Connexin 43 modulates reverse electron transfer in cardiac mitochondria from inducible knock-out Cx43Cre-ER(T)/fl mice by altering the coenzyme Q pool.

Succinate accumulates during myocardial ischemia and is rapidly oxidized during reperfusion, leading to reactive oxygen species (ROS) production through reverse electron transfer (RET) from mitochondrial complex II to complex I, and favoring cell death. Given that connexin 43 (Cx43) modulates mitochondrial ROS production, we investigated whether Cx43 influences RET using inducible knock-out Cx43Cre-ER(T)/fl mice. Oxygen consumption, ROS production, membrane potential and coenzyme Q (CoQ) pool were analyzed in subsarcolemmal (SSM, expressing Cx43) and interfibrillar (IFM) cardiac mitochondria isolated from wild-type Cx43fl/fl mice and Cx43Cre-ER(T)/fl knock-out animals treated with 4-hydroxytamoxifen (4OHT). In addition, infarct size was assessed in isolated hearts from these animals submitted to ischemia-reperfusion (IR), and treated or not with malonate, a complex II inhibitor attenuating RET. Succinate-dependent ROS production and RET were significantly lower in SSM, but not IFM, from Cx43-deficient animals. Mitochondrial membrane potential, a RET driver, was similar between groups, whereas CoQ pool (2.165 ± 0.338 vs. 4.18 ± 0.55 nmol/mg protein, p < 0.05) and its reduction state were significantly lower in Cx43-deficient animals. Isolated hearts from Cx43Cre-ER(T)/fl mice treated with 4OHT had a smaller infarct size after IR compared to Cx43fl/fl, despite similar concentration of succinate at the end of ischemia, and no additional protection by malonate. Cx43 deficiency attenuates ROS production by RET in SSM, but not IFM, and was associated with a decrease in CoQ levels and a change in its redox state. These results may partially explain the reduced infarct size observed in these animals and their lack of protection by malonate.

TRPV4 Channels Promote Pathological, but Not Physiological, Cardiac Remodeling through the Activation of Calcineurin/NFAT and TRPC6.

TRPV4 channels, which respond to mechanical activation by permeating Ca2+ into the cell, may play a pivotal role in cardiac remodeling during cardiac overload. Our study aimed to investigate TRPV4 involvement in pathological and physiological remodeling through Ca2+-dependent signaling. TRPV4 expression was assessed in heart failure (HF) models, induced by isoproterenol infusion or transverse aortic constriction, and in exercise-induced adaptive remodeling models. The impact of genetic TRPV4 inhibition on HF was studied by echocardiography, histology, gene and protein analysis, arrhythmia inducibility, Ca2+ dynamics, calcineurin (CN) activity, and NFAT nuclear translocation. TRPV4 expression exclusively increased in HF models, strongly correlating with fibrosis. Isoproterenol-administered transgenic TRPV4-/- mice did not exhibit HF features. Cardiac fibroblasts (CFb) from TRPV4+/+ animals, compared to TRPV4-/-, displayed significant TRPV4 overexpression, elevated Ca2+ influx, and enhanced CN/NFATc3 pathway activation. TRPC6 expression paralleled that of TRPV4 in all models, with no increase in TRPV4-/- mice. In cultured CFb, the activation of TRPV4 by GSK1016790A increased TRPC6 expression, which led to enhanced CN/NFATc3 activation through synergistic action of both channels. In conclusion, TRPV4 channels contribute to pathological remodeling by promoting fibrosis and inducing TRPC6 upregulation through the activation of Ca2+-dependent CN/NFATc3 signaling. These results pose TRPV4 as a primary mediator of the pathological response.

Long-Term Protective Effects of Succinate Dehydrogenase Inhibition during Reperfusion with Malonate on Post-Infarction Left Ventricular Scar and Remodeling in Mice.

