Citrate Secretion Research Articles

SlSTOP1-regulated SlHAK5 expression confers Al tolerance in tomato by facilitating citrate secretion from roots.

SENSITIVE TO PROTON RHIZOTOXICITY 1 (STOP1) is a core transcription factor that regulates the expression of aluminum (Al) resistance genes to manage Al toxicity in plants. However, the genome-wide roles of SlSTOP1 in the Al stress response of tomato (Solanum lycopersicum) remain largely unknown. Here, we report that SlSTOP1 is crucial for Al tolerance in tomato, as loss-of-function mutants of SlSTOP1 displayed hypersensitivity to Al stress. Aluminum stress had no effect on SlSTOP1 mRNA expression, but promoted accumulation of SlSTOP1 protein in the nucleus. Through integrated DNA affinity purification sequencing and RNA sequencing analysis, we identified 39 SlSTOP1-targeted Al-responsive genes, some of which are homologous to known Al resistance genes in other plant species, suggesting that these SlSTOP1-targeted genes play essential roles in Al resistance in tomato. Furthermore, using peak enrichment analysis of SlSTOP1-targeted sequences, we identified a cis-acting element bound by SlSTOP1 and validated this finding via dual-luciferase reporter and electrophoretic mobility shift assay (EMSA). Additionally, we demonstrated SlHAK5 is one of direct targets of SlSTOP1 and functionally characterized it in terms of Al stress tolerance. Compared with wild-type plants, Slhak5 mutants developed by CRISPR/Cas9 technology presented increased sensitivity to Al stress, which was associated with reduced citrate secretion from the roots. Together, our findings demonstrate that SlSTOP1 directly interacts with cis-acting elements located in the promoters of target genes involved in diverse pathways contributing to Al resistance in tomato.

Breeding for an elite malting barley cultivar with acid soil tolerance.

Barley (Hordeum vulgare L.) is the fourth most produced cereal crop in the world, but its productivity on acid soil has been restricted due to its high sensitivity to aluminum (Al) toxicity. The major gene controlling Al tolerance in barley is HvAACT1 (Al-activated citrate transporter 1), which is involved in citrate secretion from the roots for Al detoxification. Here we bred a malting barley cultivar with enhanced acid soil tolerance by introgression of a 1-kb transposon regulating the expression of HvAACT1 into an elite malting cultivar through multiple backcrossing and marker-assisted selection. The line selected showed increased expression of HvAACT1, enhanced citrate secretion from the roots and decreased Al binding to the roots. This line produced more than two to three times the grain yield compared with the original cultivar when grown on acidic soil, providing a potentially sustainable and economic way to boost productivity of malting barley cultivars in areas with acidic soil.

Current Understanding of Androgen Signaling in Prostatitis and its Treatment: A Review.

Chronic prostatitis is a highly prevalent condition that significantly impacts the quality of life and fertility of men. Because of its heterogeneous nature, there is no definitive treatment, which requires ongoing research into its etiology. Additionally, the association between prostatitis and an elevated risk of prostate cancer highlights the importance of comprehending androgen involvement in prostatitis. This paper examines the current understanding of androgen signaling in prostatitis and explores contemporary therapeutic approaches. It was reviewed Medline articles comprehensively, using keywords such as nonbacterial prostatitis, prostatitis infertility, androgen role in prostatitis, and chronic pelvic pain. Several cellular targets are linked to androgen signaling. Notably, the major tyrosine phosphatase activity (cPAcP) in normal human prostate is influenced by androgen signaling, and its serum levels inversely correlate with prostate cancer progression. Androgens also regulate membrane-associated zinc and pyruvate transporters transduction in prostate cells, suggesting promising avenues for novel drug development aimed at inhibiting these molecules to reduce cancer tumor growth. Various therapies for prostatitis have been evaluated, including antibiotics, anti-inflammatory medications (including bioflavonoids), neuromodulators, alpha-blockers, 5α-reductase inhibitors, and androgen receptor antagonists. These therapies have demonstrated varying degrees of success in ameliorating symptoms.In conclusion, aging decreases circulating T and intraprostatic DHT, altering the proper functioning of the prostate, reducing the ability of androgens to maintain normal Zn2+ levels, and diminishing the secretion of citrate, PAcP, and other proteins into the prostatic fluid. The Zn2+-transporter decreases or is absent in prostate cancer, so the pyruvate transporter activates. Consequently, the cell ATP increases, inducing tumor growth.

