Molecular Mechanism Of MdWRKY126 In Regulating Organic Acid And Sugar Content In Apple Fruit

Flavor is a crucial component of fruit quality,which is mainly determined by the content and ratio of soluble sugar and organic acid.It is of great significance to analyze the molecular regulation mechanism of fruit sugar and acid and explore the relationship between acid and sugar in fruit to improve fruit flavor and quality.The regulation of fruit acidity by Ma1 at Ma locus,the quantitative trait locus of malate in apple fruit,accounted for 17%-42%of fruit acidity.In general,apples with genotype ma1/ma1 have significantly lower acidity at ripening than those with genotype Ma1/Ma1 or Ma1/ma1.In previous studies,we found that‘BSKP’ and ‘AF’,both of the ma1ma1 genotypes,varied greatly in fruit acidity,and identified a candidate gene MdWRKY126 involved in the acidity regulation of apple fruit via transcriptomic and gene expression analysis.The present study revealed that MdWRKY126 regulated the expression of cytoplasmic malate dehydrogenase gene MdcyMDH5,thus affecting the transcriptional regulation mechanism of apple fruit acidity by bioinformatics analysis and molecular methods,and explored the metabolic regulatory network of the effect of acid changes induced by cytoplasmic malate dehydrogenase on sugar in fruit based on the changes of sugar content in MdWRKY126 and MdcyMDH5 overexpressed apple calli and MdcyMDH1 overexpressed apple fruit,for further analyzing the molecular regulation mechanism of fruit sugar and acid and providing new insights and theoretical guidance to improve apple fruit quality.The main results are as follows:1.Transcription factor MdWRKY126 regulates the organic acid accumulation in apple fruit.The determination of malate content and MdWRKY126 expression level in fruits of‘BSKP’ and ‘AF’ showed that the expression of MdWRKY126 was positively correlated with malate content in fruit.Phylogenetic analysis showed that MdWRKY126 was a homologue of Ph PH3,a transcription factor regulating acidity,reported in Petunia.MdWRKY126 overexpression and interference vectors were constructed and transformed respectively into‘Orin’ apple calli,‘Micro Tom’ tomato and instantaneously transformed into ’Fuji’ apple fruit by Agrobacterium mediated method.Compared with the control,the overexpression of MdWRKY126 significantly increased the content of malate in apple calli and fruit,while the silencing of MdWRKY126 inhibited the accumulation of organic acid in apple calli.The heterologous overexpression of MdWRKY126 in tomato also promoted the accumulation of malate and citrate in tomato fruit.These results indicated that transcription factor MdWRKY126 regulates the accumulation of apple fruit acidity.2.The cytoplasmic malate dehydrogenase gene MdcyMDH5 regulated by transcription factor MdWRKY126 was involved in the malate accumulation of apple fruit.The present study analyzed the relationship between MdWRKY126 and apple acidity related proton pump genes Md PH1 and Md PH5 based on the research progress of petunia and other species,and there was no direct interaction were observed.In order to explore the acidity regulation genes downstream of transcription factor MdWRKY126,the expression levels of malate synthesis and transport related genes in MdWRKY126 overexpressed apple calli were analyzed.The results showed that the m RNA expression levels of 3 malate dehydrogenase,5transporter and 3 proton pump genes in MdWRKY126 transgenic lines were higher than those in the control.We then examined the expression levels of the 11 malate related genes in the‘AF’ and ‘BSKP’ apples and the expression levels of six genes(MdcyMDH5,MdcyMDH1,Md MDH18,Md TDT1,MDVHA-A3-1 and Md AVP3)were high expression in ‘BSKP’ fruit and low expression in ‘AF’ fruit just like MdWRKY126.Cis-acting element prediction results showed that the promoters of MdcyMDH5,MdcyMDH1 and Md AVP3 contained cis-acting element W-box which were bound by WRKY transcription factor.It was confirmed that MdWRKY126 could directly bind the promoter of MdcyMDH5 gene and activate its transcription expression by Ch IP-PCR,LUC,Y1 H and GUS activity assay.In addition,overexpression of MdcyMDH5 increased the activity of MDH,and significantly increased the malate content in apple calli and fruit.The Md MDH5 were silenced by virus-induced gene silencing(VIGS)technique in MdWRKY126 overexpressed transgenic material,and the modification of malate content in MdWRKY126 transgenic material was restored.These results suggested that MdcyMDH5,as a direct downstream functional gene of MdWRKY126,was involved in the accumulation of malate in apple calli and fruit.3.The cytoplasmic malate dehydrogenase gene MdcyMDH regulates sucrose accumulation in apple.The present study found that malate accumulation was both increased in MdcyMDH5 and MdWRKY126 transgenic calli and fruit,and sucrose content was also significantly increased.Meanwhile,we also verified that the overexpression of another malate dehydrogenase gene MdcyMDH1 could increase the content of malate and sucrose in apple fruit.To further explore how the accumulation of malate in the cytoplasm altered sucrose accumulation in apple fruit,we analyzed the differentially expressed genes(DEGs)in the transcriptome of wild-type(WT)and MdcyMDH1 overexpressed mature fruit,and found that the expression levels of sucrose-6-phosphate synthase(SPS)genes in transgenic apple were significantly increased and the SPS activity was also significantly higher than that of the control.Similarly,the overexpression of MdcyMDH5 and MdWRKY126 also increased the expression levels of Md SPSs and SPS activity in apple calli,which might be related to the increased sucrose content in transgenic materials.In order to verify the importance of Md SPSs in the regulation of the cytoplasmic malate dehydrogenase gene on the sucrose content of apple fruit,the Md SPSB2 and Md SPSC2 were silenced by VIGS in MdcyMDH1 overexpressed apple fruit,and the modification of sucrose content in MdcyMDH1 transgenic material was restored.In addition,some significantly up-regulated starch cleavage related genes and gluconeogenesis related genes were found in DEGs of MdcyMDH1 overexpressed fruit.These results suggested that malate dehydrogenase might indirectly regulate SPS activity and related gene expression by affecting starch metabolism or gluconeogenesis pathway,and thus affect sucrose metabolism and fruit sugar content.