Beatriz Cubero

Ion Exchangers NHX1 and NHX2 Mediate Active Potassium Uptake into Vacuoles to Regulate Cell Turgor and Stomatal Function in Arabidopsis

Intracellular Na+,K+/H+ antiporters (NHXs) play central roles in maintaining ion homeostasis and pH control. Tonoplast-localized proteins NHX1 and NHX2 are critical for active K+ uptake into the vacuole, a process that is required to create osmotic potential for cell expansion and turgor regulation. These proteins are abundantly expressed in guard cells, where they contribute to stomata function. Intracellular NHX proteins are Na+,K+/H+ antiporters involved in K+ homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na+,K+/H+ antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K+. By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na+ sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K+ pool, which in turn provoked greater K+ retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function.

The AtNHX1 exchanger mediates potassium compartmentation in vacuoles of transgenic tomato

NHX-type antiporters in the tonoplast have been reported to increase the salt tolerance of various plants species, and are thought to mediate the compartmentation of Na(+) in vacuoles. However, all isoforms characterized so far catalyze both Na(+)/H(+) and K(+)/H(+) exchange. Here, we show that AtNHX1 has a critical involvement in the subcellular partitioning of K(+), which in turn affects plant K(+) nutrition and Na(+) tolerance. Transgenic tomato plants overexpressing AtNHX1 had larger K(+) vacuolar pools in all growth conditions tested, but no consistent enhancement of Na(+) accumulation was observed under salt stress. Plants overexpressing AtNHX1 have a greater capacity to retain intracellular K(+) and to withstand salt-shock. Under K(+)-limiting conditions, greater K(+) compartmentation in the vacuole occurred at the expense of the cytosolic K(+) pool, which was lower in transgenic plants. This caused the early activation of the high-affinity K(+) uptake system, enhanced K(+) uptake by roots, and increased the K(+) content in plant tissues and the xylem sap of transformed plants. Our results strongly suggest that NHX proteins are likely candidates for the H(+)-linked K(+) transport that is thought to facilitate active K(+) uptake at the tonoplast, and the partitioning of K(+) between vacuole and cytosol.

Control of vacuolar dynamics and regulation of stomatal aperture by tonoplast potassium uptake

Significance Rapid fluxes of K+ and other osmolytes in guard cells control the opening and closing of stomata and thereby gas exchange and transpiration of plants. Despite the well-established role of the plasma membrane of guard cells in stomatal function, osmolyte uptake into the cytosol represents only a transient step to the vacuole, as more than 90% of the solutes accumulate in these organelles. We show that the tonoplast-localized K+/H+ exchangers mediate the vacuolar accumulation of K+ in guard cells, and that activity of these transporters controls not only stomatal opening but also stomatal closure. We also establish vacuolar K+/H+ exchange as a critical component involved in vacuolar remodeling and the regulation of vacuolar pH during stomatal movements. Stomatal movements rely on alterations in guard cell turgor. This requires massive K+ bidirectional fluxes across the plasma and tonoplast membranes. Surprisingly, given their physiological importance, the transporters mediating the energetically uphill transport of K+ into the vacuole remain to be identified. Here, we report that, in Arabidopsis guard cells, the tonoplast-localized K+/H+ exchangers NHX1 and NHX2 are pivotal in the vacuolar accumulation of K+ and that nhx1 nhx2 mutant lines are dysfunctional in stomatal regulation. Hypomorphic and complete-loss-of-function double mutants exhibited significantly impaired stomatal opening and closure responses. Disruption of K+ accumulation in guard cells correlated with more acidic vacuoles and the disappearance of the highly dynamic remodelling of vacuolar structure associated with stomatal movements. Our results show that guard cell vacuolar accumulation of K+ is a requirement for stomatal opening and a critical component in the overall K+ homeostasis essential for stomatal closure, and suggest that vacuolar K+ fluxes are also of decisive importance in the regulation of vacuolar dynamics and luminal pH that underlie stomatal movements.