The GATA transcription factor BcWCL2 regulates citric acid secretion to maintain redox homeostasis and full virulence in Botrytis cinerea.

Botrytis cinerea is a typical necrotrophic plant pathogenic fungus which can deliberately acidify host tissues and trigger oxidative bursts therein to facilitate its virulence. The white collar complex (WCC), consisting of BcWCL1 and BcWCL2, is recognized as the primary light receptor in B. cinerea. Nevertheless, the specific mechanisms through which the WCC components, particularly BcWCL2 as a GATA transcription factor, control virulence are not yet fully understood. This study demonstrates that deletion of BcWCL2 results in the loss of light-sensitive phenotypic characteristics. Additionally, the Δbcwcl2 strain exhibits reduced secretion of citrate, delayed infection cushion development, weaker hyphal penetration, and decreased virulence. The application of exogenous citric acid was found to restore infection cushion formation, hyphal penetration, and virulence of the Δbcwcl2 strain. Transcriptome analysis at 48 h post-inoculation revealed that two citrate synthases, putative citrate transporters, hydrolytic enzymes, and reactive oxygen species scavenging-related genes were down-regulated in Δbcwcl2, whereas exogenous citric acid application restored the expression of the above genes involved in the early infection process of Δbcwcl2. Moreover, the expression of Bcvel1, a known regulator of citrate secretion, tissue acidification, and secondary metabolism, was down-regulated in Δbcwcl2 but not in Δbcwcl1. ChIP-qPCR and electrophoretic mobility shift assays revealed that BcWCL2 can bind to the promoter sequences of Bcvel1. Overexpressing Bcvel1 in Δbcwcl2 was found to rescue the mutant defects. Collectively, our findings indicate that BcWCL2 regulates the expression of the global regulator Bcvel1 to influence citrate secretion, tissue acidification, redox homeostasis, and virulence of B. cinerea.IMPORTANCEThis study illustrated the significance of the fungal blue light receptor component BcWCL2 protein in regulating citrate secretion in Botrytis cinerea. Unlike BcWCL1, BcWCL2 may contribute to redox homeostasis maintenance during infection cushion formation, ultimately proving to be essential for full virulence. It is also demonstrated that BcWCL2 can regulate the expression of Bcvel1 to influence host tissue acidification, citrate secretion, infection cushion development, and virulence. While the role of organic acids secreted by plant pathogenic fungi in fungus-host interactions has been recognized, this paper revealed the importance, regulatory mechanisms, and key transcription factors that control organic acid secretion. These understanding of the pathogenetic mechanism of plant pathogens can provide valuable insights for developing effective prevention and treatment strategies against fungal diseases.

GmMYB183, a R2R3-MYB Transcription Factor in Tamba Black Soybean (Glycine max. cv. Tamba), Conferred Aluminum Tolerance in Arabidopsis and Soybean.

Aluminum (Al) toxicity is one of the environmental stress factors that affects crop growth, development, and productivity. MYB transcription factors play crucial roles in responding to biotic or abiotic stresses. However, the roles of MYB transcription factors in Al tolerance have not been clearly elucidated. Here, we found that GmMYB183, a gene encoding a R2R3 MYB transcription factor, is involved in Al tolerance. Subcellular localization studies revealed that GmMYB183 protein is located in the nucleus, cytoplasm and cell membrane. Overexpression of GmMYB183 in Arabidopsis and soybean hairy roots enhanced plant tolerance towards Al stress compared to the wild type, with higher citrate secretion and less Al accumulation. Furthermore, we showed that GmMYB183 binds the GmMATE75 gene promoter encoding for a plasma-membrane-localized citrate transporter. Through a dual-luciferase reporter system and yeast one hybrid, the GmMYB183 protein was shown to directly activate the transcription of GmMATE75. Furthermore, the expression of GmMATE75 may depend on phosphorylation of Ser36 residues in GmMYB183 and two MYB sites in P3 segment of the GmMATE75 promoter. In conclusion, GmMYB183 conferred Al tolerance by promoting the secretion of citrate, which provides a scientific basis for further elucidating the mechanism of plant Al resistance.

