Mechanism Of CmMYBs Regulating Sucrose Accumulation In Oriental Melon Fruit

The oriental melon(Cucumis melo var.makuwa Makino)is one of the main protected cultivation melons in Northeast and North China.It is loved by consumers due to its unique aroma and sweet taste.Sugar content is one of the important indicators of fruit flavor quality,and its level directly affects the formation of quality.Ethylene is an important plant hormone,it can regulate climacteric fruit ripening and quality formation.However,the mechanism of ethylene in sucrose accumulation in melon fruit is unclear.Therefore,it is important to clarify how ethylene participates in regulating sucrose accumulation to improve fruit flavor and quality.In this study,the high sugar variety ‘HS’ and the low sugar variety ‘LW’ melon fruit was used as experimental materials.By measuring the soluble sugar(glucose,fructose,and sucrose)content,ethylene production,sugar metabolism related enzyme activity and related gene expression,it is determined that Cm SPS1 and Cm ACO1 are key genes for sucrose accumulation and ethylene synthesis.The important roles of Cm SPSP1 and Cm ACO1 in sucrose accumulation and ethylene synthesis in melon fruit were further verified by agrobacterium-mediated transient transformation technology.Using the promoter of Cm SPS1 as bait,the melon fruit yeast single hybrid c DNA library was screened and two MYB transcription factors,Cm MYB44 and Cm MYB113,were obtained.Through transcriptional activation testing and expression analysis,it is confirmed that Cm MYB44 is a transcriptional inhibitor and Cm MYB113 is a transcriptional activator.Yeast single hybrid(Y1H),EMSA,luciferase report test and GUS activity analysis proved that Cm MYB44 can directly bind to the promoter of Cm SPS1 and Cm ACO1 to negatively regulate their expression.While Cm MYB113 can directly bind to the promoter of Cm SPS1 and Cm ACO1 to positively regulate their expression.In addition,Y1 H combined with luciferase report test and GUS activity analysis demonstrated that Cm ERFI-2 negatively regulates Cm MYB44,thereby weakening the inhibitory effect of Cm MYB44 on sucrose accumulation and ethylene synthesis.Yeast two hybrid and firefly luciferase experiments have demonstrated that there is a protein level interaction between Cm MYB113 and Cm MADS26,which further regulates sugar accumulation in melon fruit.The main findings are as follows:1.By measuring the soluble solids and soluble sugar content of high sugar variety ‘HS’and the low sugar variety ‘LW’ at different development stages,it was found that the soluble solids and sucrose content in the mature ‘HS’ fruit were significantly higher than those of the‘LW’ fruit,while the glucose and fructose content had no significant difference between the two varieties,indicating that the high and low sucrose content was the main reason for the difference in sugar content between the two varieties.2.The activities of enzymes related to sugar metabolism in two oriental melon varieties at different development stages were measured.It was found that the activity of sucrose phosphate synthase(SPS)was significantly higher in mature ‘HS’ fruit than in ‘LW’ fruit.Using q RT-PCR detection,it was found that the expression of Cm SPS1 gene in ‘HS’ fruit was significantly higher than that in ‘LW’ fruit,and the expression trend of the gene was significantly positively correlated with sucrose content.In order to clarify the function of Cm SPS1 in sucrose accumulation in melon fruit,Agrobacterium-mediated gene transient overexpression and silencing were conducted in ‘HS’ and ‘LW’ fruit.It was found that the sucrose content in melon fruit overexpressing Cm SPS1 gene was significantly higher than that in the empty control,while the sucrose content in melon fruit silencing Cm SPS1 gene was significantly lower than that in the control.The above results indicated that SPS and Cm SPS1 played an important role in sucrose accumulation of oriental melon fruit.3.The ethylene production of two melon varieties during fruit ripening was measured.It was found that ‘HS’ fruit had a significant ethylene release peak at 33 days after anthesis,while ‘LW’ fruit had no significant ethylene release peak during the whole development process,which may be a non-climacteric fruit.Further,q RT-PCR was used to detect the expression levels of ACC synthase(ACS)and ACC oxidase(ACO)gene family members in the ethylene synthesis pathway.It was found that Cm ACO1/5/6 and Cm ACS1/11/12 were differentially expressed in the two varieties,and their expression in ‘HS’ was higher than that in ‘LW’.Through instantaneously overexpressing and silencing the Cm ACO1 gene in ‘HS’and ‘LW’ fruit,the results showed that Cm ACO1 is an important gene in ethylene synthesis of melon fruit.4.Using the Cm SPS1 promoter as bait,a melon fruit yeast single hybrid c DNA library was screened and Cm MYB44 was selected.Both agrobacterium-mediated transient transformation and stable genetic techniques have demonstrated that Cm MYB44 can inhibit ethylene synthesis and sucrose accumulation in melon fruit.Further analysis of Y1 H,EMSA,luciferase report test and GUS activity showed that Cm MYB44 could directly bind to the promoter of Cm SPS1 and Cm ACO1 and negatively regulated their expression.Then,the upstream regulatory factor Cm ERFI-2 of Cm MYB44 was found through screening yeast single hybrid library.This ethylene response factor attenuated the inhibitory effect of Cm MYB44 on downstream target genes Cm SPS1 and Cm ACO1 through transcriptional inhibition of Cm MYB44,thereby promoting sucrose accumulation and ethylene synthesis in oriental melon fruit.5.Two oriental melon varieties harvested at 28 days after anthesis were treated with exogenous ethylene and 1-MCP to determine soluble sugar content,ethylene production,sugar metabolism related enzyme activity,and related gene expression.The results showed that ethylene could induce the expression of Cm SPS1 and Cm ACO1 to promote sucrose accumulation and ethylene synthesis in mature ‘HS’ melon fruit,while exogenous ethylene had no significant effect on sucrose accumulation and ethylene synthesis in ‘LW’ fruit.Using the Cm SPS1 promoter as bait,a yeast single hybrid c DNA library was conducted to obtain the transcription factor Cm MYB113.Yeast single hybrid combined with GUS activity analysis demonstrated that Cm MYB113 could directly bind to the Cm SPS1 and Cm ACO1 promoters to upregulate their expression.Agrobacterium-mediated transient transformation and transgenic technology demonstrated that Cm MYB113 was actively regulating sucrose accumulation and ethylene synthesis in melon fruit.Further,yeast two hybrid and firefly luciferase tests demonstrated that Cm MYB113 and Cm MADS26 have protein level interactions,and Cm MADS26 can directly bind to the promoter of Cm ACO1 and regulate its expression.The above results indicate that Cm MYB113 is positively regulating ethylene induced sucrose accumulation in melon fruit,and the interaction between Cm MYB113 and Cm MADS26 further cooperatively regulated sucrose accumulation in oriental melon fruit.In summary,ethylene-induced transcription factor Cm ERFI-2 inhibited the transcription of Cm MYB44 in the high sugar variety ‘HS’,weakening the inhibitory effect of Cm MYB44 on downstream target genes Cm SPS1 and Cm ACO1,thereby promoting sucrose accumulation and ethylene synthesis in the oriental melon fruit.In addition,Cm MYB113 positively regulated ethylene induced sucrose accumulation in melon fruit,and the interaction of Cm MYB113 and Cm MADS26 further regulated sucrose accumulation in oriental melon fruit,improving melon fruit flavor quality.