Succinate dehydrogenase inhibition with malonate during initial reperfusion reduces myocardial infarct size in both isolated mouse hearts subjected to global ischemia and in in situ pig hearts subjected to transient coronary ligature. However, the long-term effects of acute malonate treatment are unknown. Here, we investigated whether the protective effects of succinate dehydrogenase inhibition extend to a reduction in scar size and adverse left ventricular remodeling 28 days after myocardial infarction. Initially, ten wild-type mice were subjected to 45 min of left anterior descending coronary artery (LAD) occlusion, followed by 24 h of reperfusion, and were infused during the first 15 min of reperfusion with saline with or without disodium malonate (10 mg/kg/min, 120 µL/kg/min). Malonate-treated mice depicted a significant reduction in infarct size (15.47 ± 3.40% of area at risk vs. 29.34 ± 4.44% in control animals, p < 0.05), assessed using triphenyltetrazolium chloride. Additional animals were then subjected to a 45 min LAD ligature, followed by 28 days of reperfusion. Treatment with a single dose of malonate during the first 15 min of reperfusion induced a significant reduction in scar area, measured using Picrosirius Red staining (11.94 ± 1.70% of left ventricular area (n = 5) vs. 23.25 ± 2.67% (n = 9), p < 0.05), an effect associated with improved ejection fraction 28 days after infarction, as determined using echocardiography, and an attenuated enhancement in expression of the pro-inflammatory and fibrotic markers NF-κB and Smad2/3 in remote myocardium. In conclusion, a reversible inhibition of succinate dehydrogenase with a single dose of malonate at the onset of reperfusion has long-term protective effects in mice subjected to transient coronary occlusion.

The membrane-associated protein 17 (MAP17) is up-regulated in response to empagliflozin on top of RAS blockade in experimental diabetic nephropathy.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have proven to delay diabetic kidney disease (DKD) progression on top of the standard of care with the renin-angiotensin system (RAS) blockade. The molecular mechanisms underlying the synergistic effect of SGLT2i and RAS blockers is poorly understood. We gave a SGLT2i (empagliflozin), an angiotensin-converting enzyme inhibitor (ramipril), or a combination of both drugs for 8 weeks to diabetic (db/db) mice. Vehicle-treated db/db and db/m mice were used as controls. At the end of the experiment, mice were killed, and the kidneys were saved to perform a differential high-throughput proteomic analysis by mass spectrometry using isobaric tandem mass tags (TMT labeling) that allow relative quantification of the identified proteins. The differential proteomic analysis revealed 203 proteins differentially expressed in one or more experimental groups (false discovery rate < 0.05 and Log2 fold change ≥ ±1). Fourteen were differentially expressed in the kidneys from the db/db mice treated with empagliflozin with ramipril. Among them, MAP17 was up-regulated. These findings were subsequently validated by Western blot. The combined therapy of empagliflozin and ramipril up-regulated MAP17 in the kidney of a diabetic mice model. MAP17 is a major scaffolding protein of the proximal tubular cells that places transporters together, namely SGLT2 and NHE3. Our results suggest that SGLT2i on top of RAS blockade may protect the kidney by boosting the inactivation of NHE3 via the up-regulation of key scaffolder proteins such as MAP17.

Phylogenetic and Structure-Function Analyses of ENA ATPases: A Case Study of the ENA1 Protein from the Fungus Neurospora crassa.

ENA transporters are a group of P-type ATPases that are characterized by actively moving Na+ or K+ out of the cell against their concentration gradient. The existence of these transporters was initially attributed to some fungi, although more recently they have also been identified in mosses, liverworts, and some protozoa. Given the current increase in the number of organisms whose genomes are completely sequenced, we set out to expand our knowledge about the existence of ENA in organisms belonging to other phylogenetic groups. For that, a hidden Markov model profile was constructed to identify homologous sequences to ENA proteins in protein databases. This analysis allowed us to identify the existence of ENA-type ATPases in the most primitive groups of fungi, as well as in other eukaryotic organisms not described so far. In addition, this study has allowed the identification of a possible new group of P-ATPases, initially proposed as ENA but which maintain phylogenetic distances with these proteins. Finally, this work has also addressed this study of the structure of ENA proteins, which remained unknown due to the lack of crystallographic data. For this purpose, a 3D structure prediction of the NcENA1 protein of the fungus Neurospora crassa was performed using AlphaFold2 software v2.3.1. From this structure, the electrostatic potential of the protein was analyzed. With all these data, the protein regions and the amino acids involved in the transport of Na+ or K+ ions across the membrane were proposed for the first time. Targeted mutagenesis of some of these residues has confirmed their relevant participation in the transport function of ENA proteins.

Enhanced Cardiorenal Protective Effects of Combining SGLT2 Inhibition, Endothelin Receptor Antagonism and RAS Blockade in Type 2 Diabetic Mice.

