Citrate Transport Research Articles

Reply.

Potential conflict of interest: Nothing to report. Reply: Chen et al. propose a further loop to the IL‐6/STAT3–mIndy axis1 by recalling that interleukin‐6 (IL‐6) is an important player in chronic inflammation–related hepatocellular carcinoma (HCC). Moreover, excellent work in human hepatoma cell lines showed that RNAi‐mediated knockdown of the citrate transporter mINDY was associated with a decrease in cell proliferation, colony formation, and expression of cyclin B1. Metabolically, intracellular levels of citrate, the ATP/ADP ratio, phospholipid content, ATP citrate lyase expression, and mTOR activity decreased, whereas AMPK was activated,2 validating the effect of mIndy deletion in vivo.3 We do not know whether the findings in mice, human hepatoma cell lines, and human liver samples translate into chronic, physiologically significant effects in patients with HCC. Presumably, however, IL‐6 contributes to HCC development together with a complex molecular network, including phospholipids, citrate, AMPK, and mTOR. This part of the argument by Chen et al., therefore, is pertinent and plausible. It is thus reassuring that small molecule inhibitors of mINDY are available,4 because they may serve as interesting tools for the study of mIndy and citrate in hepatic tumorigenesis. Moreover, according to the data discussed above, inhibition of mINDY may hold the potential to be effective in the treatment of HCC; however, this notion needs to be studied further. Thus far, pharmacological inhibition of mINDY has been shown to be effective in improving glucose tolerance and reducing liver fat in diet‐induced obese mice,4 effects that also protect against HCC.

Citrate, low pH and amino acid limitation induce citrate utilization in Lactococcus lactis biovar diacetylactis.

SummaryIn Lactococcus lactis subsp. lactis biovar diacetylactis, citrate transport is facilitated by the plasmid‐encoded citrate permease (CitP). In this work, we analysed regulation of citrate utilization by pH, nutrient limitation and the presence of citrate at four different levels: (i) plasmid copy number, (ii) citP transcription, (iii) citP mRNA processing and (iv) citrate utilization capacity. Citrate was supplied as cosubstrate together with lactose. The citP gene is known to be induced in cells grown at low pH. However, we demonstrated that transcription of citP was even higher in the presence of citrate (3.8‐fold compared with 2.0‐fold). The effect of citrate has been overlooked by other researchers because they determined the effect of citrate using M17 medium, which already contains 0.80 ± 0.07 mM citrate. The plasmid copy number increased in cells grown under amino acid limitation (1.6‐fold) and/or at low pH (1.4‐fold). No significant differences in citP mRNA processing were found. Citrate utilization rates increased from approximately 1 to 65 μmol min−1 gDW−1 from lowest to highest citP expression. Acetoin formation increased during growth in an acidic environment due to induction of the acetoin pathway. Quantification of the relative contributions allowed us to construct a model for regulation of citrate utilization in L. lactis biovar diacetylactis. This knowledge will help to select conditions to improve flavour formation from citrate.

Novel mechanisms for organic acid-mediated aluminium tolerance in roots and leaves of two contrasting soybean genotypes.

