Plant growth and nitrate absorption and assimilation of two sweet potato cultivars with different N tolerances in response to nitrate supply.

In sweet potato, rational nitrogen (N) assimilation and distribution are conducive to inhibiting vine overgrowth. Nitrate (NO3-) is the main N form absorbed by roots, and cultivar is an important factor affecting N utilization. Herein, a hydroponic experiment was conducted that included four NO3- concentrations of 0 (N0), 4 (N1), 8 (N2) and 16 (N3) mmol L-1 with two cultivars of Jishu26 (J26, N-sensitive) and Xushu32 (X32, N-tolerant). For J26, with increasing NO3- concentrations, the root length and root surface area significantly decreased. However, no significant differences were observed in these parameters for X32. Higher NO3- concentrations upregulated the expression levels of the genes that encode nitrate reductase (NR2), nitrite reductase (NiR2) and nitrate transporter (NRT1.1) in roots for both cultivars. The trends in the activities of NR and NiR were subject to regulation of NR2 and NiR2 transcription, respectively. For both cultivars, N2 increased the N accumulated in leaves, growth points and roots. For J26, N3 further increased the N accumulation in these organs. Under higher NO3- nutrition, compared with X32, J26 exhibited higher expression levels of the NiR2, NR2 and NRT1.1 genes, a higher influx NO3- rate in roots, and higher activities of NR and NiR in leaves and roots. Conclusively, the regulated effects of NO3- supplies on root growth and NO3- utilization were more significant for J26. Under high NO3- conditions, J26 exhibited higher capacities of NO3- absorption and distributed more N in leaves and in growth points, which may contribute to higher growth potential in shoots and more easily cause vine overgrowth.

Two new mouse alleles of Ocm and Slc26a5.

The genes Ocm (encoding oncomodulin) and Slc26a5 (encoding prestin) are expressed strongly in outer hair cells and both are involved in deafness in mice. However, it is not clear if they influence the expression of each other. In this study, we characterise the auditory phenotype resulting from two new mouse alleles, Ocmtm1e and Slc26a5tm1Cre. Each mutation leads to absence of detectable mRNA transcribed from the mutant allele, but there was no evidence that oncomodulin regulates expression of prestin or vice versa. The two mutants show distinctive patterns of auditory dysfunction. Ocmtm1e homozygotes have normal auditory brainstem response thresholds at 4 weeks old followed by progressive hearing loss starting at high frequencies, while heterozygotes show largely normal thresholds until 6 months of age, when signs of worse thresholds are detected. In contrast, Slc26a5tm1Cre homozygotes have stable but raised thresholds across all frequencies tested, 3 to 42 kHz, at least from 4 to 8 weeks old, while heterozygotes have raised thresholds at high frequencies. Distortion product otoacoustic emissions and cochlear microphonics show deficits similar to auditory brainstem responses in both mutants, suggesting that the origin of hearing impairment is in the outer hair cells. Endocochlear potentials are normal in the two mutants. Scanning electron microscopy revealed normal development of hair cells in Ocmtm1e homozygotes but scattered outer hair cell loss even at 4 weeks old when thresholds appeared normal, indicating that there is not a direct relationship between numbers of outer hair cells present and auditory thresholds.

Spinster homolog 2/S1P signaling ameliorates macrophage inflammatory response to bacterial infections by balancing PGE2 production.

