Mediated Water Transport Research Articles

Spatial variability of hydraulic traits in the root vessels of perennial herbs on the Loess Plateau of China

Plant hydraulic traits mediate water transport, photosynthesis, as well as their adaptability to drought. Studies into the adaptations of xylem hydraulics in response to environmental changes typically focused on trees and shrubs, with scarce attention given to perennial herbs. For this study, we analyzed the spatial variabilities of 9 hydraulic traits in the root vessels (root vessel traits) across species on the Loess Plateau of China. Our findings revealed that precipitation gradient, rather than temperature and elevation gradient, determined the spatial variability of the most root vessel traits. Vessel fraction, vessel area, water transfer efficiency, and hydraulic diameter increased significantly with decreases in the mean annual precipitation. The results emphasized that perennial herbs exhibited hydraulic conduction efficiency priority strategies to augment water absorption under water deficits. This was in contrast to the hydraulic safety strategies of woody trees in response to drought. In light of these findings, we recommend that priority should be given to perennial herbs in water-limited regions for current and future ecological restoration programs. The selection of the most appropriate perennial herbs for distinct sites should be further elucidated as a standard procedure in ecological restoration practice.

TIP aquaporins in Cyperus esculentus: genome-wide identification, expression profiles, subcellular localizations, and interaction patterns

BackgroundTonoplast intrinsic proteins (TIPs), which typically mediate water transport across vacuolar membranes, play an essential role in plant growth, development, and stress responses. However, their characterization in tigernut (Cyperus esculentus L.), an oil-bearing tuber plant of the Cyperaceae family, is still in the infancy.ResultsIn this study, a first genome-wide characterization of the TIP subfamily was conducted in tigernut, resulting in ten members representing five previously defined phylogenetic groups, i.e., TIP1–5. Although the gene amounts are equal to that present in two model plants Arabidopsis and rice, the group composition and/or evolution pattern were shown to be different. Except for CeTIP1;3 that has no counterpart in both Arabidopsis and rice, complex orthologous relationships of 1:1, 1:2, 1:3, 2:1, and 2:2 were observed. Expansion of the CeTIP subfamily was contributed by whole-genome duplication (WGD), transposed, and dispersed duplications. In contrast to the recent WGD-derivation of CeTIP3;1/-3;2, synteny analyses indicated that TIP4 and − 5 are old WGD repeats of TIP2, appearing sometime before monocot-eudicot divergence. Expression analysis revealed that CeTIP genes exhibit diverse expression profiles and are subjected to developmental and diurnal fluctuation regulation. Moreover, when transiently overexpressed in tobacco leaves, CeTIP1;1 was shown to locate in the vacuolar membrane and function in homo/heteromultimer, whereas CeTIP2;1 is located in the cell membrane and only function in heteromultimer. Interestingly, CeTIP1;1 could mediate the tonoplast-localization of CeTIP2;1 via protein interaction, implying complex regulatory patterns.ConclusionsOur findings provide a global view of CeTIP genes, which provide valuable information for further functional analysis and genetic improvement through manipulating key members in tigernut.

Arginine Promotes the Expression of Aquaporin-3 and Water Transport in Porcine Trophectoderm Cells Through NO- and cAMP-Dependent Mechanisms.

Dietary supplementation with L-arginine (Arg) has been shown to increase the volume of fetal fluids in gestating swine. Aquaporins (AQPs), known as water channel proteins, are essential for embryonic growth and development. It was not known if Arg mediates water transport through AQPs in porcine conceptus trophectoderm (pTr2) cells. pTr2 cells derived from pregnant gilts on day 12 of gestation were cultured in customized Arg-free Dulbecco's modified Eagle's Ham medium (DMEM) supplemented with either 0.00, 0.25, or 0.50 mM Arg. Arg treatment increased water transport and the expression of AQP3, which was abundantly expressed in pTr2 cells at both the mRNA and protein levels. Arg also increased the expression of iNOS and the synthesis of nitric oxide (NO) in pTr2 cells. The presence of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; an inhibitor of NO synthase) significantly attenuated the Arg-induced expression of AQP3. Furthermore, 0.50 mM Arg increased the concentrations of cAMP and the abundances of phosphorylated cAMP-dependent protein kinase A (PKA), phosphorylated PKA α/β/γ, and phosphorylated CREB. These effects of Arg were mimicked by Forskolin (a cell-permeable activator of adenylyl cyclase), but inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase). The results of this study demonstrate that Arg regulates AQP3 expression and promotes water transport in pTr2 cells through NO- and cAMP-dependent signaling pathways.