Aluminium (Al) toxicity is one of the most important limiting factors for crop yield in acidic soils. However, the mechanisms that confer Al tolerance still remain largely unknown. To understand the molecular mechanism that confers different tolerance to Al, we performed global transcriptome analysis to the roots and leaves of two contrasting soybean genotypes, BX10 (Al-tolerant) and BD2 (Al-sensitive) under 0 and 50 μM Al3+ treatments, respectively. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses revealed that the expression levels of the genes involved in lipid/carbohydrate metabolism and jasmonic acid (JA)-mediated signalling pathway were highly induced in the roots and leaves of both soybean genotypes. The gene encoding enzymes, including pyruvate kinase, phosphoenolpyruvate carboxylase, ATP-citrate lyase and glutamate-oxaloacetate transaminase 2, associated with organic acid metabolism were differentially expressed in the BX10 roots. In addition, the genes involved in citrate transport were differentially expressed. Among these genes, FRD3b was down-regulated only in BD2, whereas the other two multidrug and toxic compound extrusion genes were up-regulated in both soybean genotypes. These findings confirmed that BX10 roots secreted more citrate than BD2 to withstand Al stress. The gene encoding enzymes or regulators, such as lipoxygenase, 12-oxophytodienoate reductase, acyl-CoA oxidase and jasmonate ZIM-domain proteins, involved in JA biosynthesis and signalling were preferentially induced in BD2 leaves. This finding suggests that the JA defence response was activated, possibly weakening the growth of aerial parts because of excessive resource consumption and ATP biosynthesis deficiency. Our results suggest that the Al sensitivity in some soybean varieties could be attributed to the low level of citrate metabolism and exudation in the roots and the high level of JA-mediated defence response in the leaves.

Pathogenic mutations of the human mitochondrial citrate carrier SLC25A1 lead to impaired citrate export required for lipid, dolichol, ubiquinone and sterol synthesis

Missense mutations of the human mitochondrial citrate carrier, encoded by the SLC25A1 gene, lead to an autosomal recessive neurometabolic disorder characterised by neonatal-onset encephalopathy with severe muscular weakness, intractable seizures, respiratory distress, and lack of psychomotor development, often resulting in early death. Here, we have measured the effect of all twelve known pathogenic mutations on the transport activity. The results show that nine mutations abolish transport of citrate completely, whereas the other three reduce the transport rate by >70%, indicating that impaired citrate transport is the most likely primary cause of the disease. Some mutations may be detrimental to the structure of the carrier, whereas others may impair key functional elements, such as the substrate binding site and the salt bridge network on the matrix side of the carrier. To understand the consequences of impaired citrate transport on metabolism, the substrate specificity was also determined, showing that the human citrate carrier predominantly transports citrate, isocitrate, cis-aconitate, phosphoenolpyruvate and malate. Although D-2- and L-2 hydroxyglutaric aciduria is a metabolic hallmark of the disease, it is unlikely that the citrate carrier plays a significant role in the removal of hydroxyglutarate from the cytosol for oxidation to oxoglutarate in the mitochondrial matrix. In contrast, computer simulations of central metabolism predict that the export of citrate from the mitochondrion cannot be fully compensated by other pathways, restricting the cytosolic production of acetyl-CoA that is required for the synthesis of lipids, sterols, dolichols and ubiquinone, which in turn explains the severe disease phenotypes.

The longevity gene INDY (I'm Not Dead Yet) in metabolic control: Potential as pharmacological target.

The regulation of metabolic processes by the Indy (I'm Not Dead Yet) (SLC13A5/NaCT) gene was revealed through studies in Drosophila melanogaster and Caenorhabditis elegans. Reducing the expression of Indy in these species extended their life span by a mechanism resembling caloric restriction, without reducing food intake. In D. melanogaster, mutating the Indy gene reduced body fat content, insulin-like proteins and reactive oxygen species production. Subsequent studies indicated that Indy encodes a citrate transporter located on the cell plasma membrane. The transporter is highly expressed in the mammalian liver. We generated a mammalian knock out model deleting the mammalian homolog mIndy (SLC13A5). The knock out animals were protected from HFD induced obesity, fatty liver and insulin resistance. Moreover, we have shown that inducible and liver selective knock down of mIndy protects against the development of fatty liver and insulin resistance and that obese humans with type 2 diabetes and non-alcoholic fatty liver disease have increased levels of mIndy. Therefore, the transporter mINDY (NaCT) has been proposed to be an 'ideal target for the treatment of metabolic disease'. A small molecule inhibitor of the mINDY transporter has been generated, normalizing glucose levels and reducing fatty liver in a model of diet induced obese mice. Taken together, studies from lower organisms, mammals and humans suggest that mINDY (NaCT) is an attractive target for the treatment of metabolic disease.

