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Purpose Citrulline malate (CM) is a nonessential amino acid that increases exercise performance in males. However, based on physiological differences between genders, these results cannot be extrapolated to females. Therefore, the purpose of this investigation was to evaluate effects of acute CM supplementation on upper- and lower-body weightlifting performance in resistance-trained females. Methods Fifteen females (23 ± 3 years) completed two randomized, double-blind trials consuming either CM (8 g dextrose + 8 g CM) or a placebo (8 g dextrose). One hour after supplement consumption, participants performed six sets each of upper- (i.e., bench press) and lower-body (i.e., leg press) exercises to failure at 80 % of previously established one-repetition maximum. Immediately after each set, repetitions completed, heart rate and rating of perceived exertion (RPE) were recorded. Results Repeated-measures analysis of variance indicated that subjects completed significantly ( p  = .045) more repetitions throughout upper-body exercise when consuming CM versus placebo (34.1 ± 5.7 vs. 32.9 ± 6.0, respectively). When consuming CM, similar significant ( p  = .03) improvements in total repetitions completed were observed for lower-body exercise (66.7 ± 30.5 vs. 55.13 ± 20.64, respectively). Overall RPE score was significantly lower ( p  = .02) in upper-body exercise when subjects consumed CM versus placebo (7.9 ± 0.3 and 8.6 ± 0.2, respectively). The supplement consumed exhibited no significant effects on heart rate at any time point. Conclusions Acute CM supplementation in females increased upper- and lower-body resistance exercise performance and decreased RPE during upper-body exercise. These data indicate that athletes competing in sports with muscular endurance-based requirements may potentially improve performance by acutely supplementing CM.

This study investigated the effects of combined L -arginine (Arg) and citrulline-malate (CM) supplementation on aerobic, anaerobic, and high-intensity interval training in healthy, trained men. Both Arg and CM are widely marketed for their potential ergogenic effects, as Arg serves as a precursor to nitric oxide (NO), which may support vasodilation, muscle contractility, and exercise performance. Arg and CM are hypothesized to exert synergistic effects due to their complementary roles in NO synthesis. Citrulline can potentially enhance and prolong Arg availability, thereby amplifying NO-mediated vasodilation, nutrient delivery, and muscle performance during exercise. In this randomized, double-blind, placebo-controlled trial, 46 healthy, trained men aged 24.8 ± 5.0 years were divided into 3 exercise groups subjected to consuming 0.15 g/kg bodyweight of Arg and 0.1 g/kg bodyweight of CM prior. The participants were then randomly divided into three groups based on the exercise protocol:: the Wingate Anaerobic Test ( n  = 16), a 20 min CrossFit workout ‘Cindy’ ( n  = 16) consisting of continuous rounds of pull-ups, push-ups, and air squats to measure functional fitness and muscular endurance, or the Harvard Step Test ( n  = 14) a measurement of cardiovascular endurance and recovery. This design allowed for the evaluation of supplementation effects across multiple exercise modalities . The results revealed no significant improvement in performance with supplementation in comparison to placebo, except for a shorter time to reach peak power in the Wingate test. Findings suggest that the combined acute supplementation of Arg and CM, at the given dosages, may not provide substantial benefits for aerobic and anaerobic or CrossFit performance in active individuals. Future research with larger sample sizes and higher dosages, potentially adjusted for muscle mass, is recommended to determine whether chronic supplementation might yield greater ergogenic effects.

Manganese (Mn) toxicity is a major constraint limiting plant growth on acidic soils. Superior Mn tolerance in Stylosanthes spp. has been well documented, but its molecular mechanisms remain largely unknown. In this study, superior Mn tolerance in Stylosanthes guianensis was confirmed, as reflected by a high Mn toxicity threshold. Furthermore, genetic variation of Mn tolerance was evaluated using two S. guianensis genotypes, which revealed that the Fine-stem genotype had higher Mn tolerance than the TPRC2001-1 genotype, as exhibited through less reduction in dry weight under excess Mn, and accompanied by lower internal Mn concentrations. Interestingly, Mn-stimulated increases in malate concentrations and exudation rates were observed only in the Fine-stem genotype. Proteomic analysis of Fine-stem roots revealed that S. guianensis Malate Dehydrogenase1 (SgMDH1) accumulated in response to Mn toxicity. Western-blot and quantitative PCR analyses showed that Mn toxicity resulted in increased SgMDH1 accumulation only in Fine-stem roots, but not in TPRC2001-1. The function of SgMDH1-mediated malate synthesis was verified through in vitro biochemical analysis of SgMDH1 activities against oxaloacetate, as well as in vivo increased malate concentrations in yeast (Saccharomyces cerevisiae), soybean (Glycine max) hairy roots, and Arabidopsis (Arabidopsis thaliana) with SgMDH1 overexpression. Furthermore, SgMDH1 overexpression conferred Mn tolerance in Arabidopsis, which was accompanied by increased malate exudation and reduced plant Mn concentrations, suggesting that secreted malate could alleviate Mn toxicity in plants. Taken together, we conclude that the superior Mn tolerance of S. guianensis is achieved by coordination of internal and external Mn detoxification through malate synthesis and exudation, which is regulated by SgMDH1 at both transcription and protein levels.

