| The flavour of the fruit is determined by the sugar-to-acid ratio,the size of which depends on the ratio of sweetness to acidity and reflects how tasty the fruit is.Acidity is determined by the total amount of organic acids,of which there are approximately 16 in apples,with malate accounting for around 85% of the total organic acids and being the most dominant acid in the apple fruit.Malate content is influenced by biological processes such as synthesis,metabolism as well as translocation,and is regulated by key enzymes,making it a complex process.However,how transcription factors participate in malate anabolic processes by regulating the expression levels of these enzymes is still unknown.Sweetness is mainly determined by the total amount of soluble sugars.Soluble sugars in apples include sucrose,glucose and fructose,etc.Their content and ratio are important components of fruit quality.However,there is a need to further explore the key genes regulating sugar metabolism,elucidate their specific functions during fruit development,and improve the regulatory network related to sugar metabolism.Although many studies have reported the metabolic processes of sugars and acids and changes in related enzyme activities during fruit development,it is still unclear how transcription factors regulate the expression levels of sugar-and acid-related genes,and further studies are needed to identify the upstream regulators controlling malate and soluble sugar accumulation,and to elucidate the molecular mechanisms of their involvement in sugar and acid accumulation during fruit development.Therefore,this study explored the mechanisms related to the regulation of malate and soluble sugar accumulation by the apple b HLH transcription factor MdbHLH3 during fruit development,using apple fruit as the study material.The main results are as follows:1.MdbHLH3 promotes malate accumulation at mid-development in apples.WT and MdbHLH3 transgenic apple fruits were harvested at different days after bloom(10,20,30,60,90 and 120 DAB),and the anthocyanins content,transverse and vertical diameter,per fruit weight and malate content of the fruits were determined.The results showed that MdbHLH3 significantly promoted the accumulation of anthocyanins in fruits by activating the expression of anthocyanin-related genes.It was further found that overexpression of MdbHLH3 increased malate content in apple fruits at 60 and 90 days after flowering,while there were no significant changes in quality indexes such as transverse and vertical diameter as well as per fruit weight.Further RNA-seq analysis showed that MdbHLH3 activated malate-related gene expression.Analysis of the malate-related gene promoters revealed that MdbHLH3 may interact with MdcyMDH and Md ALMT9.Further,Chromatin immunoprecitation(CHIP-PCR),yeast one-hybrid(Y1H)assays and electrophoresis mobility shift assay(EMSA)experiments confirmed that MdbHLH3 directly binds to the promoter of the MdcyMDH gene.Then the transcriptional activation of the MdcyMDH gene by MdbHLH3 was confirmed by transient dual luciferase analysis,GUS staining of apple calli and recombinant protein abundance assay.Finally,transient injection into apple fruit revealed that MdbHLH3 promotes malate accumulation by positively regulating the expression level of MdcyMDH.2.MdbHLH3 regulates the accumulation of soluble sugars in apple fruits.WT and MdbHLH3 transgenic apple fruits were harvested at different days after bloom(30,60,90,120 and 135 DAB),and the contents of sorbitol,sucrose,glucose and fructose as well as the activities of related sugar metabolizing enzymes were measured in the fruits.The results showed that overexpression of MdbHLH3 promoted the accumulation of sugars in apple fruits by regulating the activity of related sugar metabolizing enzymes.Next,RNA-seq analysis showed that MdbHLH3 activated the expression of sugar-related genes.Further analysis of sugar-related genes by CHIP-PCR,Y1 H assays and EMSA experiments confirmed that MdbHLH3 directly binds to the promoter of a pyrophosphate-dependent phosphofructokinase gene,Md PFP?.Then the transcriptional activation of the Md PFP? gene by MdbHLH3 was confirmed by transient dual luciferase analysis,GUS staining of apple calli,GUS activity assay and recombinant protein abundance assay.Finally,transient injection into apple fruit revealed that MdbHLH3 promoted the accumulation of fructose 6-phosphate as well as sucrose by positively regulating the expression level of Md PFP?.3.Md PFP? regulates the accumulation of sugars in fruits.Overexpression of the Md PFP? gene in tomato plants enhanced photosynthetic carbon assimilation in leaves by affecting photosynthetic efficiency and related fluorescence parameters,significantly increasing the sucrose,fructose,and glucose contents therein.Then,by regulating the activity of sugar metabolizing enzymes as well as carbohydrate partitioning,sucrose,fructose and glucose contents in tomato fruits were also found to be significantly increased.Thus,Md PFP?affects the accumulation of soluble sugars in leaves and fruits by regulating carbohydrate uptake and partitioning.4.The apple b HLH transcription factor MdbHLH3 regulated carbohydrate redistribution.The reducing activity of cy MDH and ch MDH and the oxidizing activity of m MDH were significantly enhanced in MdbHLH3 transgenic apple plants,and the expression levels of the key genes of mitochondrial metabolism,Mdm MDH and Mdm SDH,were elevated,while leaf photosynthesis-related indicators were significantly increased,indicating that MdbHLH3 regulated the metabolism of mitochondria and chloroplasts.Then,it was found that the expression levels of two key genes in the glycoisogenic pathway,Md FBP and Md PEPCK,were significantly elevated,and overexpression of MdbHLH3 enhanced the strength of leaves as a source in the source-library relationship and promoted the accumulation of photosynthetic carbon assimilation products such as starch,sorbitol and sucrose.Further,carbon 13 isotope pulse labeling experiments finally showed that MdbHLH3 regulated carbohydrate uptake,distribution and conversion and promoted the accumulation of soluble sugars in fruits.In this study,the MdbHLH3-MdcyMDH and MdbHLH3-Md PFP? regulatory modules were combined with Md PFP? gene function and changes in physiological indicators to resolve the mechanisms involved in the regulation of malate and sugar accumulation by transcription factors and key genes,providing an important reference for the crosstalk between sugar and acid metabolism in plants and a new idea for the regulation of sugar and acid quality in horticultural crops.This study not only improved the complex regulatory network of malate and sugar accumulation,but also showed that transcription factors and key genes are involved in the regulation of source-library relationship,which is important for guiding horticultural crop production and variety improvement. |
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