Significant variation in organic acid components was detected in mature fruits of 101 apple accessions using high-performance liquid chromatography. The Malus species predominantly accumulated malic acid and citric acid, whereas wild fruits exhibited significantly higher levels of organic acid content than that in cultivated fruits. Differential accumulation patterns during fruit developmental stages was detected between malic acid and citric acid, thus suggesting a complex genetic regulation mechanism of organic acid metabolism in apple fruit. A highly positive correlation was detected between fruit total organic acid content with malic acid and citric acid content, thus suggesting that malic acid and citric acid are the principal determinants of apple fruit acidity. In contrast to malic acid, citric acid was predominantly detected in partial wild apples, while extremely low to undetectable concentrations of citric acid were observed in cultivated apple fruits; this is likely due to the genetic effects of parental characters. Our results provide vital information that could be useful for future studies on genetic analysis and improvement of organic acid accumulation in apple fruits.

The content of organic acids greatly influences the taste and storage life of fleshy fruit. Our current understanding of the molecular mechanism of organic acid accumulation in apple (Malus domestica) fruit focuses on the aluminum-activated malate transporter 9/Ma1 gene. In this study, we identified a candidate gene, MdWRKY126, for controlling fruit acidity independent of Ma1 using homozygous recessive mutants of Ma1, namely Belle de Boskoop “BSKP” and Aifeng “AF.” Analyses of transgenic apple calli and flesh and tomato (Solanum lycopersicum) fruit demonstrated that MdWRKY126 was substantially associated with malate content. MdWRKY126 was directly bound to the promoter of the cytoplasmic NAD-dependent malate dehydrogenase MdMDH5 and promoted its expression, thereby enhancing the malate content of apple fruit. In MdWRKY126 overexpressing calli, the mRNA levels of malate-associated transporters and proton pump genes also significantly increased, which contributed to the transport of malate accumulated in the cytoplasm to the vacuole. These findings demonstrated that MdWRKY126 regulates malate anabolism in the cytoplasm and coordinates the transport between cytoplasm and vacuole to regulate malate accumulation. Our study provides useful information to improve our understanding of the complex mechanism regulating apple fruit acidity.

Auxin plays important roles throughout plant growth and development. However, the mechanisms of auxin regulation of plant structure are poorly understood. In this study, we identified a transcription factor of the BARLEY B RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family in apple (Malus × domestica), MdBPC2. It was highly expressed in dwarf rootstocks and it negatively regulated auxin biosynthesis. Overexpression of MdBPC2 in apple decreased plant height, altered leaf morphology, and inhibited root system development. These phenotypes were due to reduced auxin levels and were restored reversed after exogenous IAA treatment. Silencing of MdBPC2 alone had no obvious phenotypic effect, while silencing both class I and class II BPCs in apple significantly increased auxin content in plants. Biochemical analysis demonstrated that MdBPC2 directly bound to the GAGA-rich element in the promoters of the auxin synthesis genes MdYUC2a and MdYUC6b, inhibiting their transcription and reducing auxin accumulation in MdBPC2 overexpression lines. Further studies established that MdBPC2 interacted with the polycomb group (PcG) protein LIKE HETEROCHROMATIN PROTEIN 1 (LHP1) to inhibit MdYUC2a and MdYUC6b expression via methylation of histone 3 lysine 27 (H3K27me3). Silencing MdLHP1 reversed the negative effect of MdBPC2 on auxin accumulation. Our results reveal a dwarfing mechanism in perennial woody plants involving control of auxin biosynthesis by a BPC transcription factor, suggesting its use for genetic improvement of apple rootstock.

To comprehensively understand the responses of carbohydrate metabolism and transport to different levels of nitrogen supply in growing shoot tips of crabapple (Malus hupehensis Rehd), enzyme activities and related genes involved in the sugar metabolism pathway were investigated. The nitrogen and chlorophyll content of plants increased with increasing nitrogen supply. High nitrogen application increased the net photosynthesis rate and the growth rate of shoot tips but decreased the synthesis capability of sucrose and sorbitol in mature leaves. However, the shoot tips of plants under high-nitrogen treatment had higher contents of sucrose and sorbitol than did those under low-nitrogen treatment, while the activity of sucrose phosphate synthase and aldose-6-phosphate was increased and the transporters MdSOT and MdSUT were up-regulated. Moreover, the activities of enzymes involved in sucrose and hexose metabolism (including sucrose synthase, fructokinase, and hexokinase) were enhanced in the shoot tips of plants under high-nitrogen conditions, and the expression levels of MdSUSY3 and MdHK1 were significantly up-regulated. These findings indicate that a high nitrogen supply increases the metabolic capacity of assimilatory substances in shoot tips, accelerates the efficiency of sugar utilization and eventually leads to a rapid increase in the growth of shoot tips. Our results highlight that high nitrogen increases the capacity of sugar unloading and metabolic utilization in growing shoot tissues.

The types and proportions of soluble sugar and organic acid in fruit significantly affect flavor quality. However, there are few reports on the crosstalk regulation between metabolism of organic acid and sugar in fruit. Here, we found that the overexpression of cytoplasmic malate dehydrogenase genes (MdcyMDHs) not only increased the malate content but also increased the sucrose concentration in transgenic apple calli and mature fruit. Enzyme activity assays indicated that the overexpression of MdcyMDH1 and MdcyMDH5 enhanced sucrose phosphate synthase (SPS) activity in transgenic materials. RNA-seq and expression analysis showed that the expression levels of SPS genes were up-regulated in MdcyMDH1-overexpressed apple fruit and MdcyMDH5-overexpressed apple calli. Further study showed that the inhibition of MdSPSB2 or MdSPSC2 expression in MdcyMDH1 transgenic fruit could reduce or eliminate, respectively, the positive effect of MdcyMDH1 on sucrose accumulation. Moreover, some starch cleavage-related genes (MdBAM6.1/6.2, MdBMY8.1/8.2, MdISA1) and the key gluconeogenesis-related phosphoenolpyruvate carboxykinase MdPEPCK1 gene were significantly up-regulated in the transcriptome differentially expressed genes of mature fruit overexpressing MdcyMDH1. These results indicate that alteration of malate metabolism mediated by MdcyMDH might regulate the expression of MdSPSs and SPS activity via affecting starch metabolism or gluconeogenesis, and thus accelerate sucrose synthesis and accumulation in fruit.