Advances in research of malate metabolism and regulation in fruit of horticultural crops

Abstract：Acidity is an important part of the sensory quality of fruit. Malic acid is the main organic acid in ripe fruits of tomatoes, apples, pears, and jujubes. Malic acid not only determines fruit acidity and quality but also has multiple important functions in the plant. Malate is well known as a key intermediate in the tricarboxylic acid (TCA) cycle and is imported into mitochondria as a respiratory substrate. Malate also participates the glyoxalate cycle pathway and is closely related to plant primary metabolism, carbon cycling, and carbohydrate accumulation. Malate plays an important role in regulating the osmotic potential, pH balance, and stress resistance in horticultural crops. Thus, it is of important theoretical significance and practical value for high- quality breeding programs as well as the study of the mechanism underlying malic acid biosynthesis and transport in fruits. Malic acid is synthesized in the cytoplasm, accumulated in the vacuole during the early stages of fruit development, and used as a respiratory substrate during fruit ripening. Malate accumulation is affected by synthesis, transport, and metabolism, and involves the participation of numerous catalytic enzymes. Malate metabolism is a complex biological system influenced not only by genetic factors but also by environmental factors, agronomic practices, and post-harvest treatments. In the cytoplasm of fruit, glycogen is converted to phosphoenolpyruvate (PEP) through the glycolytic pathway. PEP is carboxylated by phosphoenolpyruvate carboxylase (PEPC) to produce oxaloacetate (OAA), which is the first step of malic acid synthesis. Then, malate synthesis is catalyzed by cytosolic NAD-dependent malate dehydrogenase (cyMDH) and cytosolic NADP- dependent malic enzyme (cyME). The cyMDH is a key enzyme involved in malate synthesis and catalyzes the conversion reaction from OAA to malate, while cyME is an important malate-degrading enzyme that catalyzes the conversion of malate to pyruvate in the cytoplasm. In addition，malate accumulation is regulated by transmembrane transport between the vacuole and cytoplasm. The transmembrane transport of malic acid requires not only a proton pump to provide energy but also the assistance of channel proteins or transmembrane transporters. The main vacuolar transporters, such as the tonoplast-localized malate transporter (tDT) and aluminum- activated malate transporter (ALMT), participate in the transmembrane transport and accumulation of malate in the fruit. Among the ALMT family members, ALMT9 is the most widely studied gene. Apple Ma1 gene is a key malate transporter responsible for differences in malic acid content between wild and cultivated fruits. SlALMT9 is considered to be responsible for variation in malate content in the fruit among tomato genotypes. VvALMT9, a homolog of AtALMT9 in grapes, is a vacuolar malate channel that mediates the accumulation of malate and tartrate in the vacuoles of grape berries. Tonoplast proton pumps such as vacuolar-type H⁺-ATPase (V-ATPase, VHA), vacuolar-type H⁺-pumping pyrophosphatase (V-PPase, VHP), and P-ATPase (PHA) generate the driving force for vacuolar acidification by transporting protons across the membrane into the vacuole. In petunia flowers, the P-type proton pump genes PhPH1 and PhPH5 interact with each other and form a complex to promote vacuolar acidification. MdPH1 and MdPH5, homologs of PhPH1 and PhPH5 in apples, have been identified and shown to be involved in vacuolar acidification and malate accumulation. Another P-type proton pump gene Ma10 in apples was found to be significantly correlated with malic acid accumulation, explaining about 8% of the variation in fruit acidity phenotypes in natural apple populations. Increasing evidences showed that transcription factors, such as MYB, bHLH, WRKY, and ERF family members, participate in the regulation of malate transporters and proton pumps. In apples, MdMYB1, MdMYB44, and MdMYB73 regulate malate accumulation and vacuolar acidification in fruits by activating or repressing the promoter activities of the malate transporter and proton pump genes. Apart from MYB transcription factors, other transcription factors, such as bHLH and WRKY, are also involved in the regulation of malic acid accumulation and vacuolar acidification. In petunia, AN1 (bHLH transcription factor) can form a complex with AN11-PH4 to positively regulate vacuolar acidification and thus affects pH. In apples, MdbHLH3, a homolog of AN1 regulates malate accumulation in fruit by directly activating the expression of the malate dehydrogenase gene MdcyMDH. MdbHLH3 forms a complex with MdMYB1 to promote pulp anthocyanin and malate accumulation. In tomatoes, SlWRKY42 directly binds to the promoter of SlALMT9, repressing its transcription, and thereby inhibiting malate accumulation in tomato fruit. ZjWRKY7 transcription factor activates the transcription of ZjALMT4 by the W-box region of the high-acidity genotype in sour jujube, thereby promoting malate accumulation, whereas the binding ability was weakened in jujube. This paper summarizes the mechanism of malate accumulation in horticultural crops, such as tomato, apple, pear, and jujube, and provides an overview of the role of transporters, proton pumps, and upstream transcription factors responsible for malate accumulation and vacuolar acidification, which will provide a theoretical basis for quality breeding in horticultural crops.