Characterization of CcSTOP1; a C2H2-type transcription factor regulates Al tolerance gene in pigeonpea.

Al-responsive citrate-transporting CcMATE1 function and its regulation by CcSTOP1 were analyzed using NtSTOP1 -KD tobacco- and pigeonpea hairy roots, respectively, CcSTOP1 binding sequence of CcMATE1 showed similarity with AtALMT1 promoter. The molecular mechanisms of Aluminum (Al) tolerance in pigeonpea (Cajanus cajan) were characterized to provide information for molecular breeding. Al-inducible citrate excretion was associated with the expression of MULTIDRUGS AND TOXIC COMPOUNDS EXCLUSION (CcMATE1), which encodes a citrate transporter. Ectopic expression of CcMATE1-conferred Al tolerance to hairy roots of transgenic tobacco with the STOP1 regulation system knocked down. This gain-of-function approach clearly showed CcMATE1 was involved in Al detoxification. The expression of CcMATE1 and another Al-tolerance gene, ALUMINUM SENSITIVE 3 (CcALS3), was regulated by SENSITIVE TO PROTON RHIZOTOXICITY1 (CcSTOP1) according to loss-of-function analysis of pigeonpea hairy roots in which CcSTOP1 was suppressed. An in vitro binding assay showed that the Al-responsive CcMATE1 promoter contained the GGNVS consensus bound by CcSTOP1. Mutation of GGNVS inactivated the Al-inducible expression of CcMATE1 in pigeonpea hairy roots. This indicated that CcSTOP1 binding to the promoter is critical for CcMATE1 expression. The STOP1 binding sites of both the CcMATE1 and AtALMT1 promoters contained GGNVS and a flanking 3' sequence. The GGNVS region was identical in both CcMATE1 and AtALMT1. By contrast, the 3' flanking sequence with binding affinity to STOP1 did not show similarity. Putative STOP1 binding sites with similar structures were also found in Al-inducible MATE and ALMT1 promoters in other plant species. The characterized Al-responsive CcSTOP1 and CcMATE1 genes will help in pigeonpea breeding in acid soil tolerance.

Methane enhances aluminum resistance in alfalfa seedlings by reducing aluminum accumulation and reestablishing redox homeostasis

Methane (CH4) is emerging as a candidate of signal molecule recently. However, whether or how CH4 enhances plant adaptation to aluminum (Al)-contaminated environment is still unknown. In this report, the physiological roles and possible molecular mechanisms of CH4 in the modulation of Al toxicity in alfalfa seedlings were characterized. Our results showed that, CH4 pretreatment could alleviate Al-induced seedling growth inhibition and redox imbalance. The defensive effects of CH4 against Al toxicity including the remission of Al-induced root elongation inhibition, nutrient disorder, and relative electrolyte leakage. Moreover, contents of organic acids, including citrate, malate, and oxalate, were increased by CH4. These results were paralleled by the findings of CH4 regulated organic acids metabolism and transport genes, citrate synthase, malate dehydrogenase, aluminum-activated malate transporter, and aluminum activated citrate transporter. Consistently, Al accumulation in seedling roots was decreased after CH4 treatment. In addition, Al-induced oxidative stress was also alleviated by CH4, through the regulation of the activities of anti-oxidative enzymes, such as ascorbate peroxidase, superoxide dismutase, and peroxidase, as well as their corresponding transcripts. Our data clearly suggested that CH4 alleviates Al toxicity by reducing Al accumulation in organic acid-dependent fashion, and reestablishing redox homeostasis.