Plasma membrane intrinsic protein SlPIP1;7 promotes root growth and enhances drought stress tolerance in transgenic tomato (Solanum lycopersicum) plants

AbstractPlasma membrane intrinsic proteins (PIPs) play important roles in mediating water transport and regulating plant growth and development. In this study, the function of SlPIP1;7 in drought stress tolerance was characterized. Overexpression of SlPIP1;7 in tomato increased the primary root length and promoted the lateral root growth, whereas the shoot and root growth of SlPIP1;7‐silenced seedlings were severely retarded. Moreover, SlPIP1;7‐overexpressing plants exhibited obviously increased drought stress tolerance because they showed slight withering symptom, whereas the SlPIP1;7‐silenced plants were more sensitive to drought stress. Furthermore, the SlPIP1;7‐overexpressing plants showed higher root hydraulic conductivity (Lpr) and less cell membrane damage and produced less reactive oxygen species (ROS) accumulation under drought stress compared with wild‐type, whereas these indicators were opposite in SlPIP1;7‐silenced plants. These results suggested that overexpression of SlPIP1;7 gene promoted the root growth and the water absorption, maintained the water balance and increased the antioxidant defence ability of plants under drought stress, thereby imparting the drought stress tolerance. SlPIP1;7 is thus a valuable genetic resource to enhance stress tolerance in tomato plants.

Physiological and Transcriptomic Analyses Revealed the Implications of Abscisic Acid in Mediating the Rate-Limiting Step for Photosynthetic Carbon Dioxide Utilisation in Response to Vapour Pressure Deficit in Solanum Lycopersicum (Tomato).

The atmospheric vapour pressure deficit (VPD) has been demonstrated to be a significant environmental factor inducing plant water stress and affecting plant photosynthetic productivity. Despite this, the rate-limiting step for photosynthesis under varying VPD is still unclear. In the present study, tomato plants were cultivated under two contrasting VPD levels: high VPD (3–5 kPa) and low VPD (0.5–1.5 kPa). The effect of long-term acclimation on the short-term rapid VPD response was examined across VPD ranging from 0.5 to 4.5 kPa. Quantitative photosynthetic limitation analysis across the VPD range was performed by combining gas exchange and chlorophyll fluorescence. The potential role of abscisic acid (ABA) in mediating photosynthetic carbon dioxide (CO2) uptake across a series of VPD was evaluated by physiological and transcriptomic analyses. The rate-limiting step for photosynthetic CO2 utilisation varied with VPD elevation in tomato plants. Under low VPD conditions, stomatal and mesophyll conductance was sufficiently high for CO2 transport. With VPD elevation, plant water stress was gradually pronounced and triggered rapid ABA biosynthesis. The contribution of stomatal and mesophyll limitation to photosynthesis gradually increased with an increase in the VPD. Consequently, the low CO2 availability inside chloroplasts substantially constrained photosynthesis under high VPD conditions. The foliar ABA content was negatively correlated with stomatal and mesophyll conductance for CO2 diffusion. Transcriptomic and physiological analyses revealed that ABA was potentially involved in mediating water transport and photosynthetic CO2 uptake in response to VPD variation. The present study provided new insights into the underlying mechanism of photosynthetic depression under high VPD stress.

The expression response of plasma membrane aquaporins to salt stress in tomato plants