Given the increasing popularity of CrossFit® as a high-intensity functional training program and the potential benefits of citrulline malate (CM) in enhancing exercise performance through its role as a precursor to L-arginine and nitric oxide production, this study aimed to investigate the acute effects of CM supplementation on CrossFit® performance and cardiovascular function. Using a randomized, double-blind, placebo-controlled, cross-over design, 21 recreationally active participants (mean age 22.2 ± 2.6 years, mean body weight 75.9 ± 10.4 kg) with CrossFit® experience completed the “Cindy” workout under CM and placebo conditions. Participants consumed 4.4 g of CM or a placebo 60 min before the workout, and the performance was measured by the number of rounds completed. Secondary outcomes included heart rate response, time spent in different heart rate intensity zones, and post-exercise recovery time. The results indicated no significant difference in the number of rounds completed between the CM and placebo conditions (13.5 ± 5.2 vs. 13.8 ± 6.7 rounds, respectively; p = 0.587). However, the time spent in zone 4 (80–90% of HR max) was significantly increased in the CM condition (527 ± 395 s vs. 453 ± 334 s; p = 0.017), suggesting a potential benefit for aerobic capacity and anaerobic threshold. No significant differences in post-exercise recovery time were observed (6.6 ± 4.7 h vs. 6.9 ± 4.7 h; p = 0.475). This study highlights the need for further research with larger sample sizes, both genders, and different CM dosages to clarify these findings and better understand CM’s role in enhancing athletic performance.

• ALUMINUM-ACTIVATED MALATE TRANSPORTER1 (ALMT1)-mediated malate exudation from roots is critical for aluminium (Al) resistance in Arabidopsis. Its upstream molecular signalling regulation is not yet well understood. • The role of CALMODULIN-LIKE24 (CML24) in Al-inhibited root growth and downstream molecular regulation of ALMT1-meditaed Al resistance was investigated. • CML24 confers Al resistance demonstrated by an increased root-growth inhibition of the cml24 loss-of-function mutant under Al stress. This occurs mainly through the regulation of the ALMT1-mediated malate exudation from roots. The mutation and overexpression of CML24 leads to an elevated and reduced Al accumulation in the cell wall of roots, respectively. Al stress induced both transcript and protein abundance of CML24 in root tips, especially in the transition zone. CML24 interacts with CALMODULIN BINDING TRANSCRIPTION ACTIVATOR2 (CAMTA2) and promotes its transcriptional activity in the regulation of ALMT1 expression. This results in an enhanced malate exudation from roots and less root-growth inhibition under Al stress. Both CML24 and CAMTA2 interacted with WRKY46 suppressing the transcriptional repression of ALMT1 by WRKY46. • The study provides novel insights into understanding of the upstream molecular signalling of the ALMT1-depdendent Al resistance.

Stylosanthes (stylo) is a dominant leguminous forage in the tropics. Previous studies suggest that stylo has great potential for aluminium (Al) tolerance, but little is known about the underlying mechanism. A novel malic enzyme, SgME1, was identified from the Al‐tolerant genotype TPRC2001‐1 after 72 h Al exposure by two‐dimensional electrophoresis, and the encoding gene was cloned and characterized via heterologous expression in yeast, Arabidopsis thaliana and bean (Phaseolus vulgaris) hairy roots. Internal Al detoxification might be mainly responsible for the 72 h Al tolerance of TPRC2001‐1, as indicated by 5.8‐fold higher root malate concentrations and approximately two‐fold higher Al concentrations in roots and root symplasts of TPRC2001‐1 than those of the Al‐sensitive genotype Fine‐stem. An accompanying increase in malate secretion might also reduce a fraction of Al uptake in TPRC2001‐1. Gene and protein expression of SgME1 was only enhanced in TPRC2001‐1 after 72 h Al exposure. Overexpressing SgME1 enhanced malate synthesis and rescued yeast, A. thaliana and bean hairy roots from Al toxicity via increasing intracellular malate concentrations and/or accompanied malate exudation. These results provide strong evidence that superior Al tolerance of stylo is mainly conferred by Al‐enhanced malate synthesis, functionally controlled by SgME1.

