Mechanosensitive aquaporins.

Cellular systems must deal with mechanical forces to satisfy their physiological functions. In this context, proteins with mechanosensitive properties play a crucial role in sensing and responding to environmental changes. The discovery of aquaporins (AQPs) marked a significant breakthrough in the study of water transport. Their transport capacity and regulation features make them key players in cellular processes. To date, few AQPs have been reported to be mechanosensitive. Like mechanosensitive ion channels, AQPs respond to tension changes in the same range. However, unlike ion channels, the aquaporin's transport rate decreases as tension increases, and the molecular features of the mechanism are unknown. Nevertheless, some clues from mechanosensitive ion channels shed light on the AQP-membrane interaction. The GxxxG motif may play a critical role in the water permeation process associated with structural features in AQPs. Consequently, a possible gating mechanism triggered by membrane tension changes would involve a conformational change in the cytoplasmic extreme of the single file region of the water pathway, where glycine and histidine residues from loop B play a key role. In view of their transport capacity and their involvement in relevant processes related to mechanical forces, mechanosensitive AQPs are a fundamental piece of the puzzle for understanding cellular responses.

Endophytic fungi and drought tolerance: ecophysiological adjustment in shoot and root of an annual mesophytic host grass.

Epichloid endophytic fungi, vertically transmitted symbionts of grasses, can increase plant tolerance to biotic and abiotic stress. Our aim was to identify ecophysiological mechanisms by which the endophyte Epichloë occultans confers drought tolerance to the annual grass Lolium multiflorum Lam. Endophyte-associated or endophyte-free plants were either well-watered or subjected to water deficit. We evaluated plant biomass, root length and nitrogen concentration, and we assessed intrinsic water use efficiency (iWUE) and its components net photosynthesis and stomatal conductance, by carbon and oxygen isotope analysis of shoot tissues. Endophyte-free plants produced more biomass than endophyte-associated ones at field capacity, while water deficit strongly reduced endophyte-free plants biomass. As a result, both types of plants produced similar biomass under water restriction. Based on oxygen isotope composition of plant cellulose, stomatal conductance decreased with water deficit in both endophyte-associated and endophyte-free plants. Meanwhile, carbon isotope composition indicated that iWUE increased with water deficit only in endophyte-associated plants. Thus, the isotope data indicated that net photosynthesis decreased more strongly in endophyte-free plants under water deficit. Additionally, endophyte presence reduced root length but increased its hydraulic conductivity. In conclusion, endophytic fungi confer drought tolerance to the host grass by adjusting shoot and root physiology.

Root hydraulics adjustment is governed by a dominant cell-to-cell pathway in Beta vulgaris seedlings exposed to salt stress.

Soil salinity reduces root hydraulic conductivity (Lpr) of several plant species. However, how cellular signaling and root hydraulic properties are linked in plants that can cope with water restriction remains unclear. In this work, we exposed the halotolerant species red beet (Beta vulgaris) to increasing concentrations of NaCl to determine the components that might be critical to sustaining the capacity to adjust root hydraulics. Our strategy was to use both hydraulic and cellular approaches in hydroponically grown seedlings during the first osmotic phase of salt stress. Interestingly, Lpr presented a bimodal profile response apart from the magnitude of the imposed salt stress. As well as Lpr, the PIP2-aquaporin profile follows an unphosphorylated/phosphorylated pattern when increasing NaCl concentration while PIP1 aquaporins remain constant. Lpr also shows high sensitivity to cycloheximide. In low NaCl concentrations, Lpr was high and 70 % of its capacity could be attributed to the CHX-inhibited cell-to-cell pathway. More interestingly, roots can maintain a constant spontaneous exudated flow that is independent of the applied NaCl concentration. In conclusion, Beta vulgaris root hydraulic adjustment completely lies in a dominant cell-to-cell pathway that contributes to satisfying plant water demands.

TuMV triggers stomatal closure but reduces drought tolerance in Arabidopsis.