Aquaporin isoforms have distinct functions, and show differential responses to stress conditions among different plant species. In tomato (Solanum lycopersicum), the functions of individual aquaporin isoforms under environmental stresses remain largely unknown. As a step toward understanding their functions under stress conditions, the expression response of plasma membrane intrinsic proteins (PIPs) to salt stress was investigated. Tomato seedlings were subjected to 150 mM NaCl for 1 h and 24 h, and water uptake and SlPIP expressions were investigated. The results showed that the root hydraulic conductivity (Lpr) was decreased under salt stress. Salt stress for 1 h had no effect or increased the expressions of most of the highly expressed SlPIPs in roots and leaves. Salt stress inhibited cell-to-cell water transport. Protein phosphatase and endocytosis inhibitors partially inhibited the Lpr decrease under salt stress. After a 24-h salt stress, the expressions of most SlPIPs were decreased in both roots and leaves, and a recovery treatment in control nutrient solution demonstrated a partial or full expression restoration of most previously downregulated SlPIPs. The expressions of SlPIP isoforms highly varied in both roots and leaves, and across different stress durations. SlPIP1;1, SlPIP1;3, SlPIP1;7, SlPIP2;10 and SlPIP2;12 demonstrated consistent expression responses to salt stress and recovery treatment in both roots and leaves, implying their important roles in regulating water transport. Our results suggest that SlPIPs are involved in mediating water transport in tomato, and their regulations are associated with stress duration and tissue type. At early stage of salt stress, the inhibition of Lpr in tomato might be due to the decrease of SlPIP activity; while at later stress period, the Lpr reduction might be mainly attributed to the decrease of SlPIP expression.

P1130A CELLULAR MODEL OF HUMAN MESOTHELIUM TO STUDY OF WATER TRANSPORT DURING PERITONEAL DIALYSIS AND THE BIOCOMPATIBILITY OF INNOVATIVE GLUCOSE-SPARING SOLUTIONS.

Abstract Background and Aims The three pore model postulates that the endothelium of peritoneal capillaries is the major limiting barrier regulating water transport across peritoneal membrane during peritoneal dialysis (PD). We hypothesize that the mesothelium may represent an additional selective barrier to water diffusion in PD. We previously demonstrated that the water channel AQP1 is expressed in vivo by mesothelial cells. Here, we characterized an immortalized cell line of human mesothelium (HMC) to study the functional role of the water channel AQP1 in mediating water transport during PD and also to test the biocompatibility of glucose-sparing PD solutions (Xylocore), containing xylitol and L-carnitine as the main osmotic agents. Method Cells were grown onto porous cell culture inserts to achieve polarization. Polarization was demonstrated by expression of the tight junction markers Zo-1 and occludin. Transepithelial water transport was measured by TEA+-sensitive microelectrodes. HMC cell monolayers were exposed to PD solutions at the apical side for 8 hours. The biocompatibility of conventional versus innovative PD solutions was evaluated by MTT-test, measurement of transepithelial electrical resistance (TEER) and production of pro-inflammatory cytokines by by Luminex xMAP technology. Results HMC cells showed polarized expression of Na+/K+-ATPase and tight junctions markers but no endogenous expression of AQP1. HMC showed a low TEER (40Ω/cm2) compared to renal cells not expressing AQP1(1000Ω/cm2). However, the transepithelial water transport was comparable between the two cell types. Experiments in HMCs transfected with AQP1 cDNA, suggested that the water permeability of HMC was increased by two-fold in the presence of AQP1. Biocompatibility assays indicated that in conventional dialysis solutions glucose concentration decreased cell viability in a dose-dependent manner. Glucose concentration also strongly decreased the TEER, suggesting reduction of the barrier integrity, and increased pro-inflammatory cytokines production. Interestingly, substitution of part of the glucose with xylitol and L-carnitine minimized these effects. Conclusion These results suggest that the mesothelium may represent an additional selective barrier regulating water transport through the water channel AQP1 in PD. Importantly, we also demonstrate that the formulation of glucose-sparing PD solutions containing xylitol and L-carnitine better preserve mesothelial cells viability and may represent a useful means to prolong the dialysis life of patients undergoing peritoneal dialysis.

Claudin-15 forms a water channel through the tight junction with distinct function compared to claudin-2.

Claudin-15 is mainly expressed in the small intestine and indirectly involved in glucose absorption. Similar to claudin-2 and -10b, claudin-15 is known to form a paracellular channel for small cations. Claudin-2, but not claudin-10b, also forms water channels. Here we experimentally tested whether claudin-15 also mediates water transport and if yes, whether water transport is Na+ -coupled, as seen for claudin-2. MDCK C7 cells were stably transfected with claudin-15. Ion and water permeability were investigated in confluent monolayers of control and claudin-15-expressing cells. Water flux was induced by an osmotic or ionic gradient. Expression of claudin-15 in MDCK cells strongly increased cation permeability. The permeability ratios for monovalent cations indicated a passage of partially hydrated ions through the claudin-15 pore. Accordingly, its pore diameter was determined to be larger than that of claudin-2 and claudin-10b. Mannitol-induced water flux was elevated in claudin-15-expressing cells compared to control cells. In contrast to the Na+ -coupled water flux of claudin-2 channels, claudin-15-mediated water flux was inhibited by Na+ flux. Consequently, water flux was increased in Na+ -free solution. Likewise, Na+ flux was decreased after induction of water flux through claudin-15. Claudin-15, similar to claudin-2, forms a paracellular cation and water channel. In functional contrast to claudin-2, water and Na+ fluxes through claudin-15 inhibit each other. Claudin-15 allows Na+ to retain part of its hydration shell within the pore. This then reduces the simultaneous passage of additional water through the pore.