BACKGROUND: Mitochondria play a crucial role in shaping the macrophage inflammatory response during bacterial infections. Spinster homolog 2 (Spns2), responsible for sphingosine-1-phosphate (S1P) secretion, acts as a key regulator of mitochondrial dynamics in macrophages. However, the link between Spns2/S1P signaling and mitochondrial functions remains unclear. METHODS: Peritoneal macrophages were isolated from both wild-type and Spns2 knockout rats, followed by non-targeted metabolomics and RNA sequencing analysis to identify the potential mediators through which Spns2/S1P signaling influences the mitochondrial functions in macrophages. Various agonists and antagonists were used to modulate the activation of Spns2/S1P signaling and its downstream pathways, with the underlying mechanisms elucidated through western blotting. Mitochondrial functions were assessed using flow cytometry and oxygen consumption assays, as well as morphological analysis. The impact on inflammatory response was validated through both in vitro and in vivo sepsis models, with the specific role of macrophage-expressed Spns2 in sepsis evaluated using Spns2flox/floxLyz2-Cre mice. Additionally, the regulation of mitochondrial functions by Spns2/S1P signaling was confirmed using THP-1 cells, a human monocyte-derived macrophage model. RESULTS: In this study, we unveil prostaglandin E2 (PGE2) as a pivotal mediator involved in Spns2/S1P-mitochondrial communication. Spns2/S1P signaling suppresses PGE2 production to support malate-aspartate shuttle activity. Conversely, excessive PGE2 resulting from Spns2 deficiency impairs mitochondrial respiration, leading to intracellular lactate accumulation and increased reactive oxygen species (ROS) generation through E-type prostanoid receptor 4 activation. The overactive lactate-ROS axis contributes to the early-phase hyperinflammation during infections. Prolonged exposure to elevated PGE2 due to Spns2 deficiency culminates in subsequent immunosuppression, underscoring the dual roles of PGE2 in inflammation throughout infections. The regulation of PGE2 production by Spns2/S1P signaling appears to depend on the coordinated activation of multiple S1P receptors rather than any single one. CONCLUSIONS: These findings emphasize PGE2 as a key effector of Spns2/S1P signaling on mitochondrial dynamics in macrophages, elucidating the mechanisms through which Spns2/S1P signaling balances both early hyperinflammation and subsequent immunosuppression during bacterial infections.

Congenital hereditary endothelial dystrophy with progressive sensorineural deafness: a case report of Harboyan syndrome.

We present the case of a 37-year-old woman who underwent bilateral penetrating keratoplasty for congenital hereditary endothelial dystrophy at the age of 10 years. Over the subsequent 27 years, the patient's vision slowly deteriorated. Our examination revealed decompensation of the right corneal graft. We addressed this with regraft surgery. We then learned that the patient had been suffering from progressive hearing loss since adolescence. Tonal audiometry revealed hearing per ceptive deafness of 25 dB, which was more prominent in the left ear. Because the patterns of progressive sensorineural hearing loss and congenital hereditary endothelial dystrophy have both been linked to the same gene, slc4a11, we tested our patient for mutations in this gene. The test was positive for a heterozygous slc4a11 gene fifth exon mutation on chromosome 20p13-p12, which causes a frameshift. A combined clinical and genetic evaluation confirmed a diagnosis of Harboyan syndrome. After the genetic diagnosis of the disease, she was evaluated for the need for a hearing aid due to her hearing loss. The patient was also informed about genetic counseling.

Nitrate transporters and mechanisms of nitrate signal transduction in Arabidopsis and rice.

Nitrate (NO3 -) is a significant inorganic nitrogen source in soil, playing a crucial role in influencing crop productivity. As sessile organisms, plants have evolved complex mechanisms for nitrate uptake and response to varying soil levels. Recent advancements have enhanced our understanding of nitrate uptake and signaling pathways. This mini-review offers a comparative analysis of nitrate uptake mechanisms in Arabidopsis and rice. It also examines nitrate signal transduction, highlighting the roles of AtNRT1.1 and AtNLP7 as nitrate receptors and elucidating the OsNRT1.1B-OsSPX4-OsNLP3 cascade. Additionally, it investigates nuclear transcriptional networks that regulate nitrate-responsive genes, controlled by various transcription factors (TFs) crucial for plant development. By integrating these findings, we highlight mechanisms that may help to enhance crop nitrogen utilization.

Regulation of ClC-K/barttin by endocytosis influences distal convoluted tubule hyperplasia.

