Transcription Factor CitZAT5 Modifies Hexose Accumulation In Citrus Fruit

Sugars are fundamental substances for plant developmental processes and important indicators in fruit flavor.Citrus(Citrus)is one of the most important cultivated fruit crops in the world,whose quality is largely influenced by the proportion and content of sugars.Sugar accumulation in fruit is usually decided by the joint cooperation of sugar synthesis,transport and metabolism.Molecular research exploring the mechanism of sugar transport and metabolism could provide a deep understanding of sugar accumulation,which is,therefore,critical for the formation of fruit flavor.However,the mechanism of sugar accumulation in citrus fruit has not been deeply studied,especially at the level of transcriptional regulation.In the present study,‘Gongchuan’ and its bud sport ‘Youliang’ were used to study the mechanism of sugar metabolism and accumulation.The main findings are as follows:1.Differential soluble sugar metabolism and accumulation between ‘GC’ and‘YL’ developmental fruit.Sugar content was analyzed in ‘GC’ and ‘YL’ fruit development stage.A significantly higher hexose content was observed in ‘YL’ fruit than that in ‘GC’ fruit.These findings suggested that the difference in sweetness between mature fruits of ‘Gongchuan’ and its bud sport ‘Youliang’ is related to hexose contents.We compared the transcriptome data of ‘GC’ and ‘YL’ fruit and identified two crucially differentially expressed genes,a Su S(Sucrose Synthase)gene Cit SUS5 and a SWEET(Sugars Will Eventually be Exported Transporter)gene Cit SWEET6.2.Cit SUS5/Cit SWEET6 could promote hexose/fructose accumulation in citrus fruit.The expression level of Cit SUS5 and the Su S cleavage activity were significantly higher in ‘YL’ fruit.Transient overexpression of Cit SUS5 significantly increased the hexose content in citrus fruit.Correspondingly,the silencing of Cit SUS5 significantly reduced the hexose content of citrus fruit.Our study indicated that Cit SUS5 could promote the cleavage of sucrose to hexoses.Cit SWEET6 was highly expressed in ‘YL’ fruit.Heterologous expression of Cit SWEET6 in mutant yeast strains showed that Cit SWEET6 was a fructose transporter.Overexpression of Cit SWEET6 in citrus fruit resulted in an increase in fructose content.These results confirmed that Cit SWEET6 contributed to fructose accumulation in citrus fruit.3.Transcription factor Cit ZAT5 regulated Cit SUS5 and Cit SWEET6 expression to regulate the accumulation and composition of sugars in fruit maturing.A C2H2 transcription factor Cit ZAT5 was dug by targeting Cit SUS5 and Cit SWEET6.Cit ZAT5 was observed to be more highly expressed in ‘YL’ fruit than that in ‘GC’ fruit.In combination with dual luciferase assays,yeast one-hybrid,EMSA,yeast two-hybrid assays,Cit ZAT5 could positively regulate Cit SUS5 and Cit SWEET6 by interacting with Cit NAC47 or directly combining,thereby contribute to sucrose metabolism and fructose transportation.Furthermore,the role of Cit ZAT5 in sugar accumulation and hexose proportion was investigated by transient overexpression,VIGS and RNAi interference techniques of Cit ZAT5.In summary,higher expression level of Cit ZAT5 promoted the transcription of Cit SUS5 and Cit SWEET6,leading to the increase of hexose content in fruit maturing.This study confirmed the participation of Cit ZAT5 in regulating sugar accumulation and composition in developing fruit,and explained the molecular mechanism of different soluble sugar accumulation in ‘YL’ and ‘GC’ fruit.