Silencing of solute carrier family 13 member 5 disrupts energy homeostasis and inhibits proliferation of human hepatocarcinoma cells

The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays a key role in importing citrate from the circulation into liver cells. Recent evidence has revealed that SLC13A5 deletion protects mice from high-fat diet-induced hepatic steatosis and that mutation of the SLC13A5 orthologues in Drosophila melanogaster and Caenorhabditis elegans promotes longevity. However, despite the emerging importance of SLC13A5 in energy homeostasis, whether perturbation of SLC13A5 affects the metabolism and malignancy of hepatocellular carcinoma is unknown. Here, we sought to determine whether SLC13A5 regulates hepatic energy homeostasis and proliferation of hepatoma cells. RNAi-mediated silencing of SLC13A5 expression in two human hepatoma cell lines, HepG2 and Huh7, profoundly suppressed cell proliferation and colony formation, and induced cell cycle arrest accompanied by increased expression of cyclin-dependent kinase inhibitor p21 and decreased expression of cyclin B1. Furthermore, such suppressive effects were also observed on the growth of HepG2 cell-derived xenografts expressing SLC13A5-shRNA in nude mice. Metabolically, knockdown of SLC13A5 in HepG2 and Huh7 cells was associated with a decrease in intracellular levels of citrate, the ratio of ATP/ADP, phospholipid content, and ATP citrate lyase expression. Moreover, both in vitro and in vivo assays demonstrated that SLC13A5 depletion promotes activation of the AMP-activated protein kinase, which was accompanied by deactivation of oncogenic mechanistic target of rapamycin signaling. Together, our findings expand the role of SLC13A5 from facilitating hepatic energy homeostasis to influencing hepatoma cell proliferation and suggest a potential role of SLC13A5 in the progression of human hepatocellular carcinoma.

Abstract 1549: Prognostic significance and tumor suppressive functions of SDCT2 in renal cell carcinoma

Abstract Introduction: Five-year survival of metastatic renal cell carcinoma (mRCC) patients is < 10% and African American (AA) males have the highest incidence. Identification of the molecular determinants of mRCC and racial disparity in RCC is critical for biomarker development and targeted therapy. SDCT2 is expressed in kidney epithelial cells and is a succinate and citrate transporter, but its role has not been examined in any benign diseases or cancer. We examined SDCT2 expression in normal and RCC tissues and correlated it with clinical outcome and racial disparity. We also evaluated the biological functions and molecular signaling regulated by SDCT2 in RCC cells. Methods: Differential gene expression in the matched normal and RCC tissues (n=6/category) was evaluated by microarray analysis; results were validated by quantitative-PCR and immunoblotting in normal and RCC tissues from 53 patients (White=21; Hispanic=19; AA=13). VHL+ and VHL- RCC cells were stably transfected with a Flag-tagged SDCT2 construct. Transfectants were characterized for cell proliferation, cell cycle, motility, succinate/citrate transport and reactive oxygen species (ROS) measurement assays under normoxia and hypoxia (1% O2); cell death and senescence pathway markers were also evaluated. SDCT2 induction was evaluated following 5-azacytidine plus Trichostatin A treatment Results: SDCT2 was 63- and 100-fold downregulated in low- and high-stage RCC tissues, respectively. Q-PCR validation showed that SDCT2 levels were 40-fold downregulated in tumor tissues when compared to normal kidney (P<0.0001 Mann-Whitney test). Downregulation was 40-fold in White and Hispanic patients, but 198-fold in AA patients (P=0.0049) and also correlated with tumor stage and metastasis (P=0.009). Under hypoxia, SDCT2 expression caused over 3-fold inhibition of proliferation, cell-cycle (G1-S block), and motility in both VHL+ and VHL- cells (P<0.01), only VHL+ cells were inhibited under normoxia. SDCT2 expression induced ROS levels and succinate transport by 3-fold in RCC cells (P<0.01). SDCT2 expression induced the p16INK4a-RB pathway and apoptosis (caspase-3 and PARP activation). 5-AZA+TSA treatment caused a 50-fold induction (P<0.0001) of SDCT2 expression. Conclusion: This is the first study on a functional biomarker in RCC, SDCT2, that is a possible novel tumor suppressor gene. SDCT2 loss promotes RCC growth, survival and inhibits cellular senescence and its downregulation correlates with metastasis and racial disparity. Support: Grant NCI/NIH 5R01CA72821; 5R01CA176691 Citation Format: Andre R. Jordan, Martin Hennig, Daley S. Morera, Soum D. Lokeshwar, Asif Talukder, Lokeshwar Vinata. Prognostic significance and tumor suppressive functions of SDCT2 in renal cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1549. doi:10.1158/1538-7445.AM2017-1549

The human longevity gene homolog INDY and interleukin-6 interact in hepatic lipid metabolism.

