Specific aquaporins facilitate the diffusion of hydrogen peroxide across membranes

The metabolism of aerobic organisms continuously produces reactive oxygen species. Although potentially toxic, these compounds also function in signaling. One important feature of signaling compounds is their ability to move between different compartments, e.g. to cross membranes. Here we present evidence that aquaporins can channel hydrogen peroxide (H2O2). Twenty-four aquaporins from plants and mammals were screened in five yeast strains differing in sensitivity toward oxidative stress. Expression of human AQP8 and plant Arabidopsis TIP1;1 and TIP1;2 in yeast decreased growth and survival in the presence of H2O2. Further evidence for aquaporin-mediated H2O2 diffusion was obtained by a fluorescence assay with intact yeast cells using an intracellular reactive oxygen species-sensitive fluorescent dye. Application of silver ions (Ag+), which block aquaporin-mediated water diffusion in a fast kinetics swelling assay, also reversed both the aquaporin-dependent growth repression and the H2O2-induced fluorescence. Our results present the first molecular genetic evidence for the diffusion of H2O2 through specific members of the aquaporin family.     

The distribution of aquaporin subtypes (PIP1, PIP2 and gamma-TIP) is tissue dependent in soybean (Glycine max) root nodules

BACKGROUND AND AIMS The inner cortical cells (IC-cells) of legume root nodules have been previously shown to regulate the resistance to nodule O2 diffusion by a rapid contraction/expansion mechanism, which controls the volume of intercellular spaces and their occlusion by a liquid phase. The expression of aquaporins in IC-cells was also found to be involved in this nodule O2 diffusion mechanism. The aim of this study was to compare the expression of plasma membrane intrinsic proteins (PIP) aquaporin isoforms with tonoplast intrinsic protein (gamma-TIP) in both IC-cells and adjacent cell types. METHODS: Using immunogold labelling in ultra-thin sections of Glycine max nodules, the expression of two PIP isoforms was observed and compared with the gamma-TIP pattern. KEY RESULTS: The plasma membrane aquaporins PIP1 and PIP2 were expressed more in IC-cells and endodermis than in pericycle and infected cells. The tonoplast aquaporin gamma-TIP has shown a distribution pattern similar to that of the PIPs. CONCLUSIONS: PIPs and gamma-TIP aquaporins are highly expressed in both plasmalemma and tonoplast of nodule IC-cells. This distribution is consistent with the putative role of water fluxes associated with the regulation of nodule conductance to O2 diffusion and the subsequent ATP-dependent nitrogenase activity. In the endodermis, these aquaporins might also be involved in nutrient transport between the infected zone and vascular traces.     

Arabidopsis SNAREs SYP61 and SYP121 Coordinate the Trafficking of Plasma Membrane Aquaporin PIP2;7 to Modulate the Cell Membrane Water Permeability

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.     

Stimulus-induced downregulation of root water transport involves reactive oxygen species-activated cell signalling and plasma membrane intrinsic protein internalization

The water uptake capacity of plant roots (i.e. their hydraulic conductivity, Lp(r)) is determined in large part by aquaporins of the plasma membrane intrinsic protein (PIP) subfamily. In the present work, we investigated two stimuli, salicylic acid (SA) and salt, because of their ability to induce an accumulation of reactive oxygen species (ROS) and an inhibition of Lp(r) concomitantly in the roots of Arabidopsis plants. The inhibition of Lp(r) by SA was partially counteracted by preventing the accumulation of hydrogen peroxide (H(2)O(2)) with exogenous catalase. In addition, exogenous H(2)O(2) was able to reduce Lp(r) by up to 90% in <15 min. Based on the lack of effects of H(2)O(2) on the activity of individual aquaporins in Xenopus oocytes, and on a pharmacological dissection of the action of H(2)O(2) on Lp(r), we propose that ROS do not gate Arabidopsis root aquaporins through a direct oxidative mechanism, but rather act through cell signalling mechanisms. Expression in transgenic roots of PIP-GFP fusions and immunogold labelling indicated that external H(2)O(2) enhanced, in <15 min, the accumulation of PIPs in intracellular structures tentatively identified as vesicles and small vacuoles. Exposure of roots to SA or salt also induced an intracellular accumulation of the PIP-GFP fusion proteins, and these effects were fully counteracted by co-treatment with exogenous catalase. In conclusion, the present work identifies SA as a novel regulator of aquaporins, and delineates an ROS-dependent signalling pathway in the roots of Arabidopsis. Several abiotic and biotic stress-related stimuli potentially share this path, which involves an H(2)O(2)-induced internalization of PIPs, to downregulate root water transport.     