Compatible plant viral infections are a common cause of agricultural losses worldwide. Characterization of the physiological responses controlling plant water management under combined stresses is of great interest in the current climate change scenario. We studied the outcome of TuMV infection on stomatal closure and water balance, hormonal balance and drought tolerance in Arabidopsis. TuMV infection reduced stomatal aperture concomitantly with diminished gas exchange rate, daily water consumption and rosette initial dehydration rate. Infected plants overaccumulated salicylic acid and abscisic acid and showed altered expression levels of key ABA homeostasis genes including biosynthesis and catabolism. Also the expression of ABA signalling gene ABI2 was induced and ABCG40 (which imports ABA into guard cells) was highly induced upon infection. Hypermorfic abi2-1 mutant plants, but no other ABA or SA biosynthetic, signalling or degradation mutants tested abolished both stomatal closure and low stomatal conductance phenotypes caused by TuMV. Notwithstanding lower relative water loss during infection, plants simultaneously subjected to drought and viral stresses showed higher mortality rates than mock-inoculated drought stressed controls, alongside downregulation of drought-responsive gene RD29A. Our findings indicate that despite stomatal closure triggered by TuMV, additional phenomena diminish drought tolerance upon infection.

Plant and animal aquaporins crosstalk: what can be revealed from distinct perspectives.

Aquaporins (AQPs) can be revisited from a distinct and complementary perspective: the outcome from analyzing them from both plant and animal studies. (1) The approach in the study. Diversity found in both kingdoms contrasts with the limited number of crystal structures determined within each group. While the structure of almost half of mammal AQPs was resolved, only a few were resolved in plants. Strikingly, the animal structures resolved are mainly derived from the AQP2-lineage, due to their important roles in water homeostasis regulation in humans. The difference could be attributed to the approach: relevance in animal research is emphasized on pathology and in consequence drug screening that can lead to potential inhibitors, enhancers and/or regulators. By contrast, studies on plants have been mainly focused on the physiological role that AQPs play in growth, development and stress tolerance. (2) The transport capacity. Besides the well-described AQPs with high water transport capacity, large amount of evidence confirms that certain plant AQPs can carry a large list of small solutes. So far, animal AQP list is more restricted. In both kingdoms, there is a great amount of evidence on gas transport, although there is still an unsolved controversy around gas translocation as well as the role of the central pore of the tetramer. (3) More roles than expected. We found it remarkable that the view of AQPs as specific channels has evolved first toward simple transporters to molecules that can experience conformational changes triggered by biochemical and/or mechanical signals, turning them also into signaling components and/or behave as osmosensor molecules.

Tonoplast (BvTIP1;2) and plasma membrane (BvPIP2;1) aquaporins show different mechanosensitive properties.

Previous works proposed that aquaporins behave as mechanosensitive channels. However, principal issues about mechanosensitivity of aquaporins are not known. In this work, we characterized the mechanosensitive properties of the water channels BvTIP1;2 (TIP1) and BvPIP2;1 (PIP2) from red beet (Beta vulgaris). We simultaneously measured the mechanical behavior and the water transport rates during the osmotic response of emptied-out oocytes expressing TIP1 or PIP2. Our results indicate that TIP1 is a mechanosensitive aquaporin, whereas PIP2 is not. We found that a single exponential function between the osmotic permeability coefficient and the volumetric elastic modulus governs the mechanosensitivity of TIP1. Finally, homology modeling analysis indicates that putative residues involved in mechanosensitivity show different quantity and distribution in TIP1 and PIP2.

The Water to Solute Permeability Ratio Governs the Osmotic Volume Dynamics in Beetroot Vacuoles.

Plant cell vacuoles occupy up to 90% of the cell volume and, beyond their physiological function, are constantly subjected to water and solute exchange. The osmotic flow and vacuole volume dynamics relies on the vacuole membrane -the tonoplast- and its capacity to regulate its permeability to both water and solutes. The osmotic permeability coefficient (Pf ) is the parameter that better characterizes the water transport when submitted to an osmotic gradient. Usually, Pf determinations are made in vitro from the initial rate of volume change, when a fast (almost instantaneous) osmolality change occurs. When aquaporins are present, it is accepted that initial volume changes are only due to water movements. However, in living cells osmotic changes are not necessarily abrupt but gradually imposed. Under these conditions, water flux might not be the only relevant driving force shaping the vacuole volume response. In this study, we quantitatively investigated volume dynamics of isolated Beta vulgaris root vacuoles under progressively applied osmotic gradients at different pH, a condition that modifies the tonoplast Pf . We followed the vacuole volume changes while simultaneously determining the external osmolality time-courses and analyzing these data with mathematical modeling. Our findings indicate that vacuole volume changes, under progressively applied osmotic gradients, would not depend on the membrane elastic properties, nor on the non-osmotic volume of the vacuole, but on water and solute fluxes across the tonoplast. We found that the volume of the vacuole at the steady state is determined by the ratio of water to solute permeabilites (Pf /Ps ), which in turn is ruled by pH. The dependence of the permeability ratio on pH can be interpreted in terms of the degree of aquaporin inhibition and the consequently solute transport modulation. This is relevant in many plant organs such as root, leaves, cotyledons, or stems that perform extensive rhythmic growth movements, which very likely involve considerable cell volume changes within seconds to hours.