Targeting human mINDY may have therapeutic potential in obese patients with nonalcoholic fatty liver disease. German Clinical Trials Register: DRKS00005450. (Hepatology 2017;66:616-630).

Overexpression of BdMATE Gene Improves Aluminum Tolerance in Setaria viridis.

Acidic soils are distributed worldwide, predominantly in tropical and subtropical areas, reaching around 50% of the arable soil. This type of soil strongly reduces crop production, mainly because of the presence of aluminum, which has its solubility increased at low pH levels. A well-known physiological mechanism used by plants to cope with Al stress involves activation of membrane transporters responsible for organic acid anions secretion from the root apex to the rhizosphere, which chelate Al, preventing its absorption by roots. In sorghum, a membrane transporter gene belonging to multidrug and toxic compound extrusion (MATE) family was identified and characterized as an aluminum-activated citrate transporter gene responsible for Al tolerance in this crop. Setaria viridis is an emerging model for C4 species and it is an important model to validate some genes for further C4 crops transformation, such as sugarcane, maize, and wheat. In the present work, Setaria viridis was used as a model plant to overexpress a newly identified MATE gene from Brachypodium distachyon (BdMATE), closely related to SbMATE, for aluminum tolerance assays. Transgenic S. viridis plants overexpressing a BdMATE presented an improved Al tolerance phenotype, characterized by sustained root growth and exclusion of aluminum from the root apex in transgenic plants, as confirmed by hematoxylin assay. In addition, transgenic plants showed higher root citrate exudation into the rhizosphere, suggesting that Al tolerance improvement in these plants could be related to the chelation of the metal by the organic acid anion. These results suggest that BdMATE gene can be used to transform C4 crops of economic importance with improved aluminum tolerance.

Structural insights into the elevator-like mechanism of the sodium/citrate symporter CitS

The sodium-dependent citrate transporter of Klebsiella pneumoniae (KpCitS) belongs to the 2-hydroxycarboxylate transporter (2-HCT) family and allows the cell to use citrate as sole carbon and energy source in anaerobic conditions. Here we present crystal structures of KpCitS in citrate-bound outward-facing, citrate-bound asymmetric, and citrate-free inward-facing state. The structures reveal that the KpCitS dimerization domain remains stationary throughout the transport cycle due to a hydrogen bond network as well as extensive hydrophobic interactions. In contrast, its transport domain undergoes a ~35° rigid-body rotation and a ~17 Å translocation perpendicular to the membrane to expose the substrate-binding site alternately to either side of the membrane. Furthermore, homology models of two other 2-HCT proteins based on the KpCitS structure offer structural insights into their differences in substrate specificity at a molecular level. On the basis of our results and previous biochemical data, we propose that the activity of the 2-HCT CitS involves an elevator-like movement in which the transport domain itself traverses the lipid bilayer, carrying the substrate into the cell in a sodium-dependent manner.

INDY-A New Link to Metabolic Regulation in Animals and Humans.

