Cells respond to salt stress by osmolyte synthesis and by increase of K⁺ uptake and Na⁺ efflux at the plasma membrane and of Na⁺ accumulation at the vacuole. This cellular level of response has been approached by the molecular genetics of Saccharomyces cerevisiae. Two plasma membrane proteins, Sln1 and Sho1, operate as sensors for turgor loss under mild osmotic stress. They activate the Hog1 MAP kinase which induces several defence genes, including the ENA1 Na⁺-efflux pump. Hog1-mediated induction occurs by inactivation of the Sko1 repressor. High concentrations of Na⁺ activate the calcium-dependent protein phosphatase calcineurin, which induces the ENA1 gene via the positive transcription factor Hal8/Crz1/Tcn1. Vacuoles could operate as Na⁺ sensors through a Na⁺-Ca²⁺ exchange mechanism. The electrical potential of the plasma membrane is a major determinant of the uptake of toxic cations, including Na⁺, by non-specific leakage pathways. This biophysical parameter is modulated by the concerted activities of the proton pumping Pma1 ATPase and the Trk1,2 K⁺ uptake system. Both calcineurin and the protein kinase Hal4,5 modulate Trk1,2 and, indirectly, the membrane potential and salt tolerance. The limiting role of vacuolar cation accumulation in salt tolerance has been demonstrated by expression in yeast vacuoles of the bacterial antiporter NhaA.

Glycolysis is a central metabolic pathway that, in plants, occurs in both the cytosol and the plastids. The glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPDH) catalyzes the conversion of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate with concomitant reduction of NAD+ to NADH. Both cytosolic (GAPCs) and plastidial (GAPCps) GAPDH activities have been described. However, the in vivo functions of the plastidial isoforms remain unresolved. In this work, we have identified two Arabidopsis (Arabidopsis thaliana) chloroplast/plastid-localized GAPDH isoforms (GAPCp1 and GAPCp2). gapcp double mutants display a drastic phenotype of arrested root development, dwarfism, and sterility. In spite of their low gene expression level as compared with other GAPDHs, GAPCp down-regulation leads to altered gene expression and to drastic changes in the sugar and amino acid balance of the plant. We demonstrate that GAPCps are important for the synthesis of serine in roots. Serine supplementation to the growth medium rescues root developmental arrest and restores normal levels of carbohydrates and sugar biosynthetic activities in gapcp double mutants. We provide evidence that the phosphorylated pathway of Ser biosynthesis plays an important role in supplying serine to roots. Overall, these studies provide insights into the in vivo functions of the GAPCps in plants. Our results emphasize the importance of the plastidial glycolytic pathway, and specifically of GAPCps, in plant primary metabolism.

The regulation of intracellular ion concentrations is a fundamental property of living cells. Although many ion transporters have been identified, the systems that modulate their activity remain largely unknown. We have characterized two partially redundant genes fromSaccharomyces cerevisiae, HAL4/SAT4 andHAL5, that encode homologous protein kinases implicated in the regulation of cation uptake. Overexpression of these genes increases the tolerance of yeast cells to sodium and lithium, whereas gene disruptions result in greater cation sensitivity. These phenotypic effects of the mutations correlate with changes in cation uptake and are dependent on a functional Trk1-Trk2 potassium transport system. In addition, hal4 hal5 and trk1 trk2 mutants exhibit similar phenotypes: (i) they are deficient in potassium uptake; (ii) their growth is sensitive to a variety of toxic cations, including lithium, sodium, calcium, tetramethylammonium, hygromycin B, and low pH; and (iii) they exhibit increased uptake of methylammonium, an indicator of membrane potential. These results suggest that the Hal4 and Hal5 protein kinases activate the Trk1-Trk2 potassium transporter, increasing the influx of potassium and decreasing the membrane potential. The resulting loss in electrical driving force reduces the uptake of toxic cations and improves salt tolerance. Our data support a role for regulation of membrane potential in adaptation to salt stress that is mediated by the Hal4 and Hal5 kinases.

Membrane-delimited abscisic acid (ABA) signal transduction plays a critical role in early ABA signaling, but the molecular mechanisms linking core signaling components to the plasma membrane are unclear. We show that transient calcium-dependent interactions of PYR/PYL ABA receptors with membranes are mediated through a 10-member family of C2-domain ABA-related (CAR) proteins in Arabidopsis thaliana. Specifically, we found that PYL4 interacted in an ABA-independent manner with CAR1 in both the plasma membrane and nucleus of plant cells. CAR1 belongs to a plant-specific gene family encoding CAR1 to CAR10 proteins, and bimolecular fluorescence complementation and coimmunoprecipitation assays showed that PYL4-CAR1 as well as other PYR/PYL-CAR pairs interacted in plant cells. The crystal structure of CAR4 was solved, which revealed that, in addition to a classical calcium-dependent lipid binding C2 domain, a specific CAR signature is likely responsible for the interaction with PYR/PYL receptors and their recruitment to phospholipid vesicles. This interaction is relevant for PYR/PYL function and ABA signaling, since different car triple mutants affected in CAR1, CAR4, CAR5, and CAR9 genes showed reduced sensitivity to ABA in seedling establishment and root growth assays. In summary, we identified PYR/PYL-interacting partners that mediate a transient Ca²⁺-dependent interaction with phospholipid vesicles, which affects PYR/PYL subcellular localization and positively regulates ABA signaling.

The yeast Ppz protein phosphatases and the Hal3p inhibitory subunit are important determinants of salt tolerance, cell wall integrity and cell cycle progression. We present several lines of evidence showing that these disparate phenotypes are connected by the fact that Ppz regulates K+ transport. First, salt tolerance, cell wall integrity and cell cycle phenotypes of Ppz mutants are dependent on the Trk K+ transporters. Secondly, Ppz mutants exhibit altered activity of the Trk system, as measured by rubidium uptake. Thirdly, Ppz mutants exhibit altered intracellular K+ and pH, as expected from H+ efflux providing electrical balance during K+ uptake. Our unifying picture of Ppz phenotypes contends that activation of Trk by decreased Ppz activity results in plasma membrane depolarization (reducing uptake of toxic cations), increased intracellular K+ and turgor (compromising cell integrity), and increased intracellular pH (augmenting the expression of pH‐regulated genes and facilitating α‐factor recovery). In addition to providing a coherent explanation for all Ppz‐dependent phenotypes, our results provide evidence for a causal relationship between intracellular cation homeostasis and a potential cell cycle checkpoint.