Plastidial transporters KEA1 and KEA2 at the inner envelope membrane adjust stromal pH in the dark

Photosynthesis and carbon fixation depend critically on the regulation of pH in chloroplast compartments in the daylight and at night. While it is established that an alkaline stroma is required for carbon fixation, it is not known how alkaline stromal pH is formed, maintained or regulated. We tested whether two envelope transporters, AtKEA1 and AtKEA2, directly affected stromal pH in isolated Arabidopsis chloroplasts using the fluorescent probe 2ʹ,7ʹ-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF). External K-induced alkalinization of the stroma was observed in chloroplasts from wild-type (WT) plants but not from kea1kea2 mutants, suggesting that KEA1 and KEA2 mediate K uptake/H loss to modulate stromal pH. While light-stimulated alkalinization of the stroma was independent of KEA1 and KEA2, the rate of decay to neutral pH in the dark is delayed in kea1kea2 mutants. However, the dark-induced loss of a pH gradient across the thylakoid membrane was similar in WT and mutant chloroplasts. This indicates that proton influx from the cytosol mediated by envelope K/H antiporters contributes to adjustment of stromal pH upon light to dark transitions.This work was supported by ERDF cofinanced grants from the Spanish Agencia Estatal de Investigación, Ministerio de Economía y Competitividad/Ministerio de Ciencia e Innovación (BIO2015-65056-P, PID2019-105260GB-I00 and BIO2016-81957-REDT/AEI to KV and MPRR) and from the Consejería de Conocimiento, Investigación y Universidad, Junta de Andalucía, Spain (CVI-7558 to MPRR), and stimulated by the ERA-CAPS funded project Flux4Lives

Chloroplast envelope K+/H+ antiporters are involved in cytosol pH regulation

Variations in light intensity induce cytosol pH changes in photosynthetic tissues, providing a possible signal to adjust a variety of biochemical, physiological and developmental processes to the energy status of the cells. It was shown that these pH changes are partially due to the transport of protons in or out of the thylakoid lumen. However, the ion transporters in the chloroplast that transmit these pH changes to the cytosol are not known. KEA1 and KEA2 are K/H antiporters in the chloroplast inner envelope that adjust stromal pH in light-to-dark transitions. We previously determined that stromal pH is higher in kea1kea2 mutant cells. In this study, we now show that KEA1 and KEA2 are required to attenuate cytosol pH variations upon sudden light intensity changes in leaf mesophyll cells, showing they are important components of the light-modulated pH signalling module. The kea1kea2 mutant mesophyll cells also have a considerably less negative membrane potential. Membrane potential is dependent on the activity of the plasma membrane proton ATPase and is regulated by secondary ion transporters, mainly potassium channels in the plasma membrane. We did not find significant differences in the activity of the plasma membrane proton pump but found a strongly increased membrane permeability to protons, especially potassium, of the double mutant plasma membranes. Our results indicate that chloroplast envelope K/H antiporters not only affect chloroplast pH but also have a strong impact on cellular ion homeostasis and energization of the plasma membrane.This work was supported by grants PID2019-105260GB-I00 funded by MCIN/AEI/ 10.13039/501100011033 to KV and MPRR and Research Funds of Málaga University (0837002020 B4-2021-08) to L

Overexpression of the tomato K+/H+ antiporter LeNHX2 confers salt tolerance by improving potassium compartmentalization

• Here, the function of the tomato (Solanum lycopersicon) K +/H+ antiporter LeNHX2 was studied using 35S-driven gene overexpression of a histagged LeNHX2 protein in Arabidopsis thaliana and LeNHX2 gene silencing in tomato. • Transgenic A. thaliana plants expressed the histagged LeNHX2 both in shoots and in roots, as assayed by western blotting. Transitory expression of a green fluorescent protein (GFP) tagged protein showed that the antiporter is present in small vesicles. Internal membrane vesicles from transgenic plants displayed enhanced K+/H+ exchange activity, confirming the K+/H+ antiporter function of this enzyme. Transgenic A. thaliana plants overexpressing the histagged tomato antiporter LeNHX2 exhibited inhibited growth in the absence of K+ in the growth medium, but were more tolerant to high concentrations of Na + than untransformed controls. When grown in the presence of NaCl, transgenic plants contained lower concentrations of intracellular Na +, but more K+, as compared with untransformed controls. • Silencing of LeNHX2 in S. lycopersicon plants produced significant inhibition of plant growth and fruit and seed production as well as increased sensitivity to NaCl. • The data indicate that regulation of K+ homeostasis by LeNHX2 is essential for normal plant growth and development, and plays an important role in the response to salt stress by improving K + accumulation.Peer Reviewe

