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.

Summary: 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.

Summary: Extracellular ATP regulates higher plant growth and adaptation. The signalling events may be unique to higher plants, as they lack animal purinoceptor homologues. Although it is known that plant cytosolic free Ca²⁺can be elevated by extracellular ATP, the mechanism is unknown. Here, we have studied roots ofArabidopsis thalianato determine the events that lead to the transcriptional stress response evoked by extracellular ATP. Root cell protoplasts were used to demonstrate that signalling to elevate cytosolic free Ca²⁺is determined by ATP perception at the plasma membrane, and not at the cell wall. Imaging revealed that extracellular ATP causes the production of reactive oxygen species in intact roots, with the plasma membrane NADPH oxidase AtRBOHC being the major contributor. This resulted in the stimulation of plasma membrane Ca²⁺‐permeable channels (determined using patch‐clamp electrophysiology), which contribute to the elevation of cytosolic free Ca²⁺. Disruption of this pathway in theAtrbohCmutant impaired the extracellular ATP‐induced increase in reactive oxygen species (ROS), the activation of Ca²⁺channels, and the transcription of the MAP kinase3 gene that is known to be involved in stress responses. This study shows that higher plants, although bereft of purinoceptor homologues, could have evolved a distinct mechanism to transduce the ATP signal at the plasma membrane.

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH^•. In root cells, extracellular OH^• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH^•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH^•. An OH^•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

Bicarbonate plays a fundamental role in the cell pH status in all organisms. In autotrophs, HCO₃⁻ may further contribute to carbon concentration mechanisms (CCM). This is especially relevant in the CO₂-poor habitats of cyanobacteria, aquatic microalgae, and macrophytes. Photosynthesis of terrestrial plants can also benefit from CCM as evidenced by the evolution of C₄ and Crassulacean Acid Metabolism (CAM). The presence of HCO₃⁻ in all organisms leads to more questions regarding the mechanisms of uptake and membrane transport in these different biological systems. This review aims to provide an overview of the transport and metabolic processes related to HCO₃⁻ in microalgae, macroalgae, seagrasses, and terrestrial plants. HCO₃⁻ transport in cyanobacteria and human cells is much better documented and is included for comparison. We further comment on the metabolic roles of HCO₃⁻ in plants by focusing on the diversity and functions of carbonic anhydrases and PEP carboxylases as well as on the signaling role of CO₂/HCO₃⁻ in stomatal guard cells. Plant responses to excess soil HCO₃⁻ is briefly addressed. In conclusion, there are still considerable gaps in our knowledge of HCO₃⁻ uptake and transport in plants that hamper the development of breeding strategies for both more efficient CCM and better HCO₃⁻ tolerance in crop plants.

The stress hormone abscisic acid (ABA) induces expression of defence genes in many organs, modulates ion homeostasis and metabolism in guard cells, and inhibits germination and seedling growth. Concerning the latter effect, several mutants of Arabidopsis thaliana with improved capability for H⁺ efflux (wat1-1D, overexpression of AKT1 and ost2-1D) are less sensitive to inhibition by ABA than the wild type. This suggested that ABA could inhibit H⁺ efflux (H⁺-ATPase) and induce cytosolic acidification as a mechanism of growth inhibition. Measurements to test this hypothesis could not be done in germinating seeds and we used roots as the most convenient system. ABA inhibited the root plasma-membrane H⁺-ATPase measured in vitro (ATP hydrolysis by isolated vesicles) and in vivo (H⁺ efflux from seedling roots). This inhibition involved the core ABA signalling elements: PYR/PYL/RCAR ABA receptors, ABA-inhibited protein phosphatases (HAB1), and ABA-activated protein kinases (SnRK2.2 and SnRK2.3). Electrophysiological measurements in root epidermal cells indicated that ABA, acting through the PYR/PYL/RCAR receptors, induced membrane hyperpolarization (due to K⁺ efflux through the GORK channel) and cytosolic acidification. This acidification was not observed in the wat1-1D mutant. The mechanism of inhibition of the H⁺-ATPase by ABA and its effects on cytosolic pH and membrane potential in roots were different from those in guard cells. ABA did not affect the in vivo phosphorylation level of the known activating site (penultimate threonine) of H⁺-ATPase in roots, and SnRK2.2 phosphorylated in vitro the C-terminal regulatory domain of H⁺-ATPase while the guard-cell kinase SnRK2.6/OST1 did not.