Evidence for a link between cellular senescence and metabolic regulation is provided, through the observation that p53 represses the expression of malic enzymes, thereby regulating NADPH, lipid and glutamine metabolism; in turn, this repression further activates p53, promoting cellular senescence. Malic enzyme interactions with p53 The tumour suppressor p53 is known to regulate metabolic processes as well as cellular senescence. Peng Jiang et al . now link the two activities, showing that p53 represses the expression of the malic enzymes ME1 and ME2 and thereby regulates NADPH production, lipid and glutamine metabolism. ME1 and ME2 downregulation can, in turn, further activate p53 and thereby promote cellular senescence. ME1 and ME2 are often overexpressed in cancers and can suppress senescence and promote tumour growth. Cellular senescence both protects multicellular organisms from cancer and contributes to their ageing 1 . The pre-eminent tumour suppressor p53 has an important role in the induction and maintenance of senescence, but how it carries out this function remains poorly understood 1 , 2 , 3 . In addition, although increasing evidence supports the idea that metabolic changes underlie many cell-fate decisions and p53-mediated tumour suppression, few connections between metabolic enzymes and senescence have been established. Here we describe a new mechanism by which p53 links these functions. We show that p53 represses the expression of the tricarboxylic-acid-cycle-associated malic enzymes ME1 and ME2 in human and mouse cells. Both malic enzymes are important for NADPH production, lipogenesis and glutamine metabolism, but ME2 has a more profound effect. Through the inhibition of malic enzymes, p53 regulates cell metabolism and proliferation. Downregulation of ME1 and ME2 reciprocally activates p53 through distinct MDM2- and AMP-activated protein kinase-mediated mechanisms in a feed-forward manner, bolstering this pathway and enhancing p53 activation. Downregulation of ME1 and ME2 also modulates the outcome of p53 activation, leading to strong induction of senescence, but not apoptosis, whereas enforced expression of either malic enzyme suppresses senescence. Our findings define physiological functions of malic enzymes, demonstrate a positive-feedback mechanism that sustains p53 activation, and reveal a connection between metabolism and senescence mediated by p53.

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.

Succinic acid (SA), a dicarboxylic acid of industrial importance, can be efficiently produced by metabolically engineered Mannheimia succiniciproducens . Malate dehydrogenase (MDH) is one of the key enzymes for SA production, but has not been well characterized. Here we report biochemical and structural analyses of various MDHs and development of hyper-SA producing M. succiniciproducens by introducing the best MDH. Corynebacterium glutamicum MDH ( Cg MDH) shows the highest specific activity and least substrate inhibition, whereas M. succiniciproducens MDH ( Ms MDH) shows low specific activity at physiological pH and strong uncompetitive inhibition toward oxaloacetate ( ki of 67.4 and 588.9 μM for Ms MDH and Cg MDH, respectively). Structural comparison of the two MDHs reveals a key residue influencing the specific activity and susceptibility to substrate inhibition. A high-inoculum fed-batch fermentation of the final strain expressing cgmdh produces 134.25 g L −1 of SA with the maximum productivity of 21.3 g L −1  h −1 , demonstrating the importance of enzyme optimization in strain development. Malate dehydrogenase (MDH) is one of the key enzymes for succinic acid (SA) bioproduction. Here, the authors report biochemical and structural analyses of various MDHs to reveal amino acids influencing the specific activity and susceptibility to substrate inhibition, and achieve industrial-level SA production.

Efficient regeneration of NADPH can be a limiting factor for anabolic processes in engineered microbial cells. We tested the ability of four distinct Pyruvate-Oxaloacetate-Malate “POM” cycles composed of Saccharomyces cerevisiae pyruvate carboxylase ( PYC1 or PYC2 ), malate dehydrogenase ( ‘MDH1 or ‘MDH2 ), and malic enzyme ( sMAE1 ) to improve NADPH regeneration. Only the PYC1 , ‘MDH2 , sMAE1 combination increased the titer of fatty alcohols produced by engineered S. cerevisiae indicating that not all combinations of POM cycle enzymes could drive this pathway. Metabolomic analysis revealed that introduction of the POM cycle altered the concentration of intermediates in amino acid biosynthetic pathways and the trichloroacetic acid cycle suggesting that the POM cycle had wider effects than previously anticipated. Overexpression of the endogenous NAD + kinases UTR1 , YEF1 , and a cytosolic version of POS5 were also tested. Only expression of POS5c resulted a significant increase in fatty alcohol titer. In these minimally engineered strains, combined overexpression of the PYC1 , ‘ MDH2 , sMAE1 POM cycle and POS5c did not further increase titers. These findings indicate that more extensive metabolomic and proteomic investigations are required to identify combinations of enzymes that will yield an optimal increase in NADPH to meet anabolic demands without imposing excessive metabolic burden or disrupting pathways that might compromise bioproduct synthesis.