Evidence for the involvement of hydraulic root or shoot adjustments as mechanisms underlying water deficit tolerance in two Sorghum bicolor genotypes.

Sorghum bicolor (L.) Moench is an ancient drought-tolerant crop with potential to sustain high yields even in those environments where water is limiting. Understanding the performance of this species in early phenological stages could be a useful tool for future yield improvement programs. The aim of this work was to study the response of Sorghum seedlings under water deficit conditions in two genotypes (RedLandB2 and IS9530) that are currently employed in Argentina. Morphological and physiological traits were studied to present an integrated analysis of the shoot and root responses. Although both genotypes initially developed a conserved and indistinguishable response in terms of drought tolerance parameters (growth rate, biomass reallocation, etc.), water regulation displayed different underlying strategies. To avoid water loss, both genotypes adjusted their plant hydraulic resistance at different levels: RedLandB2 regulated shoot resistance through stomata (isohydric strategy), while IS9530 controlled root resistance (anisohydric strategy). Moreover, only in IS9530 was root hydraulic conductance restricted in the presence of HgCl2, in agreement with water movement through cell-to-cell pathways and aquaporins activity. The different responses between genotypes suggest a distinct strategy at the seedling stage and add new information that should be considered when evaluating Sorghum phenotypic plasticity in changing environments.

Natural variation of root hydraulics in Arabidopsis grown in normal and salt-stressed conditions.

To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics.

The significance of roots as hydraulic rheostats.

Roots are the primary sites of water uptake by plants. Roots also sense most of the physico-chemical parameters of the soil, perceive signals from the shoots, and adjust their growth and water transport properties accordingly. The present opinion paper discusses the significance of the variable water transport capacity (hydraulic conductance) of roots during development and in response to environmental stimuli. It is shown that root hydraulics determines water uptake intensities but also water potential gradients within the plant. It is indicated how the dynamics of root hydraulics contributes to many integrated plant nutritional and growth functions. For instance, the heterogeneity of soil water and nutrient availability and the heterogeneity of root hydraulic properties feed each other and play critical roles in root transport functions. Another important aspect is the integration of root hydraulics within the mutual interactions of roots and shoots, for co-ordinated growth and water-saving responses to drought.

Expression analysis of the first arbuscular mycorrhizal fungi aquaporin described reveals concerted gene expression between salt-stressed and nonstressed mycelium.

Roots of most plants in nature are colonized by arbuscular mycorrhizal (AM) fungi. Among the beneficial effects of this symbiosis to the host plant is the transport of water by the AM mycelium from inaccessible soil water resources to host roots. Here, an aquaporin (water channel) gene from an AM fungus (Glomus intraradices), which was named GintAQP1, is reported for the first time. From experiments in different colonized host roots growing under several environmental conditions, it seems that GintAQP1 gene expression is regulated in a compensatory way regarding host root aquaporin expression. At the same time, from in vitro experiments, it was shown that a signaling communication between NaCl-treated mycelium and untreated mycelium took place in order to regulate gene expression of both GintAQP1 and host root aquaporins. This communication could be involved in the transport of water from osmotically favorable growing mycelium or host roots to salt-stressed tissues.

Analysis of the source of heterogeneity in the osmotic response of plant membrane vesicles.

Plasma membrane vesicles have been widely employed to understand the biophysics of water movements, especially when active aquaporins are present. In general, water permeability coefficients in these preparations outcome from the analysis of the osmotic response of the vesicles by means of light scattering. As from now, this is possible by following a theoretical approach that assumes that scattered light follows a single exponential function and that this behavior is the consequence of vesicle volume changes due to an osmotic challenge. However, some experimental data do not necessarily fit to single exponentials but to double ones. It is argued that the observed double exponential behavior has two possible causes: different vesicle population in terms of permeability or in terms of size distribution. As classical models cannot identify this source of heterogeneity, a mathematical modeling approach was developed based on phenomenological equations of water transport. In the three comparative models presented here, it was assumed that water moves according to an osmotic mechanism across the vesicles, and there is no solute movement across them. Interestingly, when tested in a well described plasma membrane vesicle preparation, the application of these models indicates that the source of heterogeneity in the osmotic response is vesicles having different permeability, clearly discarding the variable size effect. In conclusion, the mathematical approach presented here allows to identify the source of heterogeneity; this information being of particular interest, especially when studying gating mechanisms triggered in water channel activity.

