protein 1)plays an important role in pigment accumulation and fruit ripening in tomato,while its defective mutant(hp1)delays fruit ripening,but the specific mechanism is unknown.A protein containing WD40 domain was predicted by bioinformatics as a tomato(Solanum lycopersicum)homolog of AtFIE in Arabidopsis thaliana,termed as SlFIE(fertilization-independent endosperm).The expression of SlFIE was more abundant in seeds and fruit at Breaker and the later stage by the expression pattern analysis of different tomato tissues.Subcellular assay indicated that SlFIE was localized in the nucleus and cytoplasm.Thus,transgenic lines of SlFIE overexpression(SlFIE-OE),gene knockout(SlFIE-CRISPR)and fruit-specific RNA interference(SlFIE-RNAi)were obtained by genetic transformation,respectively,for the functional characterization.Compared to the wild type,the time of SlFIE-OE fruits ripening was accelerated by 3days,and the post-harvest experiments of SlFIE-OE fruits at the MG(mature green)stage with or without ethylene treatment also showed the similar phenotype.And the expression level of ripening-related genes(RIN,NOR,CNR,TAGL1)and ethylene-synthesis-related genes(SlACS2,SlACS4)were significantly increased in SlFIE-OE fruit.In contrast,the time of SlFIE-CRISPR fruits ripening was delayed by 12 days.The post-harvest experiments of SlFIE-CRISPR fruits at the MG stage with or without ethylene treatment also showed the similar phenotype,and ethylene treatment shortened the ripening time of SlFIE-CRISPR fruits.In addition,the development of SlFIE-CRISPR mutant plants was stunted,and the flowering time of SlFIE-CRISPR was delayed by 56 days when compared to the wild type.The number of seeds in fruit of SlFIE-OE and SlFIE-CRISPR was decreased significantly.In addition,the weight of SlFIE-OE seeds was decreased,by contrast,that of SlFIE-CRISPR seeds was increased.In order to further explore the effect of SlFIE on tomato fruit ripening,SlFIE RNA interference(SlFIE-RNAi)plants driven by the fruit-specific promoter of TFM7 were obtained.SlFIE-RNAi fruits also showed a ripening-delay phenotype,and the expression level of ripening-related genes(RIN,NOR,CNR,TAGL1)and ethylene-synthesis-related genes(SlACS2,SlACS4)were significantly decreased in SlFIE-RNAi fruit at Breaker stage.In conclusion,SlFIE participates in the development of tomato plants,and positively regulates the fruit ripening process.In this and our previous studies,we have found both SlFIE and SlDDB1 involve fruit ripening regulation.To elucidate the underlying mechanism,co-immunoprecipitation assay was used to detect the interaction between SlFIE and SlDDB1.The results showed that SlFIE was a component of SlCul4-SlDDB1 complex,which is a an E3 ubiquitin ligase complex that mediates the ubiquitination and degradation of target proteins in eukaryotes.Meanwhile,SlHDA1 and SlHDA3 were previously proved to be involved in fruit ripening in tomato.In vivo,SlHDA1 and SlHDA3 were degraded by ubiquitinmediated to varying degrees,which was indicated that these two histone deacetylases could be degraded and inactivated by ubiquitination.The co-immunoprecipitation assay showed the interaction between SlHDA1 or SlHDA3 and SlFIE,and the degradation of SlHDA1 and SlHDA3 by the SlCul4-SlDDB1-SlFIE complex was verified by cell-free and co-immunoprecipitation experiments.Therefore,SlFIE,as a component of the SlCul4-SlDDB1 E3 ligase complex,targets SlHDA1 and SlHDA3 for ubiquitination and degradation,which subsequently influences the fruit ripening.Taken together,the functional characterization of SlFIE in tomato,an SlDDB1 interacting protein,was conducted by using genetics,molecular biology,and biochemistry approaches.These results provide an important dataset for understanding the molecular mechanism of fruit ripening and promoting molecular breeding for the crop improvement.