Treatments with sodium-glucose 2 cotransporter inhibitors (SGLT2i) or endothelin receptor antagonists (ERA) have shown cardiorenal protective effects. The present study aimed to evaluate the cardiorenal beneficial effects of the combination of SGLT2i and ERA on top of renin-angiotensin system (RAS) blockade. Type 2 diabetic mice (db/db) were treated with different combinations of an SGLT2i (empagliflozin), an ERA (atrasentan), and an angiotensin-converting enzyme inhibitor (ramipril) for 8 weeks. Vehicle-treated diabetic mice and non-diabetic mice were included as controls. Weight, blood glucose, blood pressure, and kidney and heart function were monitored during the study. Kidneys and heart were collected for histological examination and to study the intrarenal RAS. Treatment with empagliflozin alone or combined significantly decreased blood glucose compared to vehicle-treated db/db. The dual and triple therapies achieved significantly greater reductions in diastolic blood pressure than ramipril alone. Compared to vehicle-treated db/db, empagliflozin combined with ramipril or in triple therapy significantly prevented GFR increase, but only the triple combination exerted greater protection against podocyte loss. In the heart, empagliflozin alone or combined reduced cardiac isovolumetric relaxation time (IVRT) and left atrium (LA) diameter as compared to vehicle-treated db/db. However, only the triple therapy was able to reduce cardiomyocyte area. Importantly, the add-on triple therapy further enhanced the intrarenal ACE2/Ang(1-7)/Mas protective arm of the RAS. These data suggest that triple therapy with empagliflozin, atrasentan and ramipril show synergistic cardiorenal protective effects in a type 2 diabetic mouse model.

Citric Acid Cycle Metabolites Predict Infarct Size in Pigs Submitted to Transient Coronary Artery Occlusion and Treated with Succinate Dehydrogenase Inhibitors or Remote Ischemic Perconditioning.

Succinate dehydrogenase (SDH) inhibition with malonate during reperfusion reduced myocardial infarction in animals, whereas its endogenous substrate, succinate, is detected in plasma from STEMI patients. We investigated whether protection by SDH inhibition is additive to that of remote ischemic perconditioning (RIC) in pigs submitted to transient coronary artery occlusion, and whether protective maneuvers influence plasma levels of citric acid cycle metabolites. Forty pigs were submitted to 40 min coronary occlusion and reperfusion, and allocated to four groups (controls, sodium malonate 10 mmol/L, RIC, and malonate + RIC). Plasma was obtained from femoral and great cardiac veins and analyzed by LC-MS/MS. Malonate, RIC, and malonate + RIC reduced infarct size (24.67 ± 5.98, 25.29 ± 3.92 and 29.83 ± 4.62% vs. 46.47 ± 4.49% in controls, p < 0.05), but no additive effects were detected. Enhanced concentrations of succinate, fumarate, malate and citrate were observed in controls during initial reperfusion in the great cardiac vein, and most were reduced by cardioprotective maneuvers. Concentrations of succinate, fumarate, and malate significantly correlated with infarct size. In conclusion, despite the combination of SDH inhibition during reperfusion and RIC did not result in additive protection, plasma concentrations of selected citric acid cycle metabolites are attenuated by protective maneuvers, correlate with irreversible injury, and might become a prognosis tool in STEMI patients.

Atrial Fibrillation in Heart Failure Is Associated with High Levels of Circulating microRNA-199a-5p and 22-5p and a Defective Regulation of Intracellular Calcium and Cell-to-Cell Communication.

MicroRNAs (miRNAs) participate in atrial remodeling and atrial fibrillation (AF) promotion. We determined the circulating miRNA profile in patients with AF and heart failure with reduced ejection fraction (HFrEF), and its potential role in promoting the arrhythmia. In plasma of 98 patients with HFrEF (49 with AF and 49 in sinus rhythm, SR), differential miRNA expression was determined by high-throughput microarray analysis followed by replication of selected candidates. Validated miRNAs were determined in human atrial samples, and potential arrhythmogenic mechanisms studied in HL-1 cells. Circulating miR-199a-5p and miR-22-5p were significantly increased in HFrEF patients with AF versus those with HFrEF in SR. Both miRNAs, but particularly miR-199a-5p, were increased in atrial samples of patients with AF. Overexpression of both miRNAs in HL-1 cells resulted in decreased protein levels of L-type Ca2+ channel, NCX and connexin-40, leading to lower basal intracellular Ca2+ levels, fewer inward currents, a moderate reduction in Ca2+ buffering post-caffeine exposure, and a deficient cell-to-cell communication. In conclusion, circulating miR-199a-5p and miR-22-5p are higher in HFrEF patients with AF, with similar findings in human atrial samples of AF patients. Cells exposed to both miRNAs exhibited altered Ca2+ handling and defective cell-to-cell communication, both findings being potential arrhythmogenic mechanisms.