Overexpression of LeNHX2 and SlSOS2 increases salt tolerance and fruit production in double transgenic tomato plants

Transgenic tomato plants (Solanum lycopersicum L. cv. MicroTom) overexpressing both the K,Na/H antiporter LeNHX2 and the regulatory kinase SlSOS2 were produced by crossing transgenic homozygous lines overexpressing LeNHX2 and SlSOS2. LeNHX2 expression was enhanced in plants overexpressing LeNHX2 but surprisingly even more in plants overexpressing SlSOS2 with and without LeNHX2. All transgenic plants showed better NaCl tolerance than wild type controls and plants overexpressing both LeNHX2 and SlSOS2 grew better under saline conditions than plants overexpressing only one of these genes. Yield related parameters indicated that single and above all double transgenic plants performed significantly better than wild type controls. All transgenic plants produced fruits with a higher K content than wild-type plants and plants overexpressing SlSOS2 accumulated more Na in fruits than the rest of the plants when grown with NaCl. Roots, stems and leaves of transgenic plants overexpressing LeNHX2 showed a higher K content than wild type and single transgenic plants overexpressing SlSOS2. Na content in stems and leaves of NaCl treated plants was higher in SlSOS2 overexpressing plants than in wild type and LeNHX2 single transgenic plants. All transgenic lines showed a higher leaf relative water content and a higher plant water content and water use efficiency than wild type controls when both were grown in the presence of NaCl. Results in this work indicate that the joint overexpression of LeNHX2 and SlSOS2 improves growth and water status under NaCl stress, affects K and Na homeostasis and enhances fruit yield of tomato plants.This work was supported by grants from Consejería de Economía, Innovación, Ciencia y Empresa, Junta de Andalucía, Spain (CVI-7558 to MPRR), Spanish MINECO and Agencia Estatal de Investigación (BIO2015-65056-P and BIO2016-81957-REDT/AEI to KV and MPRR) and Moroccan MESRSFC and CNRST (PPR/2015/21 to MB).Peer Reviewe

Overexpression of LeNHX4 improved yield, fruit quality and salt tolerance in tomato plants (Solanum lycopersicum L.)

The function of the tomato K, Na/H antiporter LeNHX4 has been analyzed using 35S-driven gene construct for overexpressing a histagged LeNHX4 protein in Solanum lycopersicum L. Compared to wild-type plants, the expression of LeNHX4 was enhanced in most of plants transformed with a gene construct for LeNHX4 overexpression although some plants showed a decreased LeNHX4 expression. Overexpression of LeNHX4 was associated to an increased fruit size while silencing of this gene was related to a decreased fruit size. We have investigated the effect of LeNHX4 overexpression on fruit production and quality and we have also evaluated salt tolerance in two different overexpression lines by measuring proline, protein and glucose concentrations in tomato leaves grown either under control (0 mM NaCl) or saline (125 mM NaCl) conditions. Plants overexpressing LeNHX4 showed a higher amount of fruits than WT plants and accumulated higher contents of sugars and cations (Na and K). The application of 125 mM NaCl, affected negatively fruit production and quality of WT plants. However the transgenic lines overexpressing LeNXH4 increased fruit quality and yield. In relation to salt tolerance, overexpression lines showed higher levels of leaf proline, glucose and proteins under NaCl treatment. The overexpression of LeNHX4 in tomato plants, improved salinity tolerance and increased fruit yield and quality under both normal and salinity stress conditions.This work was supported by Grants from Consejería de Economía, Innovación, Ciencia y Empresa, Junta de Andalucía, Spain (CVI-7558 to MPRR), Spanish Ministry of Economy and Competitiveness and Agencia Estatal de Investigación (BIO2015-65056-P, BIO2016-81957-REDT/AEI and Programa I-COOPB+2013 Ref. COOPB20053 National Centre for Scientific and Technical Research COOPB20053). and Minister for Higher Education, Scientific Research and Executive Training (Morocco)

Deletion of the N‐terminal domain of the yeast vacuolar (Na +

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H exchanger showing Na/H and K/H antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na/H exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na and K accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.This work was supported by grants from Spanish MINECO and Agencia Estatal de Investigación (BIO2015‐65056‐P and BIO2016‐81957‐REDT/AEI to K.V. and M.P.R.R.) and from Consejería de Economía, Innovación, Ciencia y Empresa, Junta de Andalucía, Spain (CVI‐7558 to MPRR)

Proton Coupled Ion transport at the chloroplast envelope.

