Aquaporin Homologs Research Articles

Aquaporins: Multifunctional Players in Plant Growth, Development and Stress Responses

Aquaporins are small membrane proteins belonging to the group of Major Intrinsic proteins (MIPs) that facilitate the transport of water and small molecules across biological membranes. They were initially identified as water channels and later it has become clear that they also play important roles in several other physiological processes in plants. Plants have different types of aquaporins in terms of their location, gating properties, and solute specificity and can mainly divide into four major subfamilies. They are tonoplast intrinsic proteins (TIPs), nodulin26-like intrinsic proteins (NIPs), plasma membrane intrinsic proteins (PIPs), and the small basic intrinsic proteins (SIPs). All these aquaporins play a variety of physiological activities at all stages of plant growth and development. Also, they are believed to play a significant part in the plant’s defense mechanisms against biotic and abiotic stresses. This review comprehensively addresses the roles of diverse aquaporin homologs in growth and development and their response to different environmental factors.

An aquaporin and an aquaglyceroporin have roles in low temperature adaptation of mosquitoes (Anopheles sinensis).

Mosquitoes (Anopheles sinensis), widely geographically distributed in Asia including China, are the primary vector of the malaria parasite Plasmodium vivax and other parasitic diseases such as Malayan filariasis. An. sinensis can survive through low winter temperatures. Aquaporin channels are found in all life forms, where they facilitate environmental adaptation by allowing rapid trans-cellular movement of water (classical aquaporins) or water and solutes such as glycerol (aquaglyceroporins). Here, we identified and characterized 2 aquaporin (AQP) homologs in An. sinensis: AsAQP2 (An. sinensis aquaglyceroporin) and AsAQP4 (An. sinensis aquaporin). When expressed in frog (Xenopus laevis) oocytes, AsAQP2 transported water, glycerol, and urea; AsAQP4 transported only water. Water permeation through AsAQP2 and AsAQP4 was inhibited by mercuric chloride. AsAQP2 expression was slightly higher in adult female mosquitoes than in males, and AsAQP4 expression was significantly higher in adult males. The 2 AsAQPs were highly expressed in Malpighian tubules and midgut. AsAQP2 and AsAQP4 expression was up-regulated by blood feeding compared with sugar feeding. At freezing point (0°C), the AsAQP4 expression level increased and An. sinensis survival time reduced compared with those at normal temperature (26°C). At low temperature (8°C), the AsAQP2 and AsAQP4 expression levels decreased and survival time was significantly longer compared with those at 26°C. These results suggest that AsAQP2 and AsAQP4 have roles in water homeostasis during blood digestion and in low temperature adaptation of A. sinensis. Together, our results show that the 2 AQPs are important for mosquito diuresis after blood feeding and when exposed to low temperatures.

Cloning and Expression of Four Aquaporin Homologs from the Chinese Black Sleeper (Bostrychus sinensis): The Effects of Salinity Acclimation.

Several fish species are known to possess mechanisms that allow them to adapt to environments with different salinities. The aim of this study was to investigate the effects of salinity on the expression of aquaporins (aqp1a, aqp3a, aqp8a, and aqp9a) in the gills and intestines of Chinese black sleeper. After 30days of acclimation, the expression of aqp1a, aqp3a, and aqp9a in the gills was significantly higher in fish transferred to 5 ppt than in those transferred to 40 ppt seawater, whereas aqp8 expression was lower. In contrast, aqp1a, aqp3a, and aqp8a expression in the intestines was higher in fish acclimated in 40 ppt than in those acclimated in 5 ppt. During abrupt salinity acclimation, the levels of aqp1a and aqp9a in the gills varied over time in fish acclimated in 5 ppt, but not in 40 ppt. The aqp3a levels in gills were higher in the 5 ppt group after 24h than in the 40 ppt. The expression level of aqp8a in gills was higher in 40 ppt than in 5 ppt, except for that at 12h. In the intestines, expression level of aqp1a and aqp8a were significantly upregulated from 12 to 48h following acclimation in 40 ppt and aqp3a was higher in 40 ppt group than in 5 ppt, while aqp9a expression exhibited an opposite trend. These findings suggest that aqp1a, aqp3a, aqp8a and aqp9a may play a major osmoregulatory role in water transport in the gills and intestines during acclimation to different salinity environment.