Isotope fractionation during root water uptake by Acacia caven is enhanced by arbuscular mycorrhizas

A growing number of studies show a discrepancy between the isotopic composition of xylem water and plant water sources. We tested the effect of arbuscular mycorrhizal fungi (AMF) on the isotopic composition of Acacia caven xylem water. As the most common plant-fungal association, AMF might explain this isotopic mismatch. Seedlings were grown with and without AMF and irrigated with the same water. After 120 days, stem and soil samples were collected and following cryogenic distillation, H and O isotopic composition of xylem and soil water, as well as irrigation water, was measured. Xylem water of non-mycorrhizal seedlings was significantly depleted in 2H compared to soil water (differences up to −15.6‰). When AMF were present, the depletion was significantly higher and appeared for both H and O (differences up to −24.6‰ for δ2H and − 2.9‰ for δ18O between soil and xylem water). Results suggest that isotopic fractionation occurred during water uptake in this xerophytic species. To explain this, we propose an aquaporin-driven mechanism mediating water transport via transmembrane passage. Furthermore, we show for the first time, that AMF enhance the observed discrimination against heavy isotopes, probably by enforcing water passage through aquaporins. Given their ubiquity, AMF could question the fractionation-free assumption during root water uptake.

Regulation of water transport in Arabidopsis by methyl jasmonate

Following a stress event, jasmonate-dependent signaling pathway triggers a shift from growth to defense responses that are accompanied by the cessation of growth in many plants. However, the processes leading to this growth inhibition remain obscure. In this study, we provide evidence for a rapid inhibition of cell hydraulic conductivity (Lp) by methyl jasmonate (MeJA) in the roots of wild-type Arabidopsis within 0.5 h of 20 and 50 μM MeJA treatments. We also demonstrate that MeJA did not affect Lp in fad3-2 and fad7-2 Arabidopsis mutants that are deficient in jasmonate precursor, linolenic acid. The reductions of Lp in wild-type plants were accompanied by the down-regulation of several plasma membrane intrinsic protein (PIP) isoforms, and dephosphorylation. Treatments with HgCl2 did not further reduce Lp in the wild-type plants, but significantly reduced Lp in the fad3-2 and fad7-2 that had been first treated with MeJA. Continuous prolonged exposure to exogenous 50 μM MeJA inhibited the relative growth rates (RGR) of shoots and net photosynthesis (Pn) in the Arabidopsis wild-type and fad7-2 plants, but had no effect on the RGR of roots. The results demonstrated that a reduction of aquaporin (AQP)-mediated water transport was the initial target of MeJA exposure, and may contribute to the processes of growth inhibition by MeJA.

Aquaporin-4 Is Downregulated in the Basolateral Membrane of Ileum Epithelial Cells during Enterotoxigenic Escherichia coli-Induced Diarrhea in Mice.

Enterotoxigenic Escherichia coli (ETEC) are opportunistic pathogens that colonize the small intestine, produce enterotoxins and induce diarrhea. Some aquaporins (AQPs), such as AQP3 and AQP8, have been reported to participate in diarrhea by decreasing cellular influx in the gastrointestinal (GI) tract. AQP4 is another important water channel in the GI tract, but its role in ETEC-induced diarrhea has not been reported. Here, we demonstrated the potential roles of AQP4 in ETEC-induced diarrhea. Reverse transcription-polymerase chain reaction (RT-PCR) and western blotting showed that AQP4 was expressed in the mouse ileum, but not in the duodenum or jejunum while immunohistochemical staining showed that AQP4 localized to the basolateral membrane of ileum epithelial cells. Using an ETEC-induced mice diarrhea model, we demonstrated that both AQP4 mRNA level and the AQP4 protein level in the ileum decreased gradually over a time course of 7 days. These results suggest that AQP4 plays a role in the pathogenesis of ETEC-induced diarrhea by mediating water transport.

