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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.

Potassium (K

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).

It is well established that thylakoid membranes of chloroplasts convert light energy into chemical energy, yet the development of chloroplast and thylakoid membranes is poorly understood. Loss of function of the two envelope K(+)/H(+) antiporters AtKEA1 and AtKEA2 was shown previously to have negative effects on the efficiency of photosynthesis and plant growth; however, the molecular basis remained unclear. Here, we tested whether the previously described phenotypes of double mutant kea1kea2 plants are due in part to defects during early chloroplast development in Arabidopsis (Arabidopsis thaliana). We show that impaired growth and pigmentation is particularly evident in young expanding leaves of kea1kea2 mutants. In proliferating leaf zones, chloroplasts contain much lower amounts of photosynthetic complexes and chlorophyll. Strikingly, AtKEA1 and AtKEA2 proteins accumulate to high amounts in small and dividing plastids, where they are specifically localized to the two caps of the organelle separated by the fission plane. The unusually long amino-terminal domain of 550 residues that precedes the antiport domain appears to tether the full-length AtKEA2 protein to the two caps. Finally, we show that the double mutant contains 30% fewer chloroplasts per cell. Together, these results show that AtKEA1 and AtKEA2 transporters in specific microdomains of the inner envelope link local osmotic, ionic, and pH homeostasis to plastid division and thylakoid membrane formation.

KEA genes encode putative K(+) efflux antiporters that are predominantly found in algae and plants but are rare in metazoa; however, nothing is known about their functions in eukaryotic cells. Plant KEA proteins show homology to bacterial K(+) efflux (Kef) transporters, though two members in the Arabidopsis thaliana family, AtKEA1 and AtKEA2, have acquired an extra hydrophilic domain of over 500 residues at the amino terminus. We show that AtKEA2 is highly expressed in leaves, stems and flowers, but not in roots, and that an N-terminal peptide of the protein is targeted to chloroplasts in Arabidopsis cotyledons. The full-length AtKEA2 protein was inactive when expressed in yeast; however, a truncated AtKEA2 protein (AtsKEA2) lacking the N-terminal domain complemented disruption of the Na(+)(K(+))/H(+) antiporter Nhx1p to confer hygromycin resistance and tolerance to Na(+) or K(+) stress. To test transport activity, purified truncated AtKEA2 was reconstituted in proteoliposomes containing the fluorescent probe pyranine. Monovalent cations reduced an imposed pH gradient (acid inside) indicating AtsKEA2 mediated cation/H(+) exchange with preference for K(+)=Cs(+)Li(+)Na(+). When a conserved Asp(721) in transmembrane helix 6 that aligns to the cation binding Asp(164) of Escherichia coli NhaA was replaced with Ala, AtsKEA2 was completely inactivated. Mutation of a Glu(835) between transmembrane helix 8 and 9 in AtsKEA2 also resulted in loss of activity suggesting this region has a regulatory role. Thus, AtKEA2 represents the founding member of a novel group of eukaryote K(+)/H(+) antiporters that modulate monovalent cation and pH homeostasis in plant chloroplasts or plastids.

• 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.

Heterotrimeric G proteins (G-proteins) are versatile signaling elements conserved in Eukaryotes. In animals G-proteins relay signals from 7-transmembrane spanning G protein-coupled receptors (GPCRs) to intracellular downstream effectors; however, the existence of GPCRs in plants is controversial. Contrastingly, a surplus of receptor-like kinases (RLKs) provides signal recognition at the plant cell surface. It is established that G proteins are involved in plant defense and suggested that they relay signals from defense-related RLKs. However, it is unclear how the signaling is conducted, as physical interaction between the RLKs and G proteins has not been demonstrated. Using yeast split-ubiquitin system and Bimolecular Fluorescence Complementation assays, we demonstrate physical interaction between the Gα, Gγ1 and Gγ2 subunits, and the defense-related RD-type receptor like kinases CERK1, BAK1 and BIR1. At the same time, no interaction was detected with the non-RD RLK FLS2. We hypothesize that G-proteins mediate signal transduction immediately downstream of the pathogenesis-related RLKs.

The ductile-to-brittle transition (DBT) behavior of two similar Fe-Cr-Al oxide dispersion strengthened (ODS) stainless steels was analyzed following the Cottrell–Petch model. Both alloys were manufactured by mechanical alloying (MA) but by different forming routes. One was manufactured as hot rolled tube, and the other in the form of hot extruded bar. The two hot forming routes considered do not significantly influence the microstructure, but cause differences in the texture and the distribution of oxide particles. These have little influence on tensile properties; however, the DBT temperature and the upper shelf energy (USE) are significantly affected because of delamination orientation with regard to the notch plane. Whereas in hot rolled material the delaminations are parallel to the rolling surface, in the hot extruded material, they are randomly oriented because the material is transversally isotropic., Financial support of the Spanish Ministerio de Economia e Innovación (MINECO) through a Coordinated Project in the Materials Area of Plan Nacional 2013 (MAT2013-47460-C5-1) is gratefully acknowledged. We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI)

We previously demonstrated that Saccharomyces cerevisiae vnx1Δ mutant strains displayed an almost total loss of Na(+) and K(+)/H(+) antiporter activity in a vacuole-enriched fraction. However, using different in vitro transport conditions, we were able to reveal additional K(+)/H(+) antiporter activity. By disrupting genes encoding transporters potentially involved in the vnx1 mutant strain, we determined that Vcx1p is responsible for this activity. This result was further confirmed by complementation of the vnx1Δvcx1Δ nhx1Δ triple mutant with Vcx1p and its inactivated mutant Vcx1p-H303A. Like the Ca(2+)/H(+) antiporter activity catalyzed by Vcx1p, the K(+)/H(+) antiporter activity was strongly inhibited by Cd(2+) and to a lesser extend by Zn(2+). Unlike as previously observed for NHX1 or VNX1, VCX1 overexpression only marginally improved the growth of yeast strain AXT3 in the presence of high concentrations of K(+) and had no effect on hygromycin sensitivity. Subcellular localization showed that Vcx1p and Vnx1p are targeted to the vacuolar membrane, whereas Nhx1p is targeted to prevacuoles. The relative importance of Nhx1p, Vnx1p, and Vcx1p in the vacuolar accumulation of monovalent cations will be discussed.