Plant aquaporins alleviate drought tolerance in plants by modulating cellular biochemistry, root-architecture, and photosynthesis.

Water is a vital resource for plants to grow, thrive, and complete their life cycle. In recent years, drastic changes in the climate, especially drought frequency and severity, have increased, which reduces agricultural productivity worldwide. Aquaporins are membrane channels belonging to the major intrinsic protein superfamily, which play an essential role in cellular water and osmotic homeostasis of plants under both control and water deficit conditions. A genome-wide search reveals the vast availability of aquaporin isoforms, phylogenetic relationships, different families, conserved residues, chromosomal locations, and gene structure of aquaporins. Furthermore, aquaporins gating and subcellular trafficking are commonly controlled by phosphorylation, cytosolic pH, divalent cations, reactive oxygen species, and stoichiometry. Researchers have identified their involvement in regulating hydraulic conductance, root system architecture, modulation of abiotic stress-related genes, seed viability and germination, phloem loading, xylem water exit, photosynthetic parameters, and post-drought recovery. Remarkable effects following the change in aquaporin activity and/or gene expression have been observed on root water transport properties, nutrient acquisition, physiology, transpiration, stomatal aperture, gas exchange, and water use efficiency. The present review highlights the role of different aquaporin homologs under water-deficit stress condition in model and crop plants. Moreover, the opportunity and challenges encountered to explore aquaporins for engineering drought-tolerant crop plants are also discussed here.

Genome-wide identification, characterization, and expression profile of aquaporin gene family in flax (Linum usitatissimum)

Membrane intrinsic proteins (MIPs) form transmembrane channels and facilitate transport of myriad substrates across the cell membrane in many organisms. Majority of plant MIPs have water transporting ability and are commonly referred as aquaporins (AQPs). In the present study, we identified aquaporin coding genes in flax by genome-wide analysis, their structure, function and expression pattern by pan-genome exploration. Cross-genera phylogenetic analysis with known aquaporins from rice, arabidopsis, and poplar showed five subgroups of flax aquaporins representing 16 plasma membrane intrinsic proteins (PIPs), 17 tonoplast intrinsic proteins (TIPs), 13 NOD26-like intrinsic proteins (NIPs), 2 small basic intrinsic proteins (SIPs), and 3 uncharacterized intrinsic proteins (XIPs). Amongst aquaporins, PIPs contained hydrophilic aromatic arginine (ar/R) selective filter but TIP, NIP, SIP and XIP subfamilies mostly contained hydrophobic ar/R selective filter. Analysis of RNA-seq and microarray data revealed high expression of PIPs in multiple tissues, low expression of NIPs, and seed specific expression of TIP3 in flax. Exploration of aquaporin homologs in three closely related Linum species bienne, grandiflorum and leonii revealed presence of 49, 39 and 19 AQPs, respectively. The genome-wide identification of aquaporins, first in flax, provides insight to elucidate their physiological and developmental roles in flax.

The Roles of Aquaporins in Plant Stress Responses.

Aquaporins are membrane channel proteins ubiquitously present in all kingdoms of life. Although aquaporins were originally discovered as water channels, their roles in the transport of small neutral solutes, gasses, and metal ions are now well established. Plants contain the largest number and greatest diversity of aquaporin homologs with diverse subcellular localization patterns, gating properties, and solute specificity. The roles of aquaporins in physiological functions throughout plant growth and development are well known. As an integral regulator of plant–water relations, they are presumed to play an important role in plant defense responses against biotic and abiotic stressors. This review highlights involvement of various aquaporin homologs in plant stress responses against a variety of environmental stresses that disturb plant cell osmotic balance and nutrient homeostasis.

