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Summary Changes in carbohydrates and organic acids largely determine the palatability of edible tissues of horticulture crops. Elucidating the potential molecular mechanisms involved in the change in carbohydrates and organic acids, and their temporal and spatial crosstalk are key steps in understanding fruit developmental processes. Here, we used apple (Malus domestica Borkh.) as research materials and found that MdbHLH3, a basic helix–loop–helix transcription factor (bHLH TF), modulates the accumulation of malate and carbohydrates. Biochemical analyses demonstrated that MdbHLH3 directly binds to the promoter of MdcyMDH that encodes an apple cytosolic NAD‐dependent malate dehydrogenase, activating its transcriptional expression, thereby promoting malate accumulation in apple fruits. Additionally, MdbHLH3 overexpression increased the photosynthetic capacity and carbohydrate levels in apple leaves and also enhanced the carbohydrate accumulation in fruits by adjusting carbohydrate allocation from sources to sinks. Overall, our findings provide new insights into the mechanism of how the bHLH TF MdbHLH3 modulates the fruit quality. It directly regulates the expression of cytosolic malate dehydrogenase MdcyMDH to coordinate carbohydrate allocation and malate accumulation in apple.

Malate is the predominant organic acid in peach, contributing significantly to fruit sourness and overall organoleptic quality. However, comprehensive understanding of the molecular mechanisms underlying malate accumulation in response to different storage conditions remains limited. In this study, “Hujingmilu” peach was subjected to room temperature (RT, 25°C), low temperature (LT, 4°C), and LT followed by shelf life (LT‐SL, 25°C) treatments to investigate transcriptional regulation mechanisms underlying malate accumulation, with emphasis on biosynthesis, vacuolar storage, and transcription factor regulation. Results demonstrated that LT storage delayed the decline in fruit firmness and maintained higher malate content compared to RT and LT‐SL. Transcriptomic profiling indicated that the expression patterns of malate biosynthetic genes (PpPEPC1/2, PpNAD‐MDH1/2, PpNADP‐ME1) showed limited alignment with malate accumulation. In contrast, genes implicated in proton pump and malate transporter, such as PpAtpvA1/2/3/4/5, PpVp2, and PptDT1, were significantly upregulated under LT conditions, consistent with the observed malate accumulation. Furthermore, LT storage repressed the malate transcriptional repressor PpTST1 while inducing the candidate regulatory gene PpMYB62. These findings provided a comprehensive molecular framework for understanding malate modulation under varying storage conditions. This study explored malate‐accumulation mechanisms in “Hujingmilu” peaches under different storage conditions. Low temperature (LT) delayed firmness decline and maintained higher malate. Transcriptomics showed malate biosynthetic genes weakly correlate with malate levels, while proton pump and transporter genes were up‐regulated under LT. LT also affects transcription factors (TFs) such as PpTST1 and PpMYB62. Findings indicated that the vacuolar storage and transport system, along with specific TFs, plays crucial roles in the cold‐induced malate accumulation.

Summary Citrate is a common primary metabolite which often characterizes fruit flavour. The key regulators of citrate accumulation in fruit and vegetables are poorly understood. We systematically analysed the dynamic profiles of organic acid components during the development of kiwifruit (Actinidia spp.). Citrate continuously accumulated so that it became the predominate contributor to total acidity at harvest. Based on a co‐expression network analysis using different kiwifruit cultivars, an Al‐ACTIVATED MALATE TRANSPORTER gene (AcALMT1) was identified as a candidate responsible for citrate accumulation. Electrophysiological assays using expression of this gene in Xenopus oocytes revealed that AcALMT1 functions as a citrate transporter. Additionally, transient overexpression of AcALMT1 in kiwifruit significantly increased citrate content, while tissues showing higher AcALMT1 expression accumulated more citrate. The expression of AcALMT1 was highly correlated with 17 transcription factor candidates. However, dual‐luciferase and EMSA assays indicated that only the NAC transcription factor, AcNAC1, activated AcALMT1 expression via direct binding to its promoter. Targeted CRISPR‐Cas9‐induced mutagenesis of AcNAC1 in kiwifruit resulted in dramatic declines in citrate levels while malate and quinate levels were not substantially affected. Our findings show that transcriptional regulation of a major citrate transporter, by a NAC transcription factor, is responsible for citrate accumulation in kiwifruit, which has broad implications for other fruits and vegetables.