Marked decrease of aquaporin-4 protein is independent of the changes in α1-syntrophin and TRPV4 levels in response to denervation-induced muscle atrophy in vivo.

Aquaporin-4 (AQP4) is a selective water channel mediating water transport across cell membranes in skeletal muscles. Recently, it was noted that AQP4 is one of the key molecules regulating muscle morphology. Indeed, the AQP4 accumulation level was stably maintained in hypertrophied skeletal muscles. On the other hand, whether the AQP4 accumulation level is stably maintained in atrophied muscles remains poorly understood. The present study investigated the changes in the AQP4 accumulation level in the atrophied muscles at 2 weeks after denervation. As a result, the accumulation level of AQP4 in the atrophied muscle was significantly decreased compared with that in the control muscle (p < 0.05). Interestingly, the accumulation level of α1-syntrophin, which is an essential factor in regulating the stable accumulation level of AQP4, was stably maintained in the atrophied muscles. On the other hand, the accumulation level of the transient receptor potential vanilloid 4 (TRPV4), which contributes to cell volume control via interaction with AQP4, was significantly increased in the atrophied muscles compared with that in the control muscle (p < 0.05). Therefore, the present study suggested that the imbalance between the AQP4 accumulation level and skeletal muscle volume may be induced in the atrophied muscles by denervation, and the decrease in the accumulation level of AQP4 may be accompanied by defects in the functional and structural relationships with α1-syntrophin and TRPV4.

55. Aquaporins and CSF Flux Are Critical Determinants of AAV Mediated CNS Gene Transfer

The central nervous system (CNS) lacks conventional lymphatics for solute clearance. Convective currents of interstitial fluid (ISF) shunt macromolecules across the brain parenchyma into cerebrospinal fluid (CSF) compartments. Here the cargo is either reabsorbed into CNS via subarachnoid ducts or disseminated into the blood or peripheral lymph vessels. Recent studies have identified that flow of water via aquaporin (AQP) channels at the astroglial endfeet enables exchange of biomaterials between ISF and CSF. Conversely, loss of AQP expression due to aging or disease is correlated with ineffective clearance of neurodegenerative accumulations such as Amyloid β and tau. AAV mediated gene therapy of CNS disorders requires a deeper understanding of such factors and their effects on transduction efficiency and biodistribution/clearance. Here, we demonstrate that AQP mediated water transport dictates various aspects of AAV gene transfer in the CNS. We compared the CNS spread, transduction profile and systemic leakage of clinically relevant AAV vectors in wildtype (WT) or AQP knockout (AQP-/-) mice. Within minutes following intracerebroventricular (ICV) administration, AQP-/- mice exhibited highly restricted spread of fluorophore labeled AAVs when compared to wild type mice. Transgene expression was markedly increased from AAV administrations in AQP-/- mice. Further, systemic leakage and off-target transgene expression in peripheral tissues were markedly reduced for some AAV serotypes in AQP-/- mice when compared to WT mice. These results suggest that AQP-mediated water transport plays a critical role in determining the spread, transduction efficiency and systemic leakage of AAV vectors following CNS administration. We hypothesize that altered CSF flux under conditions pertinent to aging and CNS disease, can impact the residence time of AAV vectors and consequently gene transfer efficiency. Further results evaluating the CNS transport properties of AAV in mouse models of aging and disease will be presented.

Further Characterization of Anuran‐Like Aquaporins in the Urinary Bladder of Urodeles, Notopthalmus viridescens and Cynops pyrrhogaster

