Regulation Mechanisms Of Malate Content And Malate Transporter Gene MdALMT9 In Postharvest Apple Fruit

Apple is one of the important cultivated fruit trees in northern China,which has a high economic quality.The intrinsic quality of apple fruit measures its commercial quality and organic acid is one of the most important indexes to determine the flavor and quality of apple fruit.During the postharvest storage of apples,the organic acid content gradually decreased,resulting in a lighter fruit flavor in the later storage periods,and finally led to great reduce in storage quality of the apple fruits.Malate is the main organic acid in apple fruit.Up to now,the relevant researches have focused on the genetic and molecular mechanisms of malic acid accumulation in the process of apple development,and less researches have been conducted on the metabolism and regulation of malic acid during postharvest storage.The aluminum-induced malate transporter gene MdALMT9 is thought to play a key role in the formation of apple fruit acidity.It is mainly responsible for transporting malic acid into the vacuoles of pulp cells and promoting the accumulation of malic acid.However,there are few reports on the regulatory mechanism of MdALMT9,and the possible involved metabolic pathways are not clear.In this study,we explored the regulation of malate metabolism in postharvest apple fruit with different varieties（‘Cripps Pink’,‘Fuji’and‘Qinguan’）in response to GABA and Ca Cl₂ treatments and screened the suitable treatment concentrations.Meanwhile,we investigated the characteristics of malate metabolism and its response to ethylene signals in apple fruit during storage.Following the finding that MdALMT9 responded to ethylene induction,the further researches are focused on the functional characterization of MdALMT9 and the regulation mechanisms of MdALMT9 in response to ethylene signals.The main results obtained are as follows:1.10 m M GABA and 4%Ca Cl₂ were screened as suitable concentrations for postharvest treatment and both treatments significantly increased the malate concentration in’Cripps Pink’apples by directly participating in the regulation of malate metabolism.The two treatments remarkably increased the activities of the key enzymes involved in malate synthesis and inhibited the activities of the enzymes dominated malate degradation,along with the upregulated expression of genes related to malate synthesis and downregulated expression of genes related to malate degradation.Additionally,10 m M GABA and 4%Ca Cl₂ treatments significantly inhibited the ethylene production and respiration rate in apple fruits,thereby reducing the consumption of malate as a substrate for respiratory metabolism,and activated the activity of GABA pathway to induce the increase of endogenous GABA content.2. The change patterns of malate content were different in‘Cripps Pink’,‘Fuji’ and‘Qinguan’apples during storage,and ethylene treatment promoted the decline of malate content in all three different apple varieties,while 1-MCP treatment significantly delayed the consumption of malate.In addition,five differentially expressed genes,including NAD-MDH（LOC103405182）,PEPC（LOC103431561）,NADP-MEs（LOC103400711/LOC103417117）,PEPCK（LOC103412658）,involved in malate metabolism in the senescence process of apple fruit during storage were screened by RNA-seq analysis.Overall,ethylene treatment has promoted the expression levels of these genes involved in malate synthesis and degradation,indicating the decrease in malate content mainly caused by the malate degradation during postharvest storage.3. The full-length sequence of MdALMT9 gene and a promoter sequence of about 2,000 bp were cloned from the fruits and leaves of‘Fuji’apple,respectively.The MdALMT9 protein is localized on the vacuolar membrane and has six transmembrane domains.During postharvest storage,ethylene treatment significantly increased the expression of MdALMT9 during ethylene climacteric period,indicating that there was a possible regulatory relationship between ethylene signal and MdALMT9.Overexpression of MdALMT9 in tomato significantly increased the malate and citrate contents in different development stages of tomato fruit,which indicated that MdALMT9 could promote the malate and citrate accumulations by activating the malate and even citrate transport in tomato fruit.Promoter sequence analysis of MdALMT9showed that the promoter sequence contained auxin and ABA response elements and cis-acting elements in response to drought and hypoxia stress.The transient transformation of tomato showed that IAA,ABA and ethylene all enhanced the promoter activity of MdALMT9.4. The expression patterns of Md ERF017 and Md ERF017-like was highly similar to that of MdALMT9 in response to ethylene treatment,suggesting that there might be a regulatory relationship between them.The dual-luciferase assays revealed that Md ERF017 and Md ERF017-like can significantly induce the promoter activity of MdALMT9.Site mutation technology,EMSA and Y1H test showed that Md ERF017can activate promoter activities of MdALMT9 and Md ERF017 by directly combining three different GCC-box elements.Overexpression of Md ERF017 and Md ERF017-like in apple callus significantly promoted the malate accumulation via inducing the upregulated expression of genes involved in malate transport and metabolism and enhanced the resistance of apple callus to drought and Al³⁺stresses,while co-silencing of the two transcription factors induced by VIGS on apple fruit contributed to lower malate content and the downregulated expression levels of genes involved in malate transport and metabolism compared with the control.Further studies showed that Md ERF017 significantly induced the promoter activities of these genes.The above results indicated that ethylene-responsive transcription factors Md ERF017 and Md ERF017-like participate in malate accumulation by regulating the expression of a series of genes related to malate transport and metabolism,including MdALMT9,and this signaling pathway is likely to influence the plant resistance to stresses.In summary,we screened the 10 m M GABA and 4%Ca Cl₂ as suitable concentrations for postharvest treatment and its roles in regulating malate metabolism was identified.Meanwhile,the function of MdALMT9 in malate transport was identified and Md ERF017 and Md ERF017-like were found to be involved in the regulation of malate accumulation by modulating the expression of a series of genes related to malate transport and metabolism,including MdALMT9.Since ethylene treatment promote the decline of malate concentration,which was dominated by malate degradation,it is speculated that the regulation of the two ethylene-responsive transcription factors on malate accumulation is mainly involved in plant resistance to stresses.