Aging Impairs Reverse Remodeling and Recovery of Ventricular Function after Isoproterenol-Induced Cardiomyopathy.

Information about heart failure with reduced ejection fraction (HFrEF) in women and the potential effects of aging in the female heart is scarce. We investigated the vulnerability to develop HFrEF in female elderly mice compared to young animals, as well as potential differences in reverse remodeling. First, HF was induced by isoproterenol infusion (30 mg/kg/day, 28 days) in young (10-week-old) and elderly (22-month-old) female mice. In a second set of animals, mice underwent isoproterenol infusion followed by no treatment during 28 additional days. Cardiac remodeling was assessed by echocardiography, histology and gene expression of collagen-I and collagen-III. Following isoproterenol infusion, elderly mice developed similar HFrEF features compared to young animals, except for greater cell hypertrophy and tissue fibrosis. After beta-adrenergic withdrawal, young female mice experienced complete reversal of the HFrEF phenotype. Conversely, reversed remodeling was impaired in elderly animals, with no significant recovery of LV ejection fraction, cardiomyocyte hypertrophy and collagen deposition. In conclusion, chronic isoproterenol infusion is a valid HF model for elderly and young female mice and induces a similar HF phenotype in both. Elderly animals, unlike young, show impaired reverse remodeling, with persistent tissue fibrosis and cardiac dysfunction even after beta-adrenergic withdrawal.

Mapping potentials adjacent to the cavo-tricuspid isthmus ablation line during incremental pacing: A feasible and highly accurate maneuver to confirm complete CTI conduction block.

BACKGROUND: The diagnostic accuracy of incremental atrial pacing (IP) to determine complete cavo-tricuspid isthmus (CTI) block during typical atrial flutter (AFL) ablation is limited by both an extensive/nonlinear ablation and/or the presence of intra-atrial conduction delay elsewhere in the right atrium. We examined the diagnostic performance of an IP variant based on the assessment of the atrial potentials adjacent to the ablation line which aims at overcoming both limitations. METHODS: From a prospective population of 108 consecutive patients, 15 were excluded due to observation of inconclusive CTI ablation potentials precluding for a straight comparison between the IP maneuver and its variant. In the remaining 93, IP was performed from the low lateral right atrium and the coronary sinus ostium, with the ablation catheter positioned both at the CTI line and adjacent (<5 mm) to its septal and lateral aspect. The IP variant consisted of measuring the interval between the two atrial electrograms situated on the same side of the ablation line, opposite to the pacing site, a ≤10 ms increase indicating complete CTI block. RESULTS: The IP maneuver and its variant were consistent with complete CTI block in 82/93 (88%) and 87/93 (93%) patients, respectively. Four patients had AFL recurrence during follow-up: 2/4 and 4/4 had been adequately classified as incomplete block by the IP maneuver and its variant, respectively. Twenty-three patients (24%) had significant intra-atrial conduction delay elsewhere in the right atrium. The IP maneuver and its variant were suggestive of an incomplete CTI block in 11/23 and 4/23 in this setting (P = .028), with the later best predicting subsequent AFL relapses (2/12 vs 2/4, P = .01). CONCLUSIONS: The IP variant, which was designed to overcome the limitations of the conventional IP maneuver, accurately distinguishes complete from incomplete CTI block and helps to predict AFL recurrences after ablation.

The endophyte Serendipita indica reduces the sodium content of Arabidopsis plants exposed to salt stress: fungal ENA ATPases are expressed and regulated at high pH and during plant co-cultivation in salinity.

Serendipita indica (formerly Piriformospora indica) is an endophytic fungus that colonizes plant roots producing a beneficial effect on plant growth and development under optimal and suboptimal conditions provoked by any biotic or abiotic stress, such as salt stress. Salinity induces osmotic and ionic imbalances in plants, mainly by altering the Na+ and K+ contents. However, the mechanism by which Serendipita improves plant growth has yet to be elucidated. Previous works suggest that this fungus improves the plant osmotic state but not much is known about whether it participates in readjustment of the ionic imbalance in plants. Here, we report that co-cultivation with Serendipita reduces the Na+ content of Arabidopsis plants under saline conditions. Additionally, we describe the functional characterization of the two Serendipita ENA ATPases, which are homologous to the main proteins involved in the salt tolerance of other fungi. Their heterologous expression in salt-sensitive yeast mutants shows that SiENA1 is involved in Na+ and K+ efflux, while SiENA5 seems to only be involved in Na+ detoxification. Both are induced and might have a relevant function at alkaline pH, condition in which there are few chlamydospores in the mycelium, as well as during co-cultivation with plants exposed to saline conditions.