The Indy (I’m Not Dead Yet) gene encodes the fly homolog of the mammalian SLC13A5 citrate transporter. Reduced expression of the Indy gene in flies and worms extends their longevity. INDY is expressed in the plasma membrane of metabolically active tissues. Decreased expression of Indy in worms, flies, mice, and rats alters metabolism in a manner similar to calorie restriction. Reducing INDY activity prevents weight gain in flies, worms, and mice, and counteracts the negative effects of age or a high fat diet on metabolism and insulin sensitivity. The metabolic effects of reducing INDY activity are the result of reduced cytoplasmic citrate. Citrate is a key metabolite and has a central role in energy status of the cell by effecting lipid and carbohydrate metabolism and energy production. Thereby newly described drugs that reduce INDY transporting activity increase insulin sensitivity and reduce hepatic lipid levels via its effect on hepatic citrate uptake. A recent report presented the first direct link between increased hepatic levels of human INDY, insulin resistance, and non-alcoholic fatty liver disease in obese humans. Similarly increased hepatic mIndy levels were observed in non-human primates fed on a high fat diet for 2 years. This effect is mediated via the stimulatory effect of the interleukin-6/Stat3 pathway on mINDY hepatic expression. These findings make INDY a potential and very promising target for the treatment of metabolic disorders in humans.

Defective enamel and bone development in sodium-dependent citrate transporter (NaCT) Slc13a5 deficient mice.

There has been growing recognition of the essential roles of citrate in biomechanical properties of mineralized tissues, including teeth and bone. However, the sources of citrate in these tissues have not been well defined, and the contribution of citrate to the regulation of odontogenesis and osteogenesis has not been examined. Here, tooth and bone phenotypes were examined in sodium-dependent citrate transporter (NaCT) Slc13a5 deficient C57BL/6 mice at 13 and 32 weeks of age. Slc13a5 deficiency led to defective tooth development, characterized by absence of mature enamel, formation of aberrant enamel matrix, and dysplasia and hyperplasia of the enamel organ epithelium that progressed with age. These abnormalities were associated with fragile teeth with a possible predisposition to tooth abscesses. The lack of mature enamel was consistent with amelogenesis imperfecta. Furthermore, Slc13a5 deficiency led to decreased bone mineral density and impaired bone formation in 13-week-old mice but not in older mice. The findings revealed the potentially important role of citrate and Slc13a5 in the development and function of teeth and bone.

Plasma Membrane Na⁺-Coupled Citrate Transporter (SLC13A5) and Neonatal Epileptic Encephalopathy.

SLC13A5 is a Na+-coupled transporter for citrate that is expressed in the plasma membrane of specific cell types in the liver, testis, and brain. It is an electrogenic transporter with a Na+:citrate3− stoichiometry of 4:1. In humans, the Michaelis constant for SLC13A5 to transport citrate is ~600 μM, which is physiologically relevant given that the normal concentration of citrate in plasma is in the range of 150–200 μM. Li+ stimulates the transport function of human SLC13A5 at concentrations that are in the therapeutic range in patients on lithium therapy. Human SLC13A5 differs from rodent Slc13a5 in two important aspects: the affinity of the human transporter for citrate is ~30-fold less than that of the rodent transporter, thus making human SLC13A5 a low-affinity/high-capacity transporter and the rodent Slc13a5 a high-affinity/low-capacity transporter. In the liver, SLC13A5 is expressed exclusively in the sinusoidal membrane of the hepatocytes, where it plays a role in the uptake of circulating citrate from the sinusoidal blood for metabolic use. In the testis, the transporter is expressed only in spermatozoa, which is also only in the mid piece where mitochondria are located; the likely function of the transporter in spermatozoa is to mediate the uptake of citrate present at high levels in the seminal fluid for subsequent metabolism in the sperm mitochondria to generate biological energy, thereby supporting sperm motility. In the brain, the transporter is expressed mostly in neurons. As astrocytes secrete citrate into extracellular medium, the potential function of SLC13A5 in neurons is to mediate the uptake of circulating citrate and astrocyte-released citrate for subsequent metabolism. Slc13a5-knockout mice have been generated; these mice do not have any overt phenotype but are resistant to experimentally induced metabolic syndrome. Recently however, loss-of-function mutations in human SLC13A5 have been found to cause severe epilepsy and encephalopathy early in life. Interestingly, there is no evidence of epilepsy or encephalopathy in Slc13a5-knockout mice, underlining the significant differences in clinical consequences of the loss of function of this transporter between humans and mice. The markedly different biochemical features of human SLC13A5 and mouse Slc13a5 likely contribute to these differences between humans and mice with regard to the metabolic consequences of the transporter deficiency. The exact molecular mechanisms by which the functional deficiency of the citrate transporter causes epilepsy and impairs neuronal development and function remain to be elucidated, but available literature implicate both dysfunction of GABA (γ-aminobutyrate) signaling and hyperfunction of NMDA (N-methyl-d-aspartate) receptor signaling. Plausible synaptic mechanisms linking loss-of-function mutations in SLC13A5 to epilepsy are discussed.

