Functional Research Of SlMBP21 In Tomato Sepal Development And Fruit Ripening

The MADS-box gene family is important for plant development and involved in numerous aspects of plant development,such as root architecture,leaf morphology,flower identity,fruit ripening,seed maturation,abscission zone formation and so on.In tomato,the SEP subclade genes of MADS-box family include TM5,TM29,RIN,MADS1 and SlMBP21.The first four genes have been reported to play roles in flower or fruit development,while the function of MADS box gene SlMBP21 was still not clear.Hence,the function of SlMBP21 was firstly analyzed by bioinformatics analysis.Subsequently,we constructed the Sl MBP21-RNAi transgenic plant and the function of SlMBP21 in sepal development and fruit ripening was further identified in molecular level,cellular level,physiological and biochemistry level.Following are the main results of our work:(1)Phylogenetic analysis revealed that RIN,MADS1 and SlMBP21 were closest in genetic relationship and SlMBP21 might be involved in fruit ripening.The analysis of protein structure showed that SlMBP21 included Keratin-like domain and might be important for the protein complex forming.The analysis of cis-element in promoter suggested SlMBP21 promoter contains the elements related to reponse to abscisic acid,jasmonic acid,gibberellin,auxin and stress.It can be seen that SlMBP21 was involoved in these hormones signalling and plant stress risistance.(2)SlMBP21 may regulate cell expansion to control sepal size,and ethylene and auxin mediate this process.1)According to the phenotypes and statistical analysis,we foud the sepal became longer in SlMBP21-RNAi plant,compared with wild type.Tissue analysis indicated that the SlMBP21-RNAi epidermal cell was larger than wild type.Meanwhile,RNA-sequencing results revealed that the expression of the genes associated with cell expansion was affected in SlMBP21-RNAi sepal.Hence,we inferred that SlMBP21 regulated cell expansion to control sepal size.2)Statistical analysis showed that the fruit set was improved in SlMBP21-RNAi plant.3)GC and HPLC-MS analysis indicated that the ethylene production was lower and auxin content was decreased in SlMBP21-RNAi sepal.ACC and NPA treatments further revealed that SlMBP21 was involoved in ethylene and auxin pathway.Moreover,the expression of genes related to ethylene and auxin was influenced in SlMBP21-RNAi sepal.Ethylene and auxin are important factors for cell expansion regulation.Hence,ethylene and auxin may mediate the regulation of SlMBP21 to cell expansion.4)Additionally,the content of chlorophyll and the activity of ribulose-1,5-bisphosphate carboxylase/oxygenase,the key photosynthetic enzyme,were both increased in SlMBP21-RNAi sepal,which indicated the photosynthesis might be enhanced in transgenic longer sepal.Therefore,the longer sepal,with better protection and enhanced photosynthesis,may contribute to improve fruit set.(3)SlMBP21 may directly regulate ACO1 to influence ethylene biosynthesis and fruit ripening.1)During fruit ripening,the color shift of SlMBP21-RNAi fruit was obviously slower than of wild type.The hue angle was higher and total carotenoid content was decreased in SlMBP21-RNAi fruit,compared with wild type.Real time-qPCR result indicated the expression of PSY1 and β-LCY2,the key enzymes involoved in carotenoid metabolism,were altered in SlMBP21-RNAi fruit.It can be seen that downregulation of SlMBP21 affected PSY1 and β-LCY2 expression,which influenced the color shift.2)According to GC analysis,the ethylene production was lower in SlMBP21-RNAi fruit.Moreover,the expression of ACS2,ACO1,E4,E8,ETR2 and EIL4 were affected.Meanwhile,ethephon treatment could recover the color shift and the inhibition of 1-MCP was stronger in SlMBP21-RNAi fruit,suggesting lower endogenous ethylene level was contributed to the delayed color shift.We inferred that SlMBP21 was involved in ethylene pathway and act at the biosynthesis stage.3)Furthermore,SlMBP21 could activate the promoter of ACO1 as revealed by the yeast one-hybrid assay and dual-luciferase transient expression assay.4)Moreover,the expression of the ripening-related key regulators,RIN,NOR,AP2 a and ERF.B3,is obviously modulated at breaker stage in SlMBP21-RNAi fruit,suggesting SlMBP21 may interact with these factors in a complex network to regulate ethylene level,pigment metabolism and fruit ripening.In conclusion,our study identified the function and mechanism of SlMBP21 in sepal size and fruit ripening,which may provide potential application value for controlling fruit ripening and improving fruit set.