ClC-K/barttin channels are involved in the transepithelial transport of chloride in the kidney and inner ear. Their physiological role is crucial in humans because mutations in CLCNKB or BSND, encoding ClC-Kb and barttin, cause Bartter's syndrome types III and IV, respectively. In vitro experiments have shown that an amino acid change in a proline-tyrosine motif in the C-terminus of barttin stimulates ClC-K currents. The molecular mechanism of this enhancement and whether this potentiation has any in vivo relevance remains unknown. We performed electrophysiological and biochemical experiments in Xenopus oocytes and kidney cells co-expressing ClC-K and barttin constructs. We demonstrated that barttin possesses a YxxØ motif and, when mutated, increases ClC-K plasma membrane stability, resulting in larger currents. To address the impact of mutating this motif in kidney physiology, we generated a knock-in mouse. Comparing wild-type (WT) and knock-in mice under a standard diet, we could not observe any difference in ClC-K and barttin protein levels or localization, either in urinary or plasma parameters. However, under a high-sodium low-potassium diet, known to induce hyperplasia of distal convoluted tubules, knock-in mice exhibit reduced hyperplasia compared to WT mice. In summary, our in vitro and in vivo studies demonstrate that the previously identified PY motif is indeed an endocytic YxxØ motif in which mutations cause a gain of function of the channel. KEY POINTS: It is revealed by mutagenesis and functional experiments that a previously identified proline-tyrosine motif regulating ClC-K plasma membrane levels is indeed an endocytic YxxØ motif. Biochemical characterization of mutants in the YxxØ motif in Xenopus oocytes and human embryonic kidney cells indicates that mutants showed increased plasma membrane levels as a result of an increased stability, resulting in higher function of ClC-K channels. Mutation of this motif does not affect barttin protein expression and subcellular localization in vivo. Knock-in mice with a mutation in this motif, under conditions of a high-sodium low-potassium diet, exhibit less hyperplasia in the distal convoluted tubule than wild-type animals, indicating a gain of function of the channel in vivo.

A photocaged, pH-sensitive anion transporter with AND logic dual-stimuli activation.

A pH-switchable anion transporter 1 was photocaged with two photolabile groups to enhance spatiotemporal control over its chloride transport activity. Simultaneous application of light irradiation and acidic pH restores the activity of 1, while either stimulus alone results in no or very low activity. The double activation strategy described herein has potential to yield more selectively cytotoxic anionophores for future medical applications.

NRT2.1 mediates the reciprocal regulation of nitrate and NO/SNO in seedling leaves of Fraxinus mandshurica and Betula platyphylla.

Nitric oxide (NO) and S-nitrosothiol (SNO) are signal molecules and the products of nitrogen metabolism. Nitrate (NO3-) is the main nitrogen source, and nitrate transporters (NRTs) are responsible for NO3- absorption or transport. However, the interactive effect between NO3-/NRT and NO/SNO in tree plants remains ambiguous. In the present study, 25 mmol L-1 NO3- and 1 mmol L-1 NO donor sodium nitroprusside (SNP) treatment that was conducted for 24 h enhanced NO/SNO and NO3- metabolism, whereas 2.5 mmol L-1 NO3- and 80 µmol L-1 N6022 (a compound that increases SNO content) treatment reduced them in seedling leaves of Fraxinus mandshurica and Betula platyphylla. Among the nine NRT family members examined, the gene expression level of NRT2.1 had a greater response to NO/SNO and NO3- treatment in the seedling leaves of F. mandshurica and B. platyphylla. Meanwhile, FmNRT2.1 mediated NO and SNO production in seedling leaves of F. mandshurica using Agrobacterium-mediated transient transformation. These findings shed light on the reciprocal regulation between NO3- and NO/SNO in seedlings of F. mandshurica and B. platyphylla, and NRT2.1 may act as a key regulatory hub.

The mitochondrial pyruvate carrier regulates adipose glucose partitioning in female mice.