Regulation of Carbon Substrate Utilization by Cardiac Mitochondria

Mitochondria play key roles in central metabolism, not only by synthesizing ATP via oxidative phosphorylation, but also by synthesizing key intermediates that serve as anaplerotic entry points in numerous pathways. Because reactions producing and/or consuming several of the intermediates in the tricarboxylic acid cycle that are transported across the mitochondrial inner membrane (pyruvate, α-ketoglutarate, citrate) are regulated in part by calcium concentration, we hypothesize the calcium is a key regulator of entry and exit of substrates into and out of the TCA cycle. We set out to first refine this hypothesis through following time courses of consumption of various carbohydrate based substrates (including complex I substrates such as pyruvate and citrate, and the complex II substrate succinate) and matching data on rates of oxidation and NADH production to simulations of mitochondrial metabolic kinetics. Specifically, model simulations were fit to data on total NAD(P)H and oxygen consumption flux at different respiratory states and with varying inorganic phosphate concentrations. The resulting identified model is used to predict how calcium regulates carbon substrate utilization by mitochondria. These hypotheses are tested using in vitro experiments with purified mitochondria. By comparing the experimental data with citrate alone to combination of pyruvate, citrate and malate as the substrates, we conclude that transport of citrate into the matrix is limited in the absence of malate. The rate of reduction of NAD is slower with pyruvate alone as substrate compared to when malate is present. The role of glutamate dehydrogenase in the context of cardiac energetics is investigated.

Erratum to: Genome-wide exploration of silicon (Si) transporter genes, Lsi1 and Lsi2 in plants; insights into Si-accumulation status/capacity of plants.

Silicon (Si) is a nonessential, beneficial micronutrient for plants. It increases the plant stress tolerance in relation to its accumulation capacity. In this work, root Si transporter genes were characterized in 17 different plants and inferred for their Si-accumulation status. A total of 62 Si transporter genes (31 Lsi1 and 31 Lsi2) were identified in studied plants. Lsi1s were 261–324 residues protein with a MIP family domain whereas Lsi2s were 472–547 residues with a citrate transporter family domain. Lsi1s possessed characteristic sequence features that can be employed as benchmark in prediction of Si-accumulation status/capacity of the plants. Silicic acid selectivity in Lsi1s was associated with two highly conserved NPA (Asn-Pro-Ala) motifs and a Gly-Ser-Gly-Arg (GSGR) ar/R filter. Two NPA regions were present in all Lsi1 members but some Ala substituted with Ser or Val. GSGR filter was only available in the proposed high and moderate Si accumulators. In phylogeny, Lsi1s formed three clusters as low, moderate and high Si accumulators based on tree topology and availability of GSGR filter. Low-accumulators contained filters WIGR, AIGR, FAAR, WVAR and AVAR, high-accumulators only with GSGR filter, and moderate-accumulators mostly with GSGR but some with A/CSGR filters. A positive correlation was also available between sequence homology and Si-accumulation status of the tested plants. Thus, availability of GSGR selectivity filter and sequence homology degree could be used as signatures in prediction of Si-accumulation status in experimentally uncharacterized plants. Moreover, interaction partner and expression profile analyses implicated the involvement of Si transporters in plant stress tolerance.

Genome-wide exploration of silicon (Si) transporter genes, Lsi1 and Lsi2 in plants; insights into Si-accumulation status/capacity of plants.

