Lourdes Rubio

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.

Plant extracellular ATP signalling by plasma membrane NADPH oxidase and Ca2+ channels

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 of Arabidopsis thaliana to 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 the AtrbohC mutant 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.

Arabidopsis Annexin1 Mediates the Radical-Activated Plasma Membrane Ca2+- and K+-Permeable Conductance in Root Cells

The Arabidopsis thaliana root cell plasma membrane contains a calcium channel that is activated by oxidizing conditions and operates in cell growth. It was identified here as the most abundant member of the Arabidopsis annexins. These are soluble proteins that can undergo conditional attachment to or insertion into membranes. 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.

Peptidyl‐prolyl cis‐trans isomerase ROF2 modulates intracellular pH homeostasis in Arabidopsis

Intracellular pH must be kept close to neutrality to be compatible with cellular functions, but the mechanisms of pH homeostasis and the responses to intracellular acidification are mostly unknown. In the plant Arabidopsis thaliana, we found that intracellular acid stress generated by weak organic acids at normal external pH induces expression of several chaperone genes, including ROF2, which encodes a peptidyl-prolyl cis-trans isomerase of the FK506-binding protein class. Loss of function of ROF2, and especially double mutation of ROF2 and the closely related gene ROF1, results in acid sensitivity. Over-expression of ROF2 confers tolerance to intracellular acidification by increasing proton extrusion from cells. The activation of the plasma membrane proton pump (H(+) -ATPase) is indirect: over-expression of ROF2 activates K(+) uptake, causing depolarization of the plasma membrane, which activates the electrogenic H(+) pump. The depolarization of ROF2 over-expressing plants explains their tolerance to toxic cations such as lithium, norspermidine and hygromycin B, whose uptake is driven by the membrane potential. As ROF2 induction and intracellular acidification are common consequences of many stresses, this mechanism of pH homeostasis may be of general importance for stress tolerance.

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H(+) with K(+) and, to lesser extent, Na(+) . Here, we investigated the response to NaCl supply and K(+) deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K(+) the opposite was found. Analysis of mineral composition showed a higher K(+) content in roots, shoots and xylem sap of transgenic plants and no differences in Na(+) content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na(+)/H(+) and, above all, K(+)/H(+) transport activity in root intracellular membrane vesicles. Under K(+) limiting conditions, transgenic plants enhanced root expression of the high-affinity K(+) uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K(+) depletion rates and half cytosolic K(+) activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K(+) and lower cytosolic K(+) activity than untransformed plants. These results indicate the fundamental role of K(+) homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.

Sediment accumulation rate and radiological characterisation of the sediment of Palmones River estuary (southern of Spain)

Chemical analyses and radioecological methods were combined in order to estimate the sediment accumulation rate in the upper 20 cm depth of the Palmones River estuary. Organic matter, total carbon, C:N and (137)Cs vertical profiles showed changes at 13 cm depth. These changes could be associated with the decrease in river input since 1987 when a dam situated in the upper part of the estuary started to store water. Using 1987 as reference to date the sediment, accumulation rate was 1.2 cm yr(-1). As alternative method, two layer model of (210)Pb(xs) vertical distribution showed a sedimentation rate of 0.7 cm yr(-1) with a surface mixing layer of 7 cm thickness. The high ammonium, potassium and sodium content in pore water and the strong correlation between (137)Cs activities and organic matter in dry sediment suggests that (137)Cs (the only anthropogenic product detected) is mainly accumulated in the estuary associated with the particulate organic material from the catchment area.

A Ca2+-Sensitive System Mediates Low-Affinity K+ Uptake in the Absence of AKT1 in Arabidopsis Plants

K(+) acquisition by Arabidopsis roots is mainly mediated by the high-affinity K(+) transporter AtHAK5 and the inward-rectifier K(+) channel AtAKT1. This model is probably universal to plants. Mutant plants lacking these two systems (athak5,atakt1) take up K(+) and grow when the external K(+) concentration is above a certain level, indicating that an additional transport system may compensate for the absence of AtHAK5 and AtAKT1. Here we describe that this alternative system is essential for providing sufficient K(+) to sustain growth of athak5,atakt1 plants. This system is especially sensitive to Ca(2+), Mg(2+), Ba(2+) and La(3+), it transports Cs(+) and its activity is reduced by cyclic nucleotides. These results suggest that a Ca(2+)-permeable voltage-independent non-selective cation channel, probably belonging to the cyclic nucleotide gated channel (CNGC) family, may provide the pathway for K(+) uptake in athak5,atakt1 plants. The genes encoding the two members of the CNGC family that have been described as mediating root K(+) uptake, AtCNGC3 and AtCNGC10, are not up-regulated in athak5,atakt1 plants, excluding overexpression of these genes as a compensatory mechanism. On the other hand, an increased driving force for K(+) in athak5,atakt1 plants due to a hyperpolarization of the membrane potential of its root cells is also discarded. The identification of this unknown system may provide tools to improve plant K(+) nutrition in conditions where AtAKT1 functionality is reduced, such as under salinity. In addition, this system may constitute an important pathway for accumulation of toxic cations such as Cs(+) or radiocesium ((137)Cs(+)), and could play a role in phytoremediation.

A mechanism of growth inhibition by abscisic acid in germinating seeds of Arabidopsis thaliana based on inhibition of plasma membrane H+-ATPase and decreased cytosolic pH, K+, and anions

Hghlight We provide the first evidence for a mechanism of growth inhibition by ABA during germination and seedling establishment based on inhibition of PM H+-ATPase and altered pH, K+, and anion homeostasis.

Regulation of K+ Transport in Tomato Roots by the TSS1 Locus. Implications in Salt Tolerance

The tss1 tomato (Lycopersicon esculentum) mutant exhibited reduced growth in low K+ and hypersensitivity to Na+ and Li+. Increased Ca2+ in the culture medium suppressed the Na+ hypersensitivity and the growth defect on low K+ medium of tss1 seedlings. Interestingly, removing NH4+ from the growth medium suppressed all growth defects of tss1, suggesting a defective NH4+-insensitive component of K+ transport. We performed electrophysiological studies to understand the contribution of the NH4+-sensitive and -insensitive components of K+ transport in wild-type and tss1 roots. Although at 1 mm Ca2+ we found no differences in affinity for K+ uptake between wild type and tss1 in the absence of NH4+, the maximum depolarization value was about one-half in tss1, suggesting that a set of K+ transporters is inactive in the mutant. However, these transporters became active by raising the external Ca2+ concentration. In the presence of NH4+, a reduced affinity for K+ was observed in both types of seedlings, but tss1 at 1 mm Ca2+ exhibited a 2-fold higher Km than wild type did. This defect was again corrected by raising the external concentration of Ca2+. Therefore, membrane potential measurements in root cells indicated that tss1 is affected in both NH4+-sensitive and -insensitive components of K+ transport at low Ca2+ concentrations and that this defective transport is rescued by increasing the concentration of Ca2+. Our results suggest that the TSS1 gene product is part of a crucial pathway mediating the beneficial effects of Ca2+ involved in K+ nutrition and salt tolerance.