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.     

Plasma membrane intrinsic proteins from maize cluster in two sequence subgroups with differential aquaporin activity

The transport of water through membranes is regulated in part by aquaporins or water channel proteins. These proteins are members of the larger family of major intrinsic proteins (MIPs). Plant aquaporins are categorized as either tonoplast intrinsic proteins (TIPs) or plasma membrane intrinsic proteins (PIPs). Sequence analysis shows that PIPs form several subclasses. We report on the characterization of three maize (Zea mays) PIPs belonging to the PIP1 and PIP2 subfamilies (ZmPIP1a, ZmPIP1b, and ZmPIP2a). The ZmPIP2a clone has normal aquaporin activity in Xenopus laevis oocytes. ZmPIP1a and ZmPIP1b have no activity, and a review of the literature shows that most PIP1 proteins identified in other plants have no or very low activity in oocytes. Arabidopsis PIP1 proteins are the only exception. Control experiments show that this lack of activity of maize PIP1 proteins is not caused by their failure to arrive at the plasma membrane of the oocytes. ZmPIP1b also does not appear to facilitate the transport of any of the small solutes tried (glycerol, choline, ethanol, urea, and amino acids). These results are discussed in relationship to the function and regulation of the PIP family of aquaporins.     

Methylation of aquaporins in plant plasma membrane

A thorough analysis, using MS, of aquaporins expressed in plant root PM (plasma membrane) was performed, with the objective of revealing novel post-translational regulations. Here we show that the N-terminal tail of PIP (PM intrinsic protein) aquaporins can exhibit multiple modifications and is differentially processed between members of the PIP1 and PIP2 subclasses. Thus the initiating methionine was acetylated or cleaved in native PIP1 and PIP2 isoforms respectively. In addition, several residues were detected to be methylated in PIP2 aquaporins. Lys3 and Glu6 of PIP2;1, one of the most abundant aquaporins in the PM, occurred as di- and mono-methylated residues respectively. Ectopic expression in Arabidopsis suspension cells of PIP2;1, either wild-type or with altered methylation sites, revealed an interplay between methylation at the two sites. Measurements of water transport in PM vesicles purified from these cells suggested that PIP2;1 methylation does not interfere with the aquaporin intrinsic water permeability. In conclusion, the present study identifies methylation as a novel post-translational modification of aquaporins, and even plant membrane proteins, and may represent a critical advance towards the identification of new regulatory mechanisms of membrane transport.     

水孔蛋白在细胞延长、盐胁迫和光合作用中的作用

水孔蛋白属于一个高度保守的、能够进行跨生物膜水分运输的通道蛋白MIP家族。水孔蛋白作为膜水通道,在控制细胞和组织的水含量中扮演重要角色。本研究的重点是属于PIP亚家族的GhPIP1;2和属于TIP亚家族的γTIP1在植物细胞延长中的作用。使用特异基因探针的Northern杂交和实时荧光PCR技术证明GhPIP1;2和GhγTIP1主要在棉花纤维延长过程中显著表达,且最高表达量在开花后5d。在细胞延长过程中,GhPIP1;2和GhγTIP1表达显著,表明它们在促使水流迅速进入液泡这一过程中扮演重要角色。而且也研究了盐胁迫植物中钙离子对水孔蛋白的影响。分别或一起用NaCl或CaCl2处理原生质体或细胞质膜。结果发现在盐胁迫条件下,水渗透率值在原生质体和质膜颗粒中都下降了,同时PIP1水孔蛋白的含量也下降了,表明NaCl对水孔蛋白的功能和含量有抑制作用。同时也观察了Ca2+的两种不同的作用。感知胁迫的胞质中游离钙离子浓度的增加可能导致水孔蛋白的关闭。而过剩的钙离子将导致水孔蛋白的上游调控。同时实验已经证明大麦的一类水孔蛋白-HvPIP2;1有更高的水和CO2转移率。本研究的目标是确定负责转运水和CO2的关键水孔蛋白...     