OBJECTIVE: The mitochondrial pyruvate carrier (MPC) occupies a critical node in intermediary metabolism, prompting interest in its utility as a therapeutic target for the treatment of obesity and cardiometabolic disease. Dysregulated nutrient metabolism in adipose tissue is a prominent feature of obesity pathophysiology, yet the functional role of adipose MPC has not been explored. We investigated whether the MPC shapes the adaptation of adipose tissue to dietary stress in female and male mice. METHODS: The impact of pharmacological and genetic disruption of the MPC on mitochondrial pathways of triglyceride assembly (lipogenesis and glyceroneogenesis) was assessed in 3T3L1 adipocytes and murine adipose explants, combined with analyses of adipose MPC expression in metabolically compromised humans. Whole-body and adipose-specific glucose metabolism were subsequently investigated in male and female mice lacking adipocyte MPC1 (Mpc1AD-/-) and fed either standard chow, high-fat western style, or high-sucrose lipid restricted diets for 24 weeks, using a combination of radiolabeled tracers and GC/MS metabolomics. RESULTS: Treatment with UK5099 or siMPC1 impaired the synthesis of lipids and glycerol-3-phosphate from pyruvate and blunted triglyceride accumulation in 3T3L1 adipocytes, whilst MPC expression in human adipose tissue was negatively correlated with indices of whole-body and adipose tissue metabolic dysfunction. Mature adipose explants from Mpc1AD-/- mice were intrinsically incapable of incorporating pyruvate into triglycerides. In vivo, MPC deletion restricted the incorporation of circulating glucose into adipose triglycerides, but only in female mice fed a zero fat diet, and this associated with sex-specific reductions in tricarboxylic acid cycle pool sizes and compensatory transcriptional changes in lipogenic and glycerol metabolism pathways. However, whole-body adiposity and metabolic health were preserved in Mpc1AD-/- mice regardless of sex, even under conditions of zero dietary fat. CONCLUSIONS: These findings highlight the greater capacity for mitochondrially driven triglyceride assembly in adipose from female versus male mice and expose a reliance upon MPC-gated metabolism for glucose partitioning in female adipose under conditions of dietary lipid restriction.

Hexavalent chromium uptake in rice (Oryza sativa L.) mediated by sulfate and phosphate transporters OsSultr1;2 and OsPht1;1.

Chromium (Cr) soil contamination is a critical global environmental concern, with hexavalent chromium (Cr[VI]) being especially perilous due to its high mobility, bioavailability, and phytotoxicity. This poses a significant threat to the cultivation of crops, particularly rice, where the mechanisms of Cr(VI) absorption remain largely unexplored. This study uncovered a competitive interaction between Cr(VI) and essential nutrients-sulfate and phosphate during the uptake process. Notably, deficiencies in sulfate and phosphate were associated with a marked increase in Cr(VI) accumulation in rice, reaching up to 76.5 % and 77.7 %, respectively. Employing q-PCR, this study identified significant up-regulation of the sulfate transporter gene, OsSultr1;2, and the phosphate transporter gene, OsPht1;1, in response to Cr(VI) stress. Genetic knockout studies have confirmed the crucial role of OsSultr1;2 in Cr(VI) uptake, with its deletion leading to a 36.1 % to 69.6 % decrease in Cr uptake by rice roots. Similarly, the knockout of OsPht1;1 resulted in an 18.1 % to 25.7 % decrease in root Cr accumulation. These findings highlight the key role of the sulfate transporter OsSultr1;2 in Cr(VI) uptake, with phosphate transporters also contributing significantly to the process. These insights are valuable for developing rice varieties with reduced Cr(VI) accumulation, ensuring the safety of rice grain production.

Inhibition of the mitochondrial pyruvate carrier in astrocytes reduces amyloid and tau accumulation in the 3xTgAD mouse model of Alzheimer's disease.

Alzheimer's Disease (AD) is characterized by an accumulation of pathologic amyloid-beta (Aß) and Tau proteins, neuroinflammation, metabolic changes and neuronal death. Reactive astrocytes participate in these pathophysiological processes by releasing pro-inflammatory molecules and recruiting the immune system, which further reinforces inflammation and contributes to neuronal death. Besides these neurotoxic effects, astrocytes can protect neurons by providing them with high amounts of lactate as energy fuel. Astrocytes rely on aerobic glycolysis to generate lactate by reducing pyruvate, the end product of glycolysis, through lactate dehydrogenase. Consequently, limited amounts of pyruvate enter astrocytic mitochondria through the Mitochondrial Pyruvate Carrier (MPC) to be oxidized. The MPC is a heterodimer composed of two subunits MPC1 and MPC2, the function of which in astrocytes has been poorly investigated. Here, we analyzed the role of the MPC in the pathogeny of AD, knowing that a reduction in overall glucose metabolism has been associated with a drop in cognitive performances and an accumulation of Aß and Tau. We generated 3xTgAD mice in which MPC1 was knocked-out in astrocytes specifically and focused our study on the biochemical hallmarks of the disease, mainly Aß and neurofibrillary tangle production. We show that inhibition of the MPC before the onset of the disease significantly reduces the quantity of Aß and Tau aggregates in the brain of 3xTgAD mice, suggesting that acting on astrocytic glucose metabolism early on could hinder the progression of the disease.