Recent Bayesian analysis of tetrapod aquaporin gene clusters by Finn et al (PLOS1, November 26, 2014) suggests anuran‐specific aquaporins that mediate water transport across the ventral skin (AQPa2S) and urinary bladder (AQPa2U) are more closely related to AQP6 found in mammals and reptiles. Furthermore, AQP6/a2 aquaporins are syntenic with AQP5. We used RT‐PCR to show both AQP6/a2U and AQP5 homologs are expressed in the urinary bladder of the newts, Notophthalmus viridescens (nv) and Cynops pyrrhogaster (cp). Full length amino acid sequences showed 72% and 71% identity between AQP6/a2U of nv and anuran species Hyla japonica and Xenopus tropicalis (xt). Amino acid identity of AQP5 between nv and cp was 95% while identity between both urodeles and xt was 69%–70%.Consensus phosphorylation sites for PKA were identified for AQP6/a2U and AQP5 for both nv and cp. AQP5 could be immunolocalized in the apical membrane of bladder epithelial cells in rehydrating newts. These results indicate an absorptive role for AQP5 in the urodele urinary bladder and suggest that this role may originate in the ancestor of urodeles and anurans.Support or Funding InformationSupported by a Grant‐in‐Aid for Scientific Research (26440165) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Pyrabactin regulates root hydraulic properties in maize seedlings by affecting PIP aquaporins in a phosphorylation-dependent manner

Pyrabactin, an agonist of abscisic acid (ABA), has led to the isolation and characterization of pyrabactin resistance 1/pyrabactin resistance 1-like (PYR1/PYLs) ABA receptors in Arabidopsis, which has well explained ABA-mediated stomatal movement and stress-related gene expression. In addition to inducing stomatal closure and inhibiting transpiration, ABA can also enhance root hydraulic conductivity (Lpr), thus maintaining water balance under water deficiency-related stress, but its molecular mechanism remains unclear. In the present study, the root hydraulic properties of maize seedlings in response to pyrabactin were compared to those caused by ABA. Similar to ABA, lower concentration of pyrabactin induced a remarkable increase in Lpr as well as in the gene expression of the plasma membrane intrinsic protein (ZmPIP) aquaporin and in the ZmPIP2; 1/2; 2 protein abundance. The pyrabactin-induced enhancement of Lpr was abolished by H2O2 application, indicating that pyrabactin regulates Lpr by modulating ZmPIP at transcriptional, translational and post-translational (activity) level. Pyrabactin-mediated water transport and ZmPIP gene expression were phosphorylation-dependent, suggesting that ABA-PYR1-(PP2C)-protein kinase-AQP signaling pathway may be involved in this process. As we know this is the first established ABA signaling transduction pathway that mediated water transport in roots. This observation further addressed the importance of PYR1/PYLs ABA receptor in regulating plant water use efficiency from the under ground level. Except inhibiting transpiration in leaves, our result introduces the exciting possibility of application ABA agonists for regulating roots water uptake in field, with a species- and dose dependent manner.

Differential responses of plasma membrane aquaporins in mediating water transport of cucumber seedlings under osmotic and salt stresses

It has long been recognized that inhibition of plant water transport by either osmotic stress or salinity is mediated by aquaporins (AQPs), but the function and regulation of AQPs are highly variable among distinct isoforms and across different species. In this study, cucumber seedlings were subjected to polyethylene glycol (PEG) or NaCl stress for duration of 2 h or 24 h. The 2 h treatment with PEG or NaCl had non-significant effect on the expression of plasma membrane AQP (CsPIPs) in roots, indicating the decrease in hydraulic conductivity of roots (Lpr ) and root cells (Lprc ) measured in these conditions were due to changes in AQP activity. After both 2 h and 24 h PEG or NaCl exposure, the decrease in hydraulic conductivity of leaves (Kleaf ) and leaf cells (Lplc ) could be attributed to a down-regulation of the two most highly expressed isoforms, CsPIP1;2 and CsPIP2;4. In roots, both Lpr and Lprc were further reduced after 24 h PEG exposure, but partially recovered after 24 h NaCl treatment, which were consistent with changes in the expression of CsPIP genes. Overall, the results demonstrated differential responses of CsPIPs in mediating water transport of cucumber seedlings, and the regulatory mechanisms differed according to applied stresses, stress durations and specific organs.

Evidence for water-permeable channels in auditory hair cells in the leopard frog

Auditory hair cells in the amphibian papilla (APHCs) of the leopard frog, Rana pipiens pipiens, have a significantly higher permeability to water than that observed in mammalian hair cells. The insensitivity of water permeability in frog hair cells to extracellular mercury suggests that an amphibian homologue of the water channel aquaporin-4 (AQP4) may mediate water transport in these cells. Using immunocytochemistry, we show that an AQP4-like protein is found in APHCs. Rabbit anti-AQP4 antibody was used in multiple-immunohistochemical staining experiments along with AP hair cell and hair bundle markers in leopard frog and mouse tissue. AQP4 immunoreactivity was found in the basal and apical poles of the APHCs and shows uniform immunoreactivity. This study provides the first identification and localization of an AQP4-like protein in the amphibian inner ear. We also report a more direct measure of hyperosmotically-induced volume changes in APHCs that confirms previous findings. The presence of water channels in anuran APHCs constitutes a novel physiological difference between amphibian and mammalian hair cell structure and function.