Defensive slime formation in Pacific hagfish requires Ca2+- and aquaporin-mediated swelling of released mucin vesicles.

Hagfishes defend themselves from fish predators via the rapid deployment of a fibrous slime that adheres to and clogs gills. The slime transforms from a thick glandular exudate to a fully hydrated product in a fraction of a second through a process that involves the swelling and rupture of numerous mucin vesicles. Here we demonstrate that the vesicle membrane plays an important role in regulating the swelling of mucin granules, and provide evidence that the membrane contains proteins that facilitate the movement of ions and water molecules. By exposing isolated mucin vesicles to varying combinations of inorganic ions, organic compounds and membrane channel inhibitors, we found that the majority of hagfish mucin vesicles require Ca(2+) to rupture. We also show that Ca(2+)-dependent rupture can be pharmacologically inhibited, which suggests a role for Ca(2+)-activated membrane transporters. We demonstrate that the aquaporin inhibitor mercuric chloride reduces the rate of vesicle swelling by an order of magnitude, which suggests that aquaporins facilitate the influx of water during vesicle deployment. Molecular evidence of two aquaporin homologues expressed in the slime glands further supports this idea. We propose a model of hagfish slime mucin vesicle rupture that involves Ca(2+)-activated transporters and aquaporins, and suggest that the presence of these proteins is an adaptation for increasing the speed of vesicle rupture and, consequently, the speed of the sliming response of hagfishes.

Aquaporin homologs and water transport in the anal papillae of the larval mosquito, Aedes aegypti

The hemolymph osmolarity of the freshwater mosquito larvae (Aedes aegypti) is greater than that of their habitat. To combat the influx of water, larvae cycle water entering through the gut and anal papillae to the Malpighian tubules for secretion. The presence of aquaporins (AQPs, water channels) may facilitate the movement of water across these tissues. Tissue distribution of mRNA transcripts of putative aquaporins from mosquito larvae, using quantitative PCR, revealed expression of transcripts in the Malpighian tubules and anal papillae. Four putative aquaporin transcripts are expressed in the Malpighian tubules and provide a basis for further work aimed at discovering the elusive water transporters functioning during diuresis. Transcripts of putative AQPs (AaAQP4 and AaAQP1b) are expressed in the anal papillae. Immunoreactivity to a human AQP1-antibody was found in the anal papillae and mercury inhibits tritiated water uptake in isolated anal papillae. Together, the results suggest that AQP(s) could be responsible for facilitating water transport at the papillae epithelium.

Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1;1 and TgTIP1;2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1;2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H2O2. TgTIP1;1- and TgTIP1;2-mediated H2O2 conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H2O2 conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I TIPs might have a broader spectrum of substrate selectivity than group II TIPs.

The role of a group III AQP, AQP11 in intracellular organelle homeostasis

AQP11 is a member of a new aquaporin subfamily which includes many aquaporin homologs with low amino acid identities, around 20% of previously identified AQPs. Although these AQPs have unusual NPA sequences, these AQPs have a completely conserved and functionally indispensable cysteine residue downstream of the second NPA box, suggesting that they belong to a specific AQP subfamily, which we propose to name the group III AQPs. On the other hand, the NPA boxes are highly conserved in previous AQP subfamilies: the group I AQPs, original water-selective aquaporin family and the group II AQPs, aquaglyceroporin family. Currently the roles of the group III AQPs are only known with AQP11 as the disruption of intracellularly located AQP11 in mice produced huge vacuoles in the proximal tubule leading to fatal polycystic kidneys at one month old. This review focused on the classification of AQPs based on primary structures to obtain insights into the function and the role of AQPs. With the accumulation of new AQP-like sequences through genome projects, this classification will be useful to predict their functions as each group may have specific characteristics in its function, distribution and regulation.