Enhancing mitochondrial pyruvate metabolism ameliorates ischemic reperfusion injury in the heart.

The clinical therapy for treating acute myocardial infarction is primary percutaneous coronary intervention (PPCI). PPCI is effective at reperfusing the heart; however, the rapid reintroduction of blood can cause ischemia-reperfusion (I/R). Reperfusion injury is responsible for up to half of the total myocardial damage, but there are no pharmacological interventions to reduce I/R. We previously demonstrated that inhibiting monocarboxylate transporter 4 (MCT4) and redirecting pyruvate toward oxidation can blunt hypertrophy. We hypothesized that this pathway might be important during I/R. Here, we establish that the pyruvate-lactate axis plays a role in determining myocardial salvage following injury. After I/R, the mitochondrial pyruvate carrier (MPC), required for pyruvate oxidation, is upregulated in the surviving myocardium. In cardiomyocytes lacking the MPC, there was increased cell death and less salvage after I/R, which was associated with an upregulation of MCT4. To determine the importance of pyruvate oxidation, we inhibited MCT4 with a small-molecule drug (VB124) at reperfusion. This strategy normalized reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨ), and Ca2+, increased pyruvate entry to the TCA cycle, increased oxygen consumption, and improved myocardial salvage and functional outcomes following I/R. Our data suggest normalizing pyruvate-lactate metabolism by inhibiting MCT4 is a promising therapy to mitigate I/R injury.

Positive feed-forward regulation of nitrate uptake by rice roots and its molecular mechanism.

To investigate the positive feed-forward regulatory mechanism of nitrate uptake by rice, its responses to various light and carbohydrates were compared. In order to measure nitrate uptake in real time, the non-invasive method was used. The results showed that net nitrate uptake increased in the light and decreased in the dark, and finally reached a steady state after about 5 h. Based on it, carbohydrates effects could be investigated without considering light effects. After sucrose addition for 2 h, net nitrate uptake increased by about 80% without a lag, while glucose, fructose and raffinose had a slight effect with a lag and other sugars had no effect. It provided an evidence that sucrose was a positive feed-forward signal molecule of nitrate uptake by rice roots. To further analyze the effect of sucrose on the expression of high affinity nitrate transporter genes OsNRT2.1, OsNRT2.2, OsNRT2.3a and OsNRT2.3b, qRT-PCR was used to further verify after treated with 10 mM sucrose. The results revealed that these genes expression was immediately up-regulated, which indicated that these genes were post transcriptionally regulated. Further, 15N exchange dynamics analyzed N transport. It is benefit for increasing nitrate uptake by rice and improving its yield.

Genome-Wide Identification and Expression Analysis of Nitrate Transporter (NRT) Gene Family in Eucalyptus grandis.

Eucalyptus grandis is an important planted hardwood tree worldwide with fast growth and good wood performance. The nitrate transporter (NRT) gene family is a major core involved in nitrogen (N) absorption and utilization in plants, but the comprehensive characterization of NRT genes in E. grandis remains mostly elusive. In this study, a total of 75 EgNRT genes were identified from the genome of E. grandis that were distributed unevenly across ten chromosomes, except Chr9. A phylogenetic analysis showed that the EgNRT proteins could be divided into three classes, namely NRT1, NRT2 and NRT3, which contained 69, 4 and 2 members, respectively. The cis-regulatory elements in the promoter regions of EgNRT genes were mainly involved in phytohormone and stress response. The transcriptome analysis indicated that the differentially expressed genes of leaf and root in E. grandis under different N supply conditions were mainly involved in the metabolic process and plant hormone signal transduction. In addition, the transcriptome-based and RT-qPCR analysis revealed that the expression of 13 EgNRT genes, especially EgNRT1.3, EgNRT1.38, EgNRT1.39 and EgNRT1.52, was significantly upregulated in the root under low-N-supply treatment, suggesting that those genes might play a critical role in root response to nitrate deficiency. Taken together, these results would provide valuable information for characterizing the roles of EgNRTs and facilitate the clarification of the molecular mechanism underlying EgNRT-mediated N absorption and distribution in E. grandis.