H2O2 induces rapid biophysical and permeability changes in the plasma membrane of Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the diffusion rate of hydrogen peroxide (H2O2) through the plasma membrane decreases during adaptation to H2O2 by means of a mechanism that is still unknown. Here, evidence is presented that during adaptation to H2O2 the anisotropy of the plasma membrane increases. Adaptation to H2O2 was studied at several times (15min up to 90min) by applying the steady-state H2O2 delivery model. For wild-type cells, the steady-state fluorescence anisotropy increased after 30min, or 60min, when using 2-(9-anthroyloxy) stearic acid (2-AS), or diphenylhexatriene (DPH) membrane probe, respectively. Moreover, a 40% decrease in plasma membrane permeability to H2O2 was observed at 15min with a concomitant two-fold increase in catalase activity. Disruption of the ergosterol pathway, by knocking out either ERG3 or ERG6, prevents the changes in anisotropy during H2O2 adaptation. H2O2 diffusion through the plasma membrane in S. cerevisiae cells is not mediated by aquaporins since the H2O2 permeability constant is not altered in the presence of the aquaporin inhibitor mercuric chloride. Altogether, these results indicate that the regulation of the plasma membrane permeability towards H2O2 is mediated by modulation of the biophysical properties of the plasma membrane.     

Membrane transport of hydrogen peroxide

Hydrogen peroxide (H2O2) belongs to the reactive oxygen species (ROS), known as oxidants that can react with various cellular targets thereby causing cell damage or even cell death. On the other hand, recent work has demonstrated that H2O2 also functions as a signalling molecule controlling different essential processes in plants and mammals. Because of these opposing functions the cellular level of H2O2 is likely to be subjected to tight regulation via processes involved in production, distribution and removal. Substantial progress has been made exploring the formation and scavenging of H2O2, whereas little is known about how this signal molecule is transported from its site of origin to the place of action or detoxification. From work in yeast and bacteria it is clear that the diffusion of H2O2 across membranes is limited. We have now obtained direct evidence that selected aquaporin homologues from plants and mammals have the capacity to channel H2O2 across membranes. The main focus of this review is (i) to summarize the most recent evidence for a signalling role of H2O2 in various pathways in plants and mammals and (ii) to discuss the relevance of specific transport of H2O2.     

Single-molecule analysis of PIP2;1 dynamics and partitioning reveals multiple modes of Arabidopsis plasma membrane aquaporin regulation

PIP2;1 is an integral membrane protein that facilitates water transport across plasma membranes. To address the dynamics of Arabidopsis thaliana PIP2;1 at the single-molecule level as well as their role in PIP2;1 regulation, we tracked green fluorescent protein-PIP2;1 molecules by variable-angle evanescent wave microscopy and fluorescence correlation spectroscopy (FCS). Single-particle tracking analysis revealed that PIP2;1 presented four diffusion modes with large dispersion of diffusion coefficients, suggesting that partitioning and dynamics of PIP2;1 are heterogeneous and, more importantly, that PIP2;1 can move into or out of membrane microdomains. In response to salt stress, the diffusion coefficients and percentage of restricted diffusion increased, implying that PIP2;1 internalization was enhanced. This was further supported by the decrease in PIP2;1 density on plasma membranes by FCS. We additionally demonstrated that PIP2;1 internalization involves a combination of two pathways: a tyrphostin A23-sensitive clathrin-dependent pathway and a methyl-β-cyclodextrin-sensitive, membrane raft-associated pathway. The latter was efficiently stimulated under NaCl conditions. Taken together, our findings demonstrate that PIP2;1 molecules are heterogeneously distributed on the plasma membrane and that clathrin and membrane raft pathways cooperate to mediate the subcellular trafficking of PIP2;1, suggesting that the dynamic partitioning and recycling pathways might be involved in the multiple modes of regulating water permeability.     