Identification of MATE Family and Characterization of GmMATE13 and GmMATE75 in Soybean's Response to Aluminum Stress.

The multidrug and toxic compound extrusion (MATE) proteins are coding by a secondary transporter gene family, and have been identified to participate in the modulation of organic acid exudation for aluminum (Al) resistance. The soybean variety Glycine max "Tamba" (TBS) exhibits high Al tolerance. The expression patterns of MATE genes in response to Al stress in TBS and their specific functions in the context of Al stress remain elusive. In this study, 124 MATE genes were identified from the soybean genome. The RNA-Seq results revealed significant upregulation of GmMATE13 and GmMATE75 in TBS upon exposure to high-dose Al3+ treatment and both genes demonstrated sequence homology to citrate transporters of other plants. Subcellular localization showed that both proteins were located in the cell membrane. Transgenic complementation experiments of Arabidopsis mutants, atmate, with GmMATE13 or GmMATE75 genes enhanced the Al tolerance of the plant due to citrate secretion. Taken together, this study identified GmMATE13 and GmMATE75 as citrate transporter genes in TBS, which could improve citrate secretion and enhance Al tolerance. Our findings provide genetic resources for the development of plant varieties that are resistant to Al toxicity.

The transcription factor SbHY5 mediates light to promote aluminum tolerance by activating SbMATE and SbSTOP1s expression

Aluminum (Al) toxicity is a major factor limiting crop yields in acid soils. Sweet sorghum (Sorghum bicolor L.) is a high-efficient energy crop widely grown in tropical and subtropical regions of the world, where acid soil is common and Al toxicity is widespread. Here, we characterized a transcription factor SbHY5 in sweet sorghum, which mediated light to promote plant Al stress adaptation. The expression of SbHY5 was induced by Al stress and increasing light intensity. The overexpression of SbHY5 improved Al tolerance in transgenic plants, which was associated with increased citrate secretion and reduced Al content in roots. Meanwhile, SbHY5 was found to localize to the nucleus and displayed transcriptional activity. SbHY5 directly activated the expression of SbMATE, indicating that a HY5-MATE-dependent citrate secretion pathway is involved in Al tolerance in plants. SbSTOP1 was reported as a key transcription factor, regulating several Al tolerance genes. Here, inspiringly, we found that SbHY5 directly promoted the transcription of SbSTOP1, implying the existence of HY5-STOP1-Al tolerance genes-mediated regulatory pathways. Besides, SbHY5 positively regulated its own transcription. Our findings revealed a novel regulatory network in which a light signaling factor, SbHY5, confers Al tolerance in plants by modulating the expression of Al stress response genes.

Enhanced Succinate Oxidation with Mitochondrial Complex II Reactive Oxygen Species Generation in Human Prostate Cancer.

The transformation of prostatic epithelial cells to prostate cancer (PCa) has been characterized as a transition from citrate secretion to citrate oxidation, from which one would anticipate enhanced mitochondrial complex I (CI) respiratory flux. Molecular mechanisms for this transformation are attributed to declining mitochondrial zinc concentrations. The unique metabolic properties of PCa cells have become a hot research area. Several publications have provided indirect evidence based on investigations using pre-clinical models, established cell lines, and fixed or frozen tissue bank samples. However, confirmatory respiratory analysis on fresh human tissue has been hampered by multiple difficulties. Thus, few mitochondrial respiratory assessments of freshly procured human PCa tissue have been published on this question. Our objective is to document relative mitochondrial CI and complex II (CII) convergent electron flow to the Q-junction and to identify electron transport system (ETS) alterations in fresh PCa tissue. The results document a CII succinate: quinone oxidoreductase (SQR) dominant succinate oxidative flux model in the fresh non-malignant prostate tissue, which is enhanced in malignant tissue. CI NADH: ubiquinone oxidoreductase activity is impaired rather than predominant in high-grade malignant fresh prostate tissue. Given these novel findings, succinate and CII are promising targets for treating and preventing PCa.