Malic enzyme (ME; NADP⁺-dependent; EC 1.1.40) provides NADPH for lipid biosynthesis in oleaginous microorganisms. Its role in vivo depends on there being an adequate supply of NADH to drive malate dehydrogenase to convert oxaloacetate to malate as a component of a cycle of three reactions: pyruvate → oxaloacetate → malate and, by the action of ME, back to pyruvate. However, the availability of cytosolic NADH is limited and, consequently, ancillary means of producing NADPH are necessary. Stoichiometries are given for the conversion of glucose to triacylglycerols involving ME with and without the reactions of the pentose phosphate pathway (PPP) as an additional source of NADPH. Some oleaginous microorganisms (such as Yarrowia lipolytica), however, lack a cytosolic ME and, if the PPP is the sole provider of NADPH, the theoretical yield of triacylglycerol from glucose falls to 27.6 % (w/w) from 31.6 % when ME is present. An alternative route for NADPH generation via a cytosolic isocitrate dehydrogenase (NADP⁺-dependent) is then discussed.

Replication‐related protein A (RepA), encoded by the citrus chlorotic dwarf‐associated virus (CCDaV), induces hypersensitive response (HR)‐like cell death and defence responses. However, the interactions between the host plant and CCDaV‐RepA remain unclear. In this study, Citrus limon chloroplast malate dehydrogenase (ClMDH) was found to interact with CCDaV‐RepA in the nucleus. ClMDH induces perinuclear chloroplast clustering (PCC). Moreover, ClMDH suppressed HR‐like cell death and the accumulation of reactive oxygen species (ROS) induced by CCDaV‐RepA, and promoted the accumulation of CCDaV‐RepA. In addition, CCDaV‐RepA overexpression altered the subcellular localisation of ClMDH from the chloroplast to the nucleus and inhibited ClMDH‐induced PCC. These results reflected the involvement of ClMDH‐induced PCC in the host response to CCDaV infection and provide new insights into the interaction between the host and CCDaV. RepA encoded by citrus chlorotic dwarf‐associated virus inhibits Citrus limon chloroplast malate dehydrogenase (ClMDH) induced perinuclear chloroplast clustering and hijacks ClMDH to the nucleus.

Cytosolic malate dehydrogenase (MDH) is a key enzyme that regulates the interconversion between malate and oxaloacetate (OAA). However, its role in modulating storage compound accumulation in maize endosperm is largely unknown. Here, we characterized a novel naturally occurring maize mdh4‐1 mutant, which produces small, opaque kernels and exhibits reduced starch but enhanced lysine content. Map‐based cloning, functional complementation and allelism analyses identified ZmMdh4 as the causal gene. Enzymatic assays demonstrated that ZmMDH4 predominantly catalyses the conversion from OAA to malate. In comparison, the activity of the mutant enzyme, which lacks one glutamic acid (Glu), was completed abolished, demonstrating that the Glu residue was essential for ZmMDH4 function. Knocking down ZmMdh4 in vivo led to a substantial metabolic shift towards glycolysis and a dramatic disruption in the activity of the mitochondrial complex I, which was correlated with transcriptomic alterations. Taken together, these results demonstrate that ZmMdh4 regulates the balance between mitochondrial respiration and glycolysis, ATP production and endosperm development, through a yet unknown feedback regulatory mechanism in mitochondria.

Fatty acid biosynthesis in oleaginous fungi requires the supply of reducing power, NADPH, and the precursor of fatty acids, acetyl-CoA, which is generated in the cytosol being produced by ATP: citrate lyase which requires citrate to be, transported from the mitochondrion by the citrate/malate/pyruvate transporter. This transporter, which is within the mitochondrial membrane, transports cytosolic malate into the mitochondrion in exchange for mitochondrial citrate moving into the cytosol (Fig. 1). The role of malate transporter in lipid accumulation in oleaginous fungi is not fully understood, however. Therefore, the expression level of the mt gene, coding for a malate transporter, was manipulated in the oleaginous fungus Mucor circinelloides to analyze its effect on lipid accumulation. The results showed that mt overexpression increased the lipid content for about 70 % (from 13 to 22 % dry cell weight, CDW), whereas the lipid content in mt knockout mutant decreased about 27 % (from 13 to 9.5 % CDW) compared with the control strain. Furthermore, the extracellular malate concentration was decreased in the mt overexpressing strain and increased in the mt knockout strain compared with the wild-type strain. This work suggests that the malate transporter plays an important role in regulating lipid accumulation in oleaginous fungus M. circinelloides.