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K⁺ channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K⁺ channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.     

Role of a single aquaporin isoform in root water uptake

Aquaporins are ubiquitous channel proteins that facilitate the transport of water across cell membranes. Aquaporins show a typically high isoform multiplicity in plants, with 35 homologs in Arabidopsis. The integrated function of plant aquaporins and the function of each individual isoform remain poorly understood. Matrix-assisted laser desorption/ionization time-of-flight analyses suggested that Plasma Membrane Intrinsic Protein2;2 (PIP2;2) is one of the abundantly expressed aquaporin isoforms in Arabidopsis root plasma membranes. Two independent Arabidopsis knockout mutants of PIP2;2 were isolated using a PCR-based strategy from a library of plant lines mutagenized by the insertion of Agrobacterium tumefaciens T-DNA. Expression in transgenic Arabidopsis of a PIP2;2 promoter-beta-glucuronidase gene fusion indicated that PIP2;2 is expressed predominantly in roots, with a strong expression in the cortex, endodermis, and stele. The hydraulic conductivity of root cortex cells, as measured with a cell pressure probe, was reduced by 25 to 30% in the two allelic PIP2;2 mutants compared with the wild type. In addition, free exudation measurements revealed a 14% decrease, with respect to wild-type values, in the osmotic hydraulic conductivity of roots excised from the two PIP2;2 mutants. Together, our data provide evidence for the contribution of a single aquaporin gene to root water uptake and identify PIP2;2 as an aquaporin specialized in osmotic fluid transport. PIP2;2 has a close homolog, PIP2;3, showing 96.8% amino acid identity. The phenotype of PIP2;2 mutants demonstrates that, despite their high homology and isoform multiplicity, plant aquaporins have evolved with nonredundant functions.     

PIP Water Transport and Its pH Dependence Are Regulated by Tetramer Stoichiometry

Many plasma membrane channels form oligomeric assemblies, and heterooligomerization has been described as a distinctive feature of some protein families. In the particular case of plant plasma membrane aquaporins (PIPs), PIP1 and PIP2 monomers interact to form heterotetramers. However, the biological properties of the different heterotetrameric configurations formed by PIP1 and PIP2 subunits have not been addressed yet. Upon coexpression of tandem PIP2-PIP1 dimers in Xenopus oocytes, we can address, for the first time to our knowledge, the functional properties of single heterotetrameric species having 2:2 stoichiometry. We have also coexpressed PIP2-PIP1 dimers with PIP1 and PIP2 monomers to experimentally investigate the localization and biological activity of each tetrameric assembly. Our results show that PIP2-PIP1 heterotetramers can assemble with 3:1, 1:3, or 2:2 stoichiometry, depending on PIP1 and PIP2 relative expression in the cell. All PIP2-PIP1 heterotetrameric species localize at the plasma membrane and present the same water transport capacity. Furthermore, the contribution of any heterotetrameric assembly to the total water transport through the plasma membrane doubles the contribution of PIP2 homotetramers. Our results also indicate that plasma membrane water transport can be modulated by the coexistence of different tetrameric species and by intracellular pH. Moreover, all the tetrameric species present similar cooperativity behavior for proton sensing. These findings throw light on the functional properties of PIP tetramers, showing that they have flexible stoichiometry dependent on the quantity of PIP1 and PIP2 molecules available. This represents, to our knowledge, a novel regulatory mechanism to adjust water transport across the plasma membrane.     

Plant aquaporins: novel functions and regulation properties

Aquaporins are water channel proteins of intracellular and plasma membranes that play a crucial role in plant water relations. The present review focuses on the most recent findings concerning the molecular and cellular properties of plant aquaporins. The mechanisms of transport selectivity and gating (i.e. pore opening and closing) have recently been described, based on aquaporin structures at atomic resolution. Novel dynamic aspects of aquaporin subcellular localisation have been uncovered. Also, some aquaporin isoforms can transport, besides water, physiologically important molecules such as CO(2), H(2)O(2), boron or silicon. Thus, aquaporins are involved in many great functions of plants, including nutrient acquisition, carbon fixation, cell signalling and stress responses.     