Silicon (Si) is a nonessential, beneficial micronutrient for plants. It increases the plant stress tolerance in relation to its accumulation capacity. In this work, root Si transporter genes were characterized in 17 different plants and inferred for their Si-accumulation status. A total of 62 Si transporter genes (31 Lsi1 and 31 Lsi2) were identified in studied plants. Lsi1s were 261-324 residues protein with a MIP family domain whereas Lsi2s were 472-547 residues with a citrate transporter family domain. Lsi1s possessed characteristic sequence features that can be employed as benchmark in prediction of Si-accumulation status/capacity of the plants. Silicic acid selectivity in Lsi1s was associated with two highly conserved NPA (Asn-Pro-Ala) motifs and a Gly-Ser-Gly-Arg (GSGR) ar/R filter. Two NPA regions were present in all Lsi1 members but some Ala substituted with Ser or Val. GSGR filter was only available in the proposed high and moderate Si accumulators. In phylogeny, Lsi1s formed three clusters as low, moderate and high Si accumulators based on tree topology and availability of GSGR filter. Low-accumulators contained filters WIGR, AIGR, FAAR, WVAR and AVAR, high-accumulators only with GSGR filter, and moderate-accumulators mostly with GSGR but some with A/CSGR filters. A positive correlation was also available between sequence homology and Si-accumulation status of the tested plants. Thus, availability of GSGR selectivity filter and sequence homology degree could be used as signatures in prediction of Si-accumulation status in experimentally uncharacterized plants. Moreover, interaction partner and expression profile analyses implicated the involvement of Si transporters in plant stress tolerance.

Is a non-synonymous SNP in the HvAACT1 coding region associated with acidic soil tolerance in barley?

The barley HvAACT1 gene codes for a citrate transporter associated with tolerance to acidic soil. In this report, we describe a single nucleotide polymorphism (SNP) in the HvAACT1 coding region that was detected as T-1,198 (in genotypes with lower root growth on acidic soil) or G-1,198 (greater root growth) and resulted in a single amino acid change (L/V-172). Molecular dynamic analysis predicted that HvAACT1 proteins with L or V-172 were stable, although the substitution led to structural changes within the protein. To evaluate the effect of the SNP on tolerance to acidic soil, barley accessions were separated into haplotypes based on the presence of a 1 kb insertion in the HvAACT1 promoter and a 21 bp insertion/deletion. These markers and the SNP-1,198 allowed the identification of five haplotypes. Short-term soil experiments showed no difference in root growth for most of the accessions containing the 21 bp insertion and T or G-1,198. In contrast, genotypes showing both the 21 bp deletion and G-1,198, with one of them having the 1 kb insertion, showed greater root growth. These results indicate that the SNP was not advantageous or deleterious when genotypes from the same haplotype were compared. The occurrence of the SNP was highly correlated with the 21 bp insertion/deletion that, together with the 1 kb insertion, explained most of the barley tolerance to acidic soil.

Genome Analysis of a Limnobacter sp. Identified in an Anaerobic Methane-Consuming Cell Consortium

Species of Limnobacter genus are widespread in a variety of environments, yet knowledges upon their metabolic potentials and mechanisms of environmental adaptation are limited. In this study, a cell aggregate containing Limnobacter and anaerobic methanotrophic archaea (ANME) was captured from an enriched anaerobic methane oxidizing (AOM) microbial community. A genomic bin of Limnobacter was obtained and analyzed, which provides the first metabolic insights into Limnobacter from an AOM environment. This Limnobacter was found to contain genes involved in the Embden-Meyerhof pathway, the citrate cycle, citronellol degradation, and transporters of various organic substances, indicating a potentially heterotrophic lifestyle. A number of genes involved in sulfur oxidization, oxidative phosphorylation and ethanol fermentation that serve both aerobic and anaerobic purposes have been found in Limnobacter. This work suggests that in the AOM environment, Limnobacter strains may live on the organic substances produced through AOM activity and subsequently may contribute to the AOM community by providing sulfate from sulfur oxidation.