Deciphering the mechanism of malate accumulation in plants would contribute to a greater understanding of plant chemistry, which has implications for improving flavor quality in crop species and enhancing human health benefits. However, the regulation of malate metabolism is poorly understood in crops such as tomato (Solanum lycopersicum). Here, we integrated a metabolite-based genome-wide association study with linkage mapping and gene functional studies to characterize the genetics of malate accumulation in a global collection of tomato accessions with broad genetic diversity. We report that TFM6 (tomato fruit malate 6), which corresponds to Al-ACTIVATED MALATE TRANSPORTER9 (Sl-ALMT9 in tomato), is the major quantitative trait locus responsible for variation in fruit malate accumulation among tomato genotypes. A 3-bp indel in the promoter region of Sl-ALMT9 was linked to high fruit malate content. Further analysis indicated that this indel disrupts a W-box binding site in the Sl-ALMT9 promoter, which prevents binding of the WRKY transcription repressor Sl-WRKY42, thereby alleviating the repression of Sl-ALMT9 expression and promoting high fruit malate accumulation. Evolutionary analysis revealed that this highly expressed Sl-ALMT9 allele was selected for during tomato domestication. Furthermore, vacuole membrane-localized Sl-ALMT9 increases in abundance following Al treatment, thereby elevating malate transport and enhancing Al resistance.

Malic enzyme (EC 1.1.1.40) converts L-malate to pyruvate and CO₂ providing NADPH for metabolism especially for lipid biosynthesis in oleaginous microorganisms. However, its role in the oleaginous yeast, Yarrowia lipolytica, is unclear. We have cloned the malic enzyme gene (YALI0E18634g) from Y. lipolytica into pET28a, expressed it in Escherichia coli and purified the recombinant protein (YlME). YlME used NAD⁺ as the primary cofactor. Kₘ values for NAD⁺ and NADP⁺ were 0.63 and 3.9 mM, respectively. Citrate, isocitrate and α-ketoglutaric acid (>5 mM) were inhibitory while succinate (5–15 mM) increased NADP⁺- but not NAD⁺-dependent activity. To determine if fatty acid biosynthesis could be increased in Y. lipolytica by providing additional NADPH from an NADP⁺-dependent malic enzyme, the malic enzyme gene (mce2) from an oleaginous fungus, Mortierella alpina, was expressed in Y. lipolytica. No significant changes occurred in lipid content or fatty acid profiles suggesting that malic enzyme is not the main source of NADPH for lipid accumulation in Y. lipolytica.

Over the past three decades, the global prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD) has rapidly increased, leading to significant economic and clinical burdens. However, aside from resmetirom (Rezdiffra™), an oral thyroid hormone receptor-β agonist, there remains a lack of approved targeted pharmacological treatments for MASLD, emphasizing the need for more optimized therapeutic strategies. Given the limitations in the safety and efficacy of the ketogenic diet, β-hydroxybutyrate (β-OHB) has emerged as a crucial regulator in MASLD treatment, but the precise mechanisms underlying its therapeutic effects remain unclear. This study aims to investigate the therapeutic effects of β-OHB on MASLD mice and elucidate the underlying mechanisms. In this study, we demonstrate that β-OHB ameliorates lipid deposition and increases pan-β-hydroxybutyrylation (Kbhb) levels in both MASLD mice and in vitro. Additionally, β-OHB also improves excessive ROS accumulation and enhances mitochondrial respiratory capacity. Furthermore, β-OHB protects against impaired fatty acid oxidation (FAO) activity in MASLD. Proteomic analysis of β-OHB-treated mice identified a significant Kbhb modification at K239 on malate dehydrogenase 2 (MDH2), which was associated with increased MDH2 enzymatic activity. Overall, this study demonstrates β-OHB exhibits therapeutic effects on hepatic steatosis and mitochondrial dysfunction in MASLD mice. We uncover a novel mechanism where β-OHB enhances MDH2 enzymatic activity through Kbhb modification at K239, thereby maintaining mitochondrial homeostasis and alleviating lipid deposition. Graphical abstract