The Role of Soil Fungi in K+ Plant Nutrition.

K+ is an essential cation and the most abundant in plant cells. After N, its corresponding element, K, is the nutrient required in the largest amounts by plants. Despite the numerous roles of K in crop production, improvements in the uptake and efficiency of use of K have not been major focuses in conventional or transgenic breeding studies in the past. In research on the mineral nutrition of plants in general, and K in particular, this nutrient has been shown to be essential to soil-dwelling-microorganisms (fungi, bacteria, protozoa, nematodes, etc.) that form mutualistic associations and that can influence the availability of mineral nutrients for plants. Therefore, this article aims to provide an overview of the role of soil microorganisms in supplying K+ to plants, considering both the potassium-solubilizing microorganisms and the potassium-facilitating microorganisms that are in close contact with the roots of plants. These microorganisms can influence the active transporter-mediated transfer of K+. Regarding the latter group of microorganisms, special focus is placed on the role of endophytic fungus. This review also includes a discussion on productivity through sustainable agriculture.

Ion Channel Disorders and Sudden Cardiac Death.

Long QT syndrome, short QT syndrome, Brugada syndrome and catecholaminergic polymorphic ventricular tachycardia are inherited primary electrical disorders that predispose to sudden cardiac death in the absence of structural heart disease. Also known as cardiac channelopathies, primary electrical disorders respond to mutations in genes encoding cardiac ion channels and/or their regulatory proteins, which result in modifications in the cardiac action potential or in the intracellular calcium handling that lead to electrical instability and life-threatening ventricular arrhythmias. These disorders may have low penetrance and expressivity, making clinical diagnosis often challenging. However, because sudden cardiac death might be the first presenting symptom of the disease, early diagnosis becomes essential. Genetic testing might be helpful in this regard, providing a definite diagnosis in some patients. Yet important limitations still exist, with a significant proportion of patients remaining with no causative mutation identifiable after genetic testing. This review aims to provide the latest knowledge on the genetic basis of cardiac channelopathies and discuss the role of the affected proteins in the pathophysiology of each one of these diseases.

Genomic and Transcriptomic Compilation of Chloroplast Ionic Transporters of Physcomitrella patens. Study of NHAD Transporters in Na+ and K+ Homeostasis.

K+ is widely used by plant cells, whereas Na+ can easily reach toxic levels during plant growth, which typically occurs in saline environments; however, the effects and functions in the chloroplast have been only roughly estimated. Traditionally, the occurrence of ionic fluxes across the chloroplast envelope or the thylakoid membranes has been mostly deduced from physiological measurements or from knowledge of chloroplast metabolism. However, many of the proteins involved in these fluxes have not yet been characterized. Based on genomic and RNA sequencing (RNA-seq) analyses, we present a comprehensive compilation of genes encoding putative ion transporters and channels expressed in the chloroplasts of the moss Physcomitrella patens, with a special emphasis on those related to Na+ and K+ fluxes. Based on the functional characterization of nhad mutants, we also discuss the putative role of NHAD transporters in Na+ homeostasis and osmoregulation of this organelle and the putative contribution of chloroplasts to salt tolerance in this moss. We demonstrate that NaCl does not affect the chloroplast functionality in Physcomitrella despite significantly modifying expression of ionic transporters and cellular morphology, specifically the chloroplast ultrastructure, revealing a high starch accumulation. Additionally, NHAD transporters apparently do not play any essential roles in salt tolerance.

Salt intolerance in Arabidopsis: shoot and root sodium toxicity, and inhibition by sodium-plus-potassium overaccumulation.