Molecular determinants of ammonia and urea conductance in plant aquaporin homologs

Aquaporins and/or aquaglyceroporins regulate the permeability of plant membranes to water and small, uncharged molecules. Using molecular simulations with a plant plasma membrane aquaporin tetramer, the residues in the channel constriction region were identified as the crucial determinants of ammonia and urea conductance. The impact of these residues was experimentally verified using AtPIP2;1 pore mutants. Several, but not all, mutants with a NIP-like selectivity filter promoted yeast growth on urea or ammonia as sole sources of nitrogen. TIP-like mutants conducted urea but not NH 3, and a residue without direct contact to the pore lumen was critical for conduction in the mutants.

Regulation of expression of two aquaporin homologs in the intestine of the European eel: effects of seawater acclimation and cortisol treatment

Complementary DNAs encoding homologs of the mammalian aquaglyceroporins (termed AQPe) and aquaporin-1 isoforms (termed AQP1) were isolated from the European eel. The AQP amino acid sequences share 35-54% identity with other known human AQPs. Although AQPe mRNA expression was approximately equivalent along the entire length of the gut, AQP1 expression was the highest in the posterior/rectal segment. Seawater (SW) acclimation increased AQP1 mRNA abundance by 5- and 17-fold in the anterior, 14- and 23-fold in the mid-, and 9- and 7-fold in the posterior/rectal gut regions of yellow and silver eels, respectively. SW acclimation had an effect on AQPe mRNA expression only in the midintestine of silver eels, where a small but significant 1.7-fold increase in abundance was measured. Western blots using an eel AQP1-specific antibody identified the presence of a major immunoreactive 28-kDa protein, primarily within the posterior/rectal segment. A 3-wk SW transfer induced an increase in AQP1 protein abundance in all intestinal segments, with the posterior/rectal region still expressing protein levels approximately 40- and 8-fold higher than the anterior and midsegments, respectively. Strong AQP1 immunofluorescence was detected within the vascular endothelium in both freshwater (FW)- and SW-acclimated eels and in the epithelial apical brush border in the posterior/rectal gut regions of SW-acclimated eels. Cortisol infusion into FW eels had no effect on intestinal AQPe mRNA expression but induced increases in AQP1 mRNA and protein levels. These results provide evidence for the presence of a SW-induced and steroid-regulated AQP water channel pathway within the intestine of the European eel.

Aquaporin Water Channels in Central Nervous System

Aquaporins (AQPs) are a family of water-selective channels that provide a major pathway for osmotically driven water transport through cell membranes. Some members of the aquaporin family have been identified in the central nervous system (CNS). The water channel aquaporin 1 (AQP1) is restricted to the apical domain of the choroid plexus epithelial cells. The AQP4 is abundantly expressed in astrocyte foot processes and ependymocytes facing capillaries and brain-cerebrospinal fluid (CSF) interfaces, whereas AQP9 is localized in tanycytes and astrocytes processes. The mRNA for other aquaporin homologs (i.e., AQP3, 5, and 8) have been recently found in cultured astrocytes. Based on their subcellular localization and data obtained from functional studies, it is assumed that aquaporins are implicated in water movements in nervous tissue and may play a role in central osmoreception, K+ siphoning, and cerebrospinal fluid formation. There have been recent reports describing different aquaporin-responses under pathologic states leading to brain edema. The data available provide a better understanding of the mechanisms responsible for brain edema and indicate that aquaporins are potential targets for drug development.