C-terminal deletion analysis of plant plasma membrane H(+)-ATPase: yeast as a model system for solute transport across the plant plasma membrane.

The plasma membrane proton pump (H(+)-ATPase) energizes solute uptake by secondary transporters. Wild-type Arabidopsis plasma membrane H(+)-ATPase (AHA2) and truncated H(+)-ATPase lacking 38, 51, 61, 66, 77, 92, 96, and 104 C-terminal amino acids were produced in yeast. All AHA2 species were correctly targeted to the yeast plasma membrane and, in addition, accumulated in internal membranes. Removal of 38 C-terminal residues from AHA2 produced a high-affinity state of plant H(+)-ATPase with a low Km value (0.1 mM) for ATP. Removal of an additional 12 amino acids from the C terminus resulted in a significant increase in molecular activity of the enzyme. There was a close correlation between molecular activity of the various plant H(+)-ATPase species and their ability to complement mutants of the endogenous yeast plasma membrane H(+)-ATPase (pma1). This correlation demonstrates that, at least in this heterologous host, activation of H(+)-ATPase is a prerequisite for proper energization of the plasma membrane.     

Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers

Cotton fiber (Gossypium hirsutum L. and G. barbadense L.) is a good model for studies of plant cell elongation and cell wall biogenesis. Aquaporins are ancient membrane channel proteins that facilitate the permeation of water across biological membranes. We studied GhPIP1-2, encoding plasma membrane intrinsic protein, and GhgammaTIP1, encoding tonoplast intrinsic protein, during cotton fiber development. The full-length cDNAs of GhPIP1-2 and GhgammaTIP1 were obtained through 5' RACE. The deduced amino acid sequences of GhPIP1-2 and GhgammaTIP1 share high sequence identity with aquaporins from diverse plant species. Phylogenetic analysis of GhPIP1-2 and GhgammaTIP1 with other plant aquaporins showed that GhPIP1-2 belongs to the PIP1 group of the PIP subfamily and GhgammaTIP1 belongs to the gammaTIP group of the TIP subfamily. GhPIP1-2 and GhgammaTIP1 contain three and two introns, respectively. Genomic Southern blot analysis indicated that GhPIP1-2 and GhgammaTIP1 have several copies and multiple homologous genes in allotetraploid cotton. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1-2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post-anthesis. Moreover, expression patterns of the two genes in G. hirsutum and G. barbadense are similar, whereas the expression levels in G. barbadense are much lower than that in G. hirsutum. The high and preferential expression of GhPIP1-2 and GhgammaTIP1 during fiber cell elongation suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion.     

PIP1 aquaporins,sterols, and osmotic water permeability of plasma membranes from etiolated pea seedlings

Plasma membrane isolated from microsomal membranes of pea seedling root and shoot cells by means of aqueous two-phase polymer system was separated by flotation in discontinuous OptiPrep gradient into “light” (≤1.146 g/cm³) and “heavy” (≥1.146 g/cm³) fractions. Osmotic water permeability of plasma membrane and its two fractions was investigated by inducing transmembrane osmotic gradient on the vesicle membrane and recording the kinetics of vesicle osmotic shrinkage by the stopped-flow method. Rate constants of osmotic shrinkage and coefficients of osmotic water permeability of the membranes were estimated on the basis of the kinetic curve approximation by exponential dependencies and using electron microscope data on vesicles sizes. In plasma membrane and its fractions the content of sterols and PIP1 aquaporins was determined. It was found that in “light” PM fractions from both roots and shoots the content of PIP1 aquaporins and sterols was higher and the osmotic water permeability coefficient was lower than in “heavy” fractions of plasma membrane. The results indicate that plasma membrane of roots and shoots is heterogeneous in osmotic water permeability. This heterogeneity may be related with the presence of microdomains with different content of aquaporins and sterols in the membrane.