Myostatin inhibits insulin-like growth factor 1-dependent citrate secretion and osteogenesis via nicotinamide adenine dinucleotide phosphate oxidase-4 in a mouse mesenchymal stem cell line.

Citrate is an indispensable component of bone. Reduced levels of citrate in bone and serum are reported in the elderly and in osteoporosis patients. Myostatin (Mstn) is implicated in skeletal homeostasis, but its effects on osteogenesis remain incompletely understood. Nox4 has critical roles in bone homeostasis. TGF-β/Mstn-associated Smad2/3 signaling has been linked to Nox4 expression. Insulin-like growth factor (IGF-1) has been shown to counteract many regulatory effects of Mstn. However, the crosstalk among Mstn, IGF-1, and Nox4 is not well understood; the interactive effects of those factors on citrate secretion, osteogenic differentiation, and bone remodeling remain unclear. In this study, we demonstrated that osteogenic differentiation induced an IGF-1-dependent upregulation of citrate secretion that was suppressed by Mstn. Inhibition of Nox4 prevented Mstn-induced reduction of citrate secretion. In addition, Mstn reduced bone nodule formation; these changes were prevented by Nox4 inhibition. Moreover, Mstn increased the ratio of RANKL to OPG mRNAs to favor osteoclast activation. These results indicate that Mstn negatively regulates osteogenesis by increasing levels of Nox4, which reduced IGF-1 expression, citrate secretion, and bone mineralization while also altering the RANKL to OPG ratio. These findings provide new and highly relevant insights into the osseous effects of myostatin.

Mesenchymal stromal cells cultured in physiological conditions sustain citrate secretion with glutamate anaplerosis

Bone marrow mesenchymal stromal cells (MSCs) have immunomodulatory and regenerative potential. However, culture conditions govern their metabolic processes and therapeutic efficacy. Here we show that culturing donor-derived MSCs in Plasmax™, a physiological medium with the concentrations of nutrients found in human plasma, supports their proliferation and stemness, and prevents the nutritional stress induced by the conventional medium DMEM. The quantification of the exchange rates of metabolites between cells and medium, untargeted metabolomics, stable isotope tracing and transcriptomic analysis, performed at physiologically relevant oxygen concentrations (1%O2), reveal that MSCs rely on a high rate of glucose to lactate conversion, coupled with parallel anaplerotic fluxes from glutamine and glutamate to support citrate synthesis and secretion. These distinctive traits of MSCs shape the metabolic microenvironment of the bone marrow niche and can influence nutrient cross-talks under physiological and pathological conditions.

The genes controlling normal function of citrate and spermine secretion are lost in aggressive prostate cancer and prostate model systems

SummaryHigh secretion of the metabolites citrate and spermine is a unique hallmark for normal prostate epithelial cells, and is reduced in aggressive prostate cancer. However, the identity of the genes controlling this biological process is mostly unknown. In this study, we have created a gene signature of 150 genes connected to citrate and spermine secretion in the prostate. We have computationally integrated metabolic measurements with multiple transcriptomics datasets from the public domain, including 3826 tissue samples from prostate and prostate cancer. The accuracy of the signature is validated by its unique enrichment in prostate samples and prostate epithelial tissue compartments. The signature highlights genes AZGP1, ANPEP and metallothioneins with zinc-binding properties not previously studied in the prostate, and the expression of these genes are reduced in more aggressive cancer lesions. However, the absence of signature enrichment in common prostate model systems can make it challenging to study these genes mechanistically.