Mechanisms and Effects of Retention of Over‐Expressed Aquaporin AtPIP2;1 in the Endoplasmic Reticulum

Plasma membrane intrinsic proteins (PIPs) are aquaporins that mediate water transport across the plant plasma membrane (PM). The present work addresses, using Arabidopsis AtPIP2;1 as a model, the mechanisms and significance of trafficking of newly synthesized PIPs from the endoplasmic reticulum (ER) to the Golgi apparatus. A functional diacidic export motif (Asp4-Val5-Glu6) was identified in the N-terminal tail of AtPIP2;1, using expression in transgenic Arabidopsis of site-directed mutants tagged with the green fluorescent protein (GFP). Confocal fluorescence imaging and a novel fluorescence recovery after photobleaching application based on the distinct diffusion of PM and intracellular AtPIP2;1-GFP forms revealed a retention in the ER of diacidic mutated forms, but with quantitative differences. Thus, the individual role of the two acidic Asp4 and Glu6 residues was established. In addition, expression in transgenic Arabidopsis of ER-retained AtPIP2;1-GFP constructs reduced the root hydraulic conductivity. Co-expression of AtPIP2;1-GFP and AtPIP1;4-mCherry constructs suggested that ER-retained AtPIP2;1-GFP may interact with other PIPs to hamper their trafficking to the PM, thereby contributing to inhibition of root cell hydraulic conductivity.

Urinary aquaporin-2 is elevated in infant atopic dermatitis

Conflicts of interest: none declared. Madam, One of the key features of atopic dermatitis (AD) is increased transepidermal water loss (TEWL),1, 2 a phenomenon attributable partly to impaired barrier function of the skin due to some genetic defects such as filaggrin gene (FLG) mutations, or to inflammatory status in AD.1 Remarkable loss of body fluid would induce a series of systemic regulatory reactions. The functional antidiuretic hormone (ADH)–aquaporin (AQP)‐2 axis is a major regulatory system to keep water balance. AQP‐2 mediates water transport across the apical plasma membrane of the renal collecting ducts. Kidney AQP‐2 expression can be estimated quantitatively by urinary excretion of AQP‐2. Moreover, the amount of urinary AQP‐2 (u‐AQP‐2) is positively correlated with the plasma levels of ADH in normal subjects.3 Increased serum ADH was detected in severe AD, a result possibly due to a dehydrated state caused by increased TEWL in these patients.2 Therefore, it is possible that u‐AQP‐2 is altered in AD. We conducted a case–control study to measure the u‐AQP‐2 level in paediatric patients with AD, and assessed its correlation with several clinical and laboratory parameters.

Effect of ethanol on fluxes of water and protons across the plasma membrane of Saccharomyces cerevisiae

Plasma membrane integrity, ability to transport substrates and maintenance of homeostasis represent obligatory requirements for efficient ethanol production by Saccharomyces cerevisiae. The effect of ethanol on water diffusion through the bilayer and on mediated water movements was evaluated by stopped flow spectroscopy. Ethanol stimulated water diffusion and inhibited mediated water transport. In a strain overexpressing AQY1, the activation energy for water transport increased progressively (from 5.9 to 12.7 kcal mol(-1)) for increasing ethanol concentrations (up to 12% v/v), indicating that mediated water transport lost importance as compared with water diffusion through the bilayer. The effect of ethanol on proton movements (inward by passive diffusion and outward through the PMA1 H(+)-ATPase) was evaluated by measuring the rate of extracellular alcalinization and acidification of unbuffered cell suspensions at different temperatures. Above 10% ethanol, H(+) diffusion was strongly increased at 30 degrees C, but no effect was observed at 20 degrees C up to 12%, indicating the existence of a threshold above which ethanol has a marked effect. On H(+) extrusion, ethanol had no effect at 20 degrees C, but induced a monotonous decrease at higher temperatures. Our results support the view that above a threshold of ethanol concentration, the membrane structure is disrupted, becoming very leaky to H(+).