Nutrient levels control root growth responses to high ambient temperature in plants.

Global warming will lead to significantly increased temperatures on earth. Plants respond to high ambient temperature with altered developmental and growth programs, termed thermomorphogenesis. Here we show that thermomorphogenesis is conserved in Arabidopsis, soybean, and rice and that it is linked to a decrease in the levels of the two macronutrients nitrogen and phosphorus. We also find that low external levels of these nutrients abolish root growth responses to high ambient temperature. We show that in Arabidopsis, this suppression is due to the function of the transcription factor ELONGATED HYPOCOTYL 5 (HY5) and its transcriptional regulation of the transceptor NITRATE TRANSPORTER 1.1 (NRT1.1). Soybean and Rice homologs of these genes are expressed consistently with a conserved role in regulating temperature responses in a nitrogen and phosphorus level dependent manner. Overall, our data show that root thermomorphogenesis is a conserved feature in species of the two major groups of angiosperms, monocots and dicots, that it leads to a reduction of nutrient levels in the plant, and that it is dependent on environmental nitrogen and phosphorus supply, a regulatory process mediated by the HY5-NRT1.1 module.

Novel Proteins of the High-Affinity Nitrate Transporter Family NRT2, SaNRT2.1 and SaNRT2.5, from the Euhalophyte Suaeda altissima: Molecular Cloning and Expression Analysis.

Two genes of nitrate transporters SaNRT2.1 and SaNRT2.5, putative orthologs of high-affinity nitrate transporter genes AtNRT2.1 and AtNRT2.5 from Arabidopsis thaliana, were cloned from the euhalophyte Suaeda altissima. Phylogenetic bioinformatic analysis demonstrated that the proteins SaNRT2.1 and SaNRT2.5 exhibited higher levels of homology to the corresponding proteins from the plants of family Amaranthaceae; the similarity of amino acid sequences between proteins SaNRT2.1 and SaNRT2.5 was lower (54%). Both SaNRT2.1 and SaNRT2.5 are integral membrane proteins forming 12 transmembrane helices as predicted by topological modeling. An attempt to demonstrate nitrate transporting activity of SaNRT2.1 or SaNRT2.5 by heterologous expression of the genes in the yeast Hansenula (Ogataea) polymorpha mutant strain Δynt1 lacking the only yeast nitrate transporter was not successful. The expression patterns of SaNRT2.1 and SaNRT2.5 were studied in S. altissima plants that were grown in hydroponics under either low (0.5 mM) or high (15 mM) nitrate and salinity from 0 to 750 mM NaCl. The growth of the plants was strongly inhibited by low nitrogen supply while stimulated by NaCl; it peaked at 250 mM NaCl for high nitrate and at 500 mM NaCl for low nitrate. Under low nitrate supply, nitrate contents in S. altissima roots, leaves and stems were reduced but increased in leaves and stems as salinity in the medium increased. Potassium contents remained stable under salinity treatment from 250 to 750 mM NaCl. Quantitative real-time PCR demonstrated that without salinity, SaNRT2.1 was expressed in all organs, its expression was not influenced by nitrate supply, while SaNRT2.5 was expressed exclusively in roots-its expression rose about 10-fold under low nitrate. Salinity increased expression of both SaNRT2.1 and SaNRT2.5 under low nitrate. SaNRT2.1 peaked in roots at 500 mM NaCl with 15-fold increase; SaNRT2.5 peaked in roots at 500 mM NaCl with 150-fold increase. It is suggested that SaNRT2.5 ensures effective nitrate uptake by roots and functions as an essential high-affinity nitrate transporter to support growth of adult S. altissima plants under nitrogen deficiency.

Human mitochondrial carriers of the SLC25 family function as monomers exchanging substrates with a ping-pong kinetic mechanism.