Immunolocalization of Aquaporin Homologs in Mouse Lacrimal Glands

It was shown recently that the aquaporin family of water channels exists in lacrimal gland cells. To determine the localization of aquaporin homologs and the pathophysiological modification of aquaporins by pilocarpine, a muscarinic agonist, we performed immunohistochemistry and immunoblotting in mouse lacrimal glands. By immunohistochemistry, aquaporin-4 (AQP4) and aquaporin-5 (AQP5) were found to be the lacrimal glands. AQP5 immunolabeling was detected in the apical membranes of the acinus and duct cells, while AQP4 was expressed in the basolateral membranes only. The tear secretion of mice systemically treated with pilocarpine was significantly (2.5-fold) higher than that of the saline-treated controls. The antibody to the AQP5 carboxy terminus showed high immunoreactivity on the apical membrane in the pilocarpine-treated lacrimal glands but not on that of saline-treated controls. However, the antibody to the extracellular domain of AQP5 showed similar immunolabeling in both groups of animals. In contrast, the immunoreactivity of AQP4 was not affected by pilocarpine stimulation. As shown by western blot analysis, the expression level of AQP5 on the apical membrane in the pilocarpine-stimulated lacrimal glands was not significantly different compared with the saline-treated controls. We conclude that AQP4 and AQP5 water channels are expressed on mouse lacrimal gland cells, with greater expression of AQP4 on the basolateral membrane and of AQP5 on the apical membrane. Furthermore, the AQP5 carboxy terminus region may undergo pathophysiological modification when tear secretion is increased by pilocarpine stimulation.

Water and Glycerol Permeabilities of Aquaporins 1–5 and MIP Determined Quantitatively by Expression of Epitope-tagged Constructs inXenopus Oocytes

The goal of this study was to compare single channel water and glycerol permeabilities of mammalian aquaporins (AQP) 1-5 and the major intrinsic protein of lens fiber (MIP). Each of the six cloned cDNAs from rat was left untagged or was epitope-tagged with c-Myc or FLAG at either the N or C terminus so that results would not depend on epitope identity or location. The constructs were expressed in Xenopus oocytes for measurement of osmotic water permeability (Pf), [3H]glycerol uptake, and protein expression. Each of the 30 epitope-tagged constructs was expressed strongly at the oocyte plasma membrane. The 10-min uptake of [3H]glycerol was increased significantly (range of 4.5-8-fold over control) in oocytes expressing untagged AQP3 (GLIP) and each of the four tagged AQP3 constructs; [3H]glycerol uptake was not increased in oocytes expressing AQP1, AQP2, AQP4, AQP5, or MIP. In oocytes microinjected with 5 ng of cRNA, average Pf values (in cm/s x 10(-3)) were 0.67 +/- 0.06 (control), 19 +/- 2 (AQP1), 10 +/- 1 (AQP2), 8 +/- 2 (AQP3), 29 +/- 1 (AQP4), 10 +/- 1 (AQP5), and 1.3 +/- 0.2 (MIP), and they were relatively insensitive to the presence, identity, or location of the epitope tag. Pf values were not affected by protein kinase A or C activation. After normalization for plasma membrane expression by immunoprecipitation of microdissected plasma membranes, single channel water permeabilities (pf, referenced to the AQP1 pf of 6 x 10(-14) cm3/s) were (in cm3/s x 10(-14)) 3.3 +/- 0.2 (AQP2), 2.1 +/- 0.3 (AQP3), 24 +/- 0.6 (AQP4), 5.0 +/- 0.4 (AQP5), and 0.25 +/- 0.05 (MIP); pf values were insensitive to epitope identity and location. These results indicate very different intrinsic water permeabilities for the mammalian aquaporin homologs, with the pf value for AQP4 remarkably higher than those for the others. The pf values establish limits on aquaporin tissue densities required for physiological function and suggest significant structural and functional differences among the aquaporins.

ABH and Colton blood group antigens on Aquaporin-1, the human red cell water channel protein

The recent identification of the red cell water transporter (AQP1) has led to the identification of the "aquaporins", a new class of membrane proteins which function as water-selective transport proteins and are involved in many physiological processes. Identification of the chromosomal localization of the corresponding gene led to the recognition that AQP1 is the structural basis of the Colton blood group antigens. Analysis of individuals with the Colton null phenotype led to the recognition that homozygosity for knockout mutations in the corresponding gene is exceedingly rare but is without a significant clinical phenotype, predicting a redundancy in expression of other aquaporin homologs. These studies demonstrate the importance which molecular studies in red cell blood group antigens may play in diverse areas of biomedical research. Moreover, they provide another example that blood group antigens may be polymorphisms in functionally important proteins on the red cell surface.