A fruit-specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria x ananassa) fruit ripening.

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

Plasma membrane of Beta vulgaris storage root shows high water channel activity regulated by cytoplasmic pH and a dual range of calcium concentrations.

Plasma membrane vesicles isolated by two-phase partitioning from the storage root of Beta vulgaris show atypically high water permeability that is equivalent only to those reported for active aquaporins in tonoplast or animal red cells (Pf=542 microm s(-1)). The values were determined from the shrinking kinetics measured by stopped-flow light scattering. This high Pf was only partially inhibited by mercury (HgCl2) but showed low activation energy (Ea) consistent with water permeation through water channels. To study short-term regulation of water transport that could be the result of channel gating, the effects of pH, divalent cations, and protection against dephosphorylation were tested. The high Pf observed at pH 8.3 was dramatically reduced by medium acidification. Moreover, intra-vesicular acidification (corresponding to the cytoplasmic face of the membrane) shut down the aquaporins. De-phosphorylation was discounted as a regulatory mechanism in this preparation. On the other hand, among divalent cations, only calcium showed a clear effect on aquaporin activity, with two distinct ranges of sensitivity to free Ca2+ concentration (pCa 8 and pCa 4). Since the normal cytoplasmic free Ca2+ sits between these ranges it allows for the possibility of changes in Ca2+ to finely up- or down-regulate water channel activity. The calcium effect is predominantly on the cytoplasmic face, and inhibition corresponds to an increase in the activation energy for water transport. In conclusion, these findings establish both cytoplasmic pH and Ca2+ as important regulatory factors involved in aquaporin gating.

Tonoplast vesicles of Beta vulgaris storage root show functional aquaporins regulated by protons.

BACKGROUND INFORMATION: Water is crucial for plant development and growth, and its transport pathways inside a plant are an ongoing topic for study. Plants express a large number of membrane intrinsic proteins whose role is now being re-evaluated by considering not only the control of the overall plant water balance but also in adaptation to environmental challenges that may affect their physiology. In particular, we focused our work on water movements across the root cell TP (tonoplast), the delimiting membrane of the vacuole. This major organelle plays a central role in osmoregulation. RESULTS: An enriched fraction of TP vesicles from Beta vulgaris (red beet) storage roots obtained by a conventional method was used to characterize its water permeability properties by means of the stopped-flow technique. The preparation showed high water permeability (485 microm x s(-1)), consistent with values reported in the literature. The water permeability was strongly blocked by HgCl(2) (reduced to 16%) and its energy activation was low. These observations allow us to postulate the presence of functional water channels in this preparation. Moreover, Western-blot analysis demonstrated the presence of a tonoplast intrinsic protein. With the purpose of studying the regulation of water channels, TP vesicles were exposed to different acidic pH media. When the pH of a medium was low (pH 5.6), the water permeability exhibited a 42% inhibition. CONCLUSIONS: Our findings prove that although almost all water channels present in the TP vesicles of B. vulgaris root are sensitive to HgCl(2), not all are inhibited by pH. This interesting selectivity to acidification of the medium could play a role in adapting the water balance in the cell-to-cell pathway.

Cytosolic pH regulates root water transport during anoxic stress through gating of aquaporins.

Flooding of soils results in acute oxygen deprivation (anoxia) of plant roots during winter in temperate latitudes, or after irrigation, and is a major problem for agriculture. One early response of plants to anoxia and other environmental stresses is downregulation of water uptake due to inhibition of the water permeability (hydraulic conductivity) of roots (Lp(r)). Root water uptake is mediated largely by water channel proteins (aquaporins) of the plasma membrane intrinsic protein (PIP) subgroup. These aquaporins may mediate stress-induced inhibition of Lp(r) but the mechanisms involved are unknown. Here we delineate the whole-root and cell bases for inhibition of water uptake by anoxia and link them to cytosol acidosis. We also uncover a molecular mechanism for aquaporin gating by cytosolic pH. Because it is conserved in all PIPs, this mechanism provides a basis for explaining the inhibition of Lp(r) by anoxia and possibly other stresses. More generally, our work opens new routes to explore pH-dependent cell signalling processes leading to regulation of water transport in plant tissues or in animal epithelia.