Carbon sources and pathways for citrate secreted by human prostate cancer cells determined by NMR tracing and metabolic modeling

Prostate epithelial cells have the unique capacity to secrete large amounts of citrate, but the carbon sources and metabolic pathways that maintain this production are not well known. We mapped potential pathways for citrate carbons in the human prostate cancer metastasis cell lines LNCaP and VCaP, for which we first established that they secrete citrate (For LNCaP 5.6 ± 0.9 nmol/h per 106 cells). Using 13C-labeled substrates, we traced the incorporation of 13C into citrate by NMR of extracellular fluid. Our results provide direct evidence that glucose is a main carbon source for secreted citrate. We also demonstrate that carbons from supplied glutamine flow via oxidative Krebs cycle and reductive carboxylation routes to positions in secreted citrate but likely do not contribute to its net synthesis. The potential anaplerotic carbon sources aspartate and asparagine did not contribute to citrate carbons. We developed a quantitative metabolic model employing the 13C distribution in extracellular citrate after 13C glucose and pyruvate application to assess intracellular pathways of carbons for secreted citrate. From this model, it was estimated that in LNCaP about 21% of pyruvate entering the Krebs cycle is converted via pyruvate carboxylase as an anaplerotic route at a rate more than sufficient to compensate carbon loss of this cycle by citrate secretion. This model provides an estimation of the fraction of molecules, including citrate, leaving the Krebs cycle at every turn. The measured ratios of 13C atoms at different positions in extracellular citrate may serve as biomarkers for (malignant) epithelial cell metabolism.

The Interplay Between Prostate Cancer Genomics, Metabolism, and the Epigenome: Perspectives and Future Prospects.

Prostate cancer is a high-incidence cancer, often detected late in life. The prostate gland is an accessory gland that secretes citrate; an impaired citrate secretion reflects imbalances in the activity of enzymes in the TCA Cycle in mitochondria. Profiling studies on prostate tumours have identified significant metabolite, proteomic, and transcriptional modulations with an increased mitochondrial metabolic activity associated with localised prostate cancer. Here, we focus on the androgen receptor, c-Myc, phosphatase and tensin Homolog deleted on chromosome 10 (PTEN), and p53 as amongst the best-characterised genomic drivers of prostate cancer implicated in metabolic dysregulation and prostate cancer progression. We outline their impact on metabolic function before discussing how this may affect metabolite pools and in turn chromatin structure and the epigenome. We reflect on some recent literature indicating that mitochondrial mutations and OGlcNAcylation may also contribute to this crosstalk. Finally, we discuss the technological challenges of assessing crosstalk given the significant differences in the spatial sensitivity and throughput of genomic and metabolomic profiling approaches.

Loss of LaMATE impairs isoflavonoid release from cluster roots of phosphorus-deficient white lupin.

White lupin (Lupinus albus L.) forms brush-like root structures called cluster roots under phosphorus-deficient conditions. Clusters secrete citrate and other organic compounds to mobilize sparingly soluble soil phosphates. In the context of aluminum toxicity tolerance mechanisms in other species, citrate is released via a subgroup of MATE/DTX proteins (multidrug and toxic compound extrusion/detoxification). White lupin contains 56 MATE/DTX genes. Many of these are closely related to gene orthologs with known substrates in other species. LaMATE is a marker gene for functional, mature clusters and is, together with its close homolog LaMATE3, a candidate for the citrate release. Both were highest expressed in mature clusters and when expressed in oocytes, induced inward-rectifying currents that were likely carried by endogenous channels. No citrate efflux was associated with LaMATE and LaMATE3 expression in oocytes. Furthermore, citrate secretion was largely unaffected in P-deficient composite mutant plants with genome-edited or RNAi-silenced LaMATE in roots. Moderately lower concentrations of citrate and malate in the root tissue and consequently less organic acid anion secretion and lower malate in the xylem sap were identified. Interestingly, however, less genistein was consistently found in mutant exudates, opening the possibility that LaMATE is involved in isoflavonoid release.