MAIN CONCLUSION: Arabidopsis plants in NaCl suffering half growth inhibition do not suffer osmotic stress and seldom shoot Na (+) toxicity; overaccumulation of Na (+) plus K (+) might trigger the inhibition. It is widely assumed that salinity inhibits plant growth by osmotic stress and shoot Na(+) toxicity. This study aims to examine the growth inhibition of Arabidopsis thaliana by NaCl concentrations that allow the completion of the life cycle. Unaffected Col-0 wild-type plants were used to define nontoxic Na(+) contents; Na(+) toxicities in shoots and roots were analyzed in hkt1 and sos1 mutants, respectively. The growth inhibition of Col-0 plants at 40 mM Na(+) was mild and equivalent to that produced by 8 and 4 mM Na(+) in hkt1 and sos1 plants, respectively. Therefore, these mutants allowed to study the toxicity of Na(+) in the absence of an osmotic challenge. Col-0 and Ts-1 accessions showed very different Na(+) contents but similar growth inhibitions; Ts-1 plants showed very high leaf Na(+) contents but no symptoms of Na(+) toxicity. Ak-1, C24, and Fei-0 plants were highly affected by NaCl showing evident symptoms of shoot Na(+) toxicity. Increasing K(+) in isotonic NaCl/KCl combinations dramatically decreased the Na(+) content in all Arabidopsis accessions and eliminated the signs of Na(+) toxicity in most of them but did not relieve growth inhibition. This suggested that the dominant inhibition in these conditions was either osmotic or of an ionic nature unspecific for Na(+) or K(+). Col-0 and Ts-1 plants growing in sorbitol showed a clear osmotic stress characterized by a notable decrease of their water content, but this response did not occur in NaCl. Overaccumulation of Na(+) plus K(+) might trigger growth reduction in NaCl-treated plants.

Burning the Gap: Electrical and Anatomical Basis of the Incremental Pacing Maneuver for Cavotricuspid Isthmus Block Assessment.

INTRODUCTION: The incremental pacing (IP) maneuver is a highly specific technique that improves the ability to confirm complete CTI conduction block during typical atrial flutter (AFL) ablation, and reduces long-term AFL recurrences. The purpose of this study is to assess the performance of new catheters equipped with additional high precision bipoles (AHPB) to allow the visualization of the cavotricuspid isthmus (CTI) conduction gap and to compare them with the IP maneuver. METHODS AND RESULTS: Twenty consecutive patients undergoing catheter ablation of the CTI for AFL were included. The IP maneuver confirmed functional versus complete CTI block. Local electrogram analysis using AHPB was then used to assess the presence or absence of gaps across the CTI line. Mean age was 67 years and 80% were male. At the end of the procedure CTI block was achieved in all patients. A transient stage of functional CTI block was observed in 40%. In all cases a continuous fragmented electrogram was present between the double potentials in the CTI in the AHPB channels. In contrast, no electrogram was observed between the CTI double potentials in any of the 20 patients once complete block was confirmed by the IP maneuver. When both techniques were compared a significant association and correlation were observed (chi-square <0.01, Spearman's rho = 1, P < 0.01). CONCLUSION: Catheters equipped with AHPB can aid in the assessment of complete CTI block during AFL ablation procedures by detecting conduction gaps that correlate with incomplete functional block diagnosed by the IP maneuver.

HKT2;2/1, a K⁺-permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism.

We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na⁺ concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na⁺ and K⁺, even at high external Na⁺ concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K⁺ uptake under saline conditions.

The transportome of the endophyte Serendipita indica in free life and symbiosis with Arabidopsis and its expression in moderate salinity.

Serendipita indica is an endophytic root symbiont fungus that enhances the growth of various plants under different stress conditions, including salinity. Here, the functional characterization of two fungal Na+/H+ antiporters, SiNHA1 and SiNHX1 has been carried out to study their putative role in saline tolerance. Although their gene expression does not respond specifically to saline conditions, they could contribute, together with the previously characterized Na+ efflux systems SiENA1 and SiENA5, to relieve Na+ from the S. indica cytosol under this stressed condition. In parallel, an in-silico study has been carried out to define its complete transportome. To further investigate the repertoire of transporters expressed in free-living cells of S. indica and during plant infection under saline conditions, a comprehensive RNA-seq approach was taken. Interestingly, SiENA5 was the only gene significantly induced under free-living conditions in response to moderate salinity at all the tested time points, revealing that it is one of the main salt-responsive genes of S. indica. In addition, the symbiosis with Arabidopsis thaliana also induced SiENA5 gene expression, but significant changes were only detected after long periods of infection, indicating that the association with the plant somehow buffers and protects the fungus against the external stress. Moreover, the significant and strongest induction of the homologous gene SiENA1 occurred during symbiosis, regardless the exposure to salinity. The obtained results suggest a novel and relevant role of these two proteins during the establishment and maintenance of fungus-plant interaction.