Póster presentado en Plant Biology Europe 2023 (PBE 2023), Jul 2023, Marseille, FranceGrant PID2019-105260GB-I00 funded by MCIN/AEI/ 10.13039/50110001103

Deletion of the N-terminal domain of the yeast vacuolar (Na+,K+)/H+ antiporter Vnx1p improves salt tolerance in yeast and transgenic Arabidopsis

Cation/proton antiporters play a major role in the control of cytosolic ion concentrations in prokaryotes and eukaryotes organisms. In yeast, we previously demonstrated that Vnx1p is a vacuolar monovalent cation/H exchanger showing Na/H and K/H antiporter activity. We have also shown that disruption of VNX1 results in an almost complete abolishment of vacuolar Na/H exchange, but yeast cells overexpressing the complete protein do not show improved salinity tolerance. In this study, we have identified an autoinhibitory N-terminal domain and have engineered a constitutively activated version of Vnx1p, by removing this domain. Contrary to the wild type protein, the activated protein has a pronounced effect on yeast salt tolerance and vacuolar pH. Expression of this truncated VNX1 gene also improves Arabidopsis salt tolerance and increases Na and K accumulation of salt grown plants thus suggesting a biotechnological potential of activated Vnx1p to improve salt tolerance of crop plants.This work was supported by grants from Spanish MINECO and Agencia Estatal de Investigación (BIO2015‐65056‐P and BIO2016‐81957‐REDT/AEI to K.V. and M.P.R.R.) and from Consejería de Economía, Innovación, Ciencia y Empresa, Junta de Andalucía, Spain (CVI‐7558 to MPRR)

Loss of function of the chloroplast membrane K+/H+ antiporters AtKEA1 and AtKEA2 alters the ROS and NO metabolism but promotes drought stress resilience

Potassium (K) exerts key physiological functions such as osmoregulation, stomatal movement, membrane transport, protein synthesis and photosynthesis among others. Previously, it was demonstrated in Arabidopsis thaliana that the loss of function of the chloroplast K Efflux Antiporters KEA1 and KEA2, located in the inner envelope membrane, provokes inefficient photosynthesis. Therefore, the main goal of this study was to evaluate the potential impact of the loss of function of those cation transport systems in the metabolism of reactive oxygen and nitrogen species (ROS and RNS). Using 14-day-old seedlings from Arabidopsis double knock-out kea1kea2 mutants, ROS metabolism and NO content in roots and green cotyledons were studied at the biochemical level. The loss of function of AtKEA1 and AtKEA2 did not cause oxidative stress but it provoked an alteration of the ROS homeostasis affecting some ROS-generating enzymes. These included glycolate oxidase (GOX) and NADPH-dependent superoxide generation activity, enzymatic and non-enzymatic antioxidants and both NADP-isocitrate dehydrogenase and NADP-malic enzyme activities. NO content, analyzed by confocal laser scanning microscopy (CLSM), was negatively affected in both photosynthetic and non-photosynthetic organs in kea1kea2 mutant seedlings. Furthermore, the S-nitrosoglutathione reductase (GSNOR) protein expression and activity were downregulated in kea1kea2 mutants, whereas the tyrosine nitrated protein profile, analyzed by immunoblot, was unaffected but the relative expression of each immunoreactive band changed. Moreover, kea1kea2 mutants showed an increased photorespiratory pathway and stomata closure, thus promoting a higher resilience to drought stress. Data suggest that the chloroplast osmotic balance and integrity maintained by AtKEA1 and AtKEA2 are necessary to keep the balance of ROS/RNS metabolism. Moreover, these data open new questions about how endogenous NO generation might be affected by the K/H transport located in the chloroplasts.The projects PID 2019-103924 GB-I00 (MICIT) and P18-FR-1359 (Plan Andaluz de Investigación, Desarrollo e Innovación), Spain, financed the research by ASM, SGG, JMP, and FJC, and the grant BIO 2015–65056 P (MINECO, Spain) that of MNAS, PRR and KV