Members of the SLC25 mitochondrial carrier family link cytosolic and mitochondrial metabolism and support cellular maintenance and growth by transporting compounds across the mitochondrial inner membrane. Their monomeric or dimeric state and kinetic mechanism have been a matter of long-standing debate. It is believed by some that they exist as homodimers and transport substrates with a sequential kinetic mechanism, forming a ternary complex where both exchanged substrates are bound simultaneously. Some studies, in contrast, have provided evidence indicating that the mitochondrial ADP/ATP carrier (SLC25A4) functions as a monomer, has a single substrate binding site, and operates with a ping-pong kinetic mechanism, whereby ADP is imported before ATP is exported. Here we reanalyze the oligomeric state and kinetic properties of the human mitochondrial citrate carrier (SLC25A1), dicarboxylate carrier (SLC25A10), oxoglutarate carrier (SLC25A11), and aspartate/glutamate carrier (SLC25A13), all previously reported to be dimers with a sequential kinetic mechanism. We demonstrate that they are monomers, except for dimeric SLC25A13, and operate with a ping-pong kinetic mechanism in which the substrate import and export steps occur consecutively. These observations are consistent with a common transport mechanism, based on a functional monomer, in which a single central substrate-binding site is alternately accessible.

NtARF11 positively regulates cadmium tolerance in tobacco by inhibiting expression of the nitrate transporter NtNRT1.1.

Heavy metal cadmium (Cd) is widespread in contaminated soil and an important factor limiting plant growth. NO3- (nitrate) affects Cd uptake and thus changes Cd tolerance in plants; however, the underlying molecular regulatory mechanisms have not yet been elucidated. Here, we analyzed a novel gene, NtARF11 (auxin response factor), which regulates Cd tolerance in tobacco via the NO3- uptake pathway, through experiments with NtARF11-knockout and NtARF11-overexpression transgenic tobacco lines. NtARF11 was highly expressed under Cd stress in tobacco plants. Under Cd stress, overexpression of NtARF11 enhanced Cd tolerance in tobacco compared to that in wild-type tobacco, as shown by the low Cd concentration, high chlorophyll concentration, and low accumulation of reactive oxygen species in NtARF11-overexpressing tobacco. Moreover, low NO3- concentrations were observed in NtARF11-overexpressing tobacco plants. Further analyses revealed direct binding of NtARF11 to the promoter of the nitrate transporter NtNRT1.1, thereby negatively regulating its expression in tobacco. Notably, NtNRT1.1 knockout reduced NO3- uptake, which resulted in low Cd concentrations in tobacco. Altogether, these results demonstrate that the NtARF11-NtNRT1.1 module functions as a positive regulator of Cd tolerance by reducing the Cd uptake in tobacco, providing new insights for improving Cd tolerance of plants through genetic engineering.

Engineering the cyanobacterial ATP-driven BCT1 bicarbonate transporter for functional targeting to C3 plant chloroplasts.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO2-concentrating mechanism. BCT1 could enhance photosynthetic CO2 assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO2-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO2 compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO2-concentrating mechanism components to improve plant photosynthesis.

Mitochondrial citrate transporters Ctp1-Yhm2 and respiratory chain: A coordinated functional connection in Saccharomyces cerevisiae metabolism.

The mitochondrial inner membrane contains some hydrophobic proteins that mediate the exchange of metabolites between the mitochondrial matrix and the cytosol. Ctp1 and Yhm2 are two carrier proteins in the yeast Saccharomyces cerevisiae responsible for the transport of citrate, a tricarboxylate involved in several metabolic pathways. Since these proteins also contribute to respiratory metabolism, in this study we investigated for the first time whether changes in citrate transport can affect the structural organization and functional properties of respiratory complexes. Through experiments in yeast mutant cells in which the gene encoding Ctp1 or Yhm2 was deleted, we found that in the absence of either mitochondrial citrate transporter, mitochondrial respiration was impaired. Structural analysis of the respiratory complexes III and IV revealed different expression levels of the catalytic and supernumerary subunits in the Δctp1 and Δyhm2 strains. In addition, Δyhm2 mitochondria appeared to be more sensitive than Δctp1 to the oxidative damage. Our results provide the first evidence for a coordinated modulation of mitochondrial citrate transport and respiratory chain activity in S. cerevisiae metabolism.