Global Transcriptional Response of Aspergillus niger to Blocked Active Citrate Export through Deletion of the Exporter Gene.

Aspergillus niger is the major industrial citrate producer worldwide. Export as well as uptake of citric acid are believed to occur by active, proton-dependent, symport systems. Both are major bottlenecks for industrial citrate production. Therefore, we assessed the consequences of deleting the citT gene encoding the A. niger citrate exporter, effectively blocking active citrate export. We followed the consumption of glucose and citrate as carbon sources, monitored the secretion of organic acids and carried out a thorough transcriptome pathway enrichment analysis. Under controlled cultivation conditions that normally promote citrate secretion, the knock-out strain secreted negligible amounts of citrate. Blocking active citrate export in this way led to a reduced glucose uptake and a reduced expression of high-affinity glucose transporter genes, mstG and mstH. The glyoxylate shunt was strongly activated and an increased expression of the OAH gene was observed, resulting in a more than two-fold higher concentration of oxalate in the medium. Deletion of citT did not affect citrate uptake suggesting that citrate export and citrate uptake are uncoupled from the system.

Corrigendum: Protective Role of Melatonin Against Postmenopausal Bone Loss via Enhancing Citrate Secretion in Osteoblasts.

Corrigendum: Protective Role of Melatonin Against Postmenopausal Bone Loss via Enhancing Citrate Secretion in Osteoblasts.

Targeting Mitochondrial Metabolism in Prostate Cancer with Triterpenoids.

Metabolic reprogramming is a hallmark of malignancy. It implements profound metabolic changes to sustain cancer cell survival and proliferation. Although the Warburg effect is a common feature of metabolic reprogramming, recent studies have revealed that tumor cells also depend on mitochondrial metabolism. Due to the essential role of mitochondria in metabolism and cell survival, targeting mitochondria in cancer cells is an attractive therapeutic strategy. However, the metabolic flexibility of cancer cells may enable the upregulation of compensatory pathways, such as glycolysis, to support cancer cell survival when mitochondrial metabolism is inhibited. Thus, compounds capable of targeting both mitochondrial metabolism and glycolysis may help overcome such resistance mechanisms. Normal prostate epithelial cells have a distinct metabolism as they use glucose to sustain physiological citrate secretion. During the transformation process, prostate cancer cells consume citrate to mainly power oxidative phosphorylation and fuel lipogenesis. A growing number of studies have assessed the impact of triterpenoids on prostate cancer metabolism, underlining their ability to hit different metabolic targets. In this review, we critically assess the metabolic transformations occurring in prostate cancer cells. We will then address the opportunities and challenges in using triterpenoids as modulators of prostate cancer cell metabolism.

OsGERLP: A novel aluminum tolerance rice gene isolated from a local cultivar in Indonesia

There is a decrease in the land available for rice cultivation due to the rapid conversion to urban uses. Subsequently, acid soil could be an alternative land cultivating rice, but will require the use of aluminum (Al)-tolerant rice varieties. This Al tolerance trait is genetically controlled, and there is a need to discover more genes needed to develop Al-tolerant rice. Therefore, the objective of this study was to clone and characterize a novel Al tolerance gene isolated from a local cultivar of Indonesian rice. The gene cloning was conducted based on the rye/rice microsynteny relationship. In addition, the root growth and gene expression analyses were performed to verify the role of the gene on Al tolerance in gene-silenced rice and in overexpressed transgenic tobacco. The results showed an Al tolerance candidate gene, OsGERLP, was successfully cloned from rice cv. Hawara Bunar, with its gene encoding a protein similar to a bacterial ribosomal L32 protein. Additionally, the analysis showed that low gene expression caused the gene-silenced rice to be sensitive to Al, while high expression induced the Al tolerance in transgenic tobacco. Furthermore, it was discovered that the gene expression level in both plants was in line with the lower expression of the OsFRDL4 gene in the silenced rice and the high expression of the MATE gene in transgenic tobacco also with the higher citrate secretion from transgenic tobacco roots. In conclusion, the OsGERLP gene could act as a regulator for other Al tolerance genes, with the potential to develop Al-tolerant rice varieties.