Mutants of the Arabidopsis thaliana Cation/H+ Antiporter AtNHX1 Conferring Increased Salt Tolerance in Yeast . The Endosome/prevacuolar compartment is a target for salt toxicity

Mutants of the plant cation/H+ antiporter AtNHX1 that confer greater halotolerance were generated by random mutagenesis and selected in yeast by phenotypic complementation. The amino acid substitutions that were selected were conservative and occurred in the second half of the membrane-associated N terminus. AtNHX1 complemented the lack of endogenous ScNHX1 in endosomal protein trafficking assays. Growth enhancement on hygromycin B and vanadate media agreed with a generally improved endosomal/prevacuolar function of the mutated proteins. In vivo measurements by 31P NMR revealed that wild-type and mutant AtNHX1 transporters did not affect cytosolic or vacuolar pH. Surprisingly, when yeast cells were challenged with lithium, a tracer for sodium, the main effect of the mutations in AtNHX1 was a reduction in the amount of compartmentalized lithium. When purified and reconstituted into proteoliposomes or assayed in intact vacuoles isolated from yeast cells, a representative mutant transporter (V318I) showed a greater cation discrimination favoring potassium transport over that of sodium or lithium. Together, our data suggest that the endosome/prevacuolar compartment is a target for salt toxicity. Poisoning by toxic cations in the endosome/prevacuolar compartment is detrimental for cell functions, but it can be alleviated by improving the discrimination of transported alkali cations by the resident cation/H+ antiporter.

The Phosphate Transporter PHT4;6 Is a Determinant of Salt Tolerance that Is Localized to the Golgi Apparatus of Arabidopsis

Insertion mutations that disrupt the function of PHT4;6 (At5g44370) cause NaCl hypersensitivity of Arabidopsis seedlings that is characterized by reduced growth of the primary root, enhanced lateral branching, and swelling of root tips. Mutant phenotypes were exacerbated by sucrose, but not by equiosmolar concentrations of mannitol, and attenuated by low inorganic phosphate in the medium. Protein PHT4;6 belongs to the Major Facilitator Superfamily of permeases that shares significant sequence similarity to mammalian type-I Pi transporters and vesicular glutamate transporters, and is a member of the PHT4 family of putative intracellular phosphate transporters of plants. PHT4;6 localizes to the Golgi membrane and transport studies indicate that PHT4;6 facilitates the selective transport of Pi but not of chloride or inorganic anions. Phenotypic similarities with other mutants displaying root swelling suggest that PHT4;6 likely functions in protein N-glycosylation and cell wall biosynthesis, which are essential for salt tolerance. Together, our results indicate that PHT4;6 transports Pi out of the Golgi lumenal space for the re-cycling of the Pi released from glycosylation processes.

Cellular Responses of the Lichen Circinaria gyrosa in Mars-Like Conditions

Lichens are extremely resistant organisms that colonize harsh climatic areas, some of them defined as “Mars-analog sites.” There still remain many unsolved questions as to how lichens survive under such extreme conditions. Several studies have been performed to test the resistance of various lichen species under space and in simulated Mars-like conditions. The results led to the proposal that Circinaria gyrosa (Lecanoromycetes, Ascomycota) is one of the most durable astrobiological model lichens. However, although C. gyrosa has been exposed to Mars-like environmental conditions while in a latent state, it has not been exposed in its physiologically active mode. We hypothesize that the astrobiological test system “Circinaria gyrosa,” could be able to be physiologically active and to survive under Mars-like conditions in a simulation chamber, based on previous studies performed at dessicated-dormant stage under simulated Mars-like conditions, that showed a complete recover of the PSII activity (Sánchez et al., 2012). Epifluorescence and confocal laser scanning microscopy (CLSM) showed that living algal cells were more abundant in samples exposed to niche conditions, which simulated the conditions in micro-fissures and micro-caves close to the surface that have limited scattered or time-dependent light exposure, than in samples exposed to full UV radiation. The medulla was not structurally affected, suggesting that the niche exposure conditions did not disturb the lichen thalli structure and morphology as revealed by field emission scanning electron microscopy (FESEM). In addition, changes in the lichen thalli chemical composition were determined by analytical pyrolysis. The chromatograms resulting from analytical pyrolysis at 500°C revealed that lichen samples exposed to niche conditions and full UV radiation consisted primarily of glycosidic compounds, lipids, and sterols, which are typical constituents of the cell walls. However, specific differences could be detected and used as markers of the UV-induced damage to the lichen membranes. Based on its viability responses after rehydration, our study shows that the test lichen survived the 30-day incubation in the Mars chamber particularly under niche conditions. However, the photobiont was not able to photosynthesize under the Mars-like conditions, which indicates that the surface of Mars is not a habitable place for C. gyrosa.