Fine-tuning mitochondrial activity in Yarrowia lipolytica for citrate overproduction

Yarrowia lipolytica is a non-conventional yeast with promising industrial potentials for lipids and citrate production. It is also widely used for studying mitochondrial respiration due to a respiratory chain like those of mammalian cells. In this study we used a genome-scale model (GEM) of Y. lipolytica metabolism and performed a dynamic Flux Balance Analysis (dFBA) algorithm to analyze and identify metabolic levers associated with citrate optimization. Analysis of fluxes at stationary growth phase showed that carbon flux derived from glucose is rewired to citric acid production and lipid accumulation, whereas the oxidative phosphorylation (OxPhos) shifted to the alternative respiration mode through alternative oxidase (AOX) protein. Simulations of optimized citrate secretion flux resulted in a pronounced lipid oxidation along with reactive oxygen species (ROS) generation and AOX flux inhibition. Then, we experimentally challenged AOX inhibition by adding n-Propyl Gallate (nPG), a specific AOX inhibitor, on Y. lipolytica batch cultures at stationary phase. Our results showed a twofold overproduction of citrate (20.5 g/L) when nPG is added compared to 10.9 g/L under control condition (no nPG addition). These results suggest that ROS management, especially through AOX activity, has a pivotal role on citrate/lipid flux balance in Y. lipolytica. All taken together, we thus provide for the first time, a key for the understanding of a predominant metabolic mechanism favoring citrate overproduction in Y. lipolytica at the expense of lipids accumulation.

Metabolic acclimation supports higher aluminium-induced secretion of citrate and malate in an aluminium-tolerant hybrid clone of Eucalyptus

BackgroundEucalyptus is the main plantation wood species, mostly grown in aluminized acid soils. To understand the response of Eucalyptus clones to aluminum (Al) toxicity, the Al-tolerant Eucalyptus grandis × E. urophylla clone GL-9 (designated “G9”) and the Al-sensitive E. urophylla clone GL-4 (designated “W4”) were employed to investigate the production and secretion of citrate and malate by roots.ResultsEucalyptus seedlings in hydroponics were exposed to the presence or absence of 4.4 mM Al at pH 4.0 for 24 h. The protein synthesis inhibitor cycloheximide (CHM) and anion channel blocker phenylglyoxal (PG) were applied to explore possible pathways involved in organic acid secretion. The secretion of malate and citrate was earlier and greater in G9 than in W4, corresponding to less Al accumulation in G9. The concentration of Al in G9 roots peaked after 1 h and decreased afterwards, corresponding with a rapid induction of malate secretion. A time-lag of about 6 h in citrate efflux in G9 was followed by robust secretion to support continuous Al-detoxification. Malate secretion alone may alleviate Al toxicity because the peaks of Al accumulation and malate secretion were simultaneous in W4, which did not secrete appreciable citrate. Enhanced activities of citrate synthase (CS) and phosphoenolpyruvate carboxylase (PEPC), and reduced activities of isocitrate dehydrogenase (IDH), aconitase (ACO) and malic enzyme (ME) were closely associated with the greater secretion of citrate in G9. PG effectively inhibited citrate and malate secretion in both Eucalyptus clones. CHM also inhibited malate and citrate secretion in G9, and citrate secretion in W4, but notably did not affect malate secretion in W4.ConclusionsG9 immediately secrete malate from roots, which had an initial effect on Al-detoxification, followed by time-delayed citrate secretion. Pre-existing anion channel protein first contributed to malate secretion, while synthesis of carrier protein appeared to be needed for citrate excretion. The changes of organic acid concentrations in response to Al can be achieved by enhanced CS and PEPC activities, but was supported by changes in the activities of other enzymes involved in organic acid metabolism. The above information may help to further explore genes related to Al-tolerance in Eucalyptus.