The role of aquaporins in root water uptake

The capacity of roots to take up water is determined in part by the resistance of living tissues to radial water flow. Both the apoplastic and cell-to-cell paths mediate water transport in these tissues but the contribution of cell membranes to the latter path has long been difficult to estimate. Aquaporins are water channel proteins that are expressed in various membrane compartments of plant cells, including the plasma and vacuolar membranes. Plant aquaporins are encoded by a large multigene family, with 35 members in Arabidopsis thaliana, and many of these aquaporins show a cell-specific expression pattern in the root. Mercury acts as an efficient blocker of most aquaporins and has been used to demonstrate the significant contribution of water channels to overall root water transport. Aquaporin-rich membranes may be needed to facilitate intense water flow across root tissues and may represent critical points where an efficient and spatially restricted control of water uptake can be exerted. Roots, in particular, show a remarkable capacity to alter their water permeability over the short term (i.e. in a few hours to less than 2-3 d) in response to many stimuli, such as day/night cycles, nutrient deficiency or stress. Recent data suggest that these rapid changes can be mostly accounted for by changes in cell membrane permeability and are mediated by aquaporins. Although the processes that allow perception of environmental changes by root cells and subsequent aquaporin regulation are nearly unknown, the study of root aquaporins provides an interesting model to understand the regulation of water transport in plants and sheds light on the basic mechanisms of water uptake by roots.     

The aquaporin gene family of the ectomycorrhizal fungus Laccaria bicolor: lessons for symbiotic functions

Soil humidity and bulk water transport are essential for nutrient mobilization. Ectomycorrhizal fungi, bridging soil and fine roots of woody plants, are capable of modulating both by being integrated into water movement driven by plant transpiration and the nocturnal hydraulic lift. Aquaporins are integral membrane proteins that function as gradient-driven water and/or solute channels. Seven aquaporins were identified in the genome of the ectomycorrhizal basidiomycete Laccaria bicolor and their role in fungal transfer processes was analyzed. Heterologous expression in Xenopus laevis oocytes revealed relevant water permeabilities for three aquaporins. In fungal mycelia, expression of the corresponding genes was high compared with other members of the gene family, indicating the significance of the respective proteins for plasma membrane water permeability. As growth temperature and ectomycorrhiza formation modified gene expression profiles of these water-conducting aquaporins, specific roles in those aspects of fungal physiology are suggested. Two aquaporins, which were highly expressed in ectomycorrhizas, conferred plasma membrane ammonia permeability in yeast. This indicates that these proteins are an integral part of ectomycorrhizal fungus-based plant nitrogen nutrition in symbiosis.     

Aquaporins and unloading of phloem-imported water in coats of developing bean seeds

Nutrients are imported into developing legume seeds by mass flow through the phloem, and reach developing embryos following secretion from their symplasmically isolated coats. To sustain homeostasis of seed coat water relations, phloem-delivered nutrients and water must exit seed coats at rates commensurate with those of import through the phloem. In this context, coats of developing French bean seeds were screened for expression of aquaporin genes resulting in cloning PvPIP1;1, PvPIP2;2 and PvPIP2;3. These genes were differentially expressed in all vegetative organs, but exhibited their strongest expression in seed coats. In seed coats, expression was localized to cells of the nutrient-unloading pathway. Transport properties of the PvPIPs were characterized by expression in Xenopus oocytes. Only PvPIP2;3 showed significant water channel activity (Pos = 150-200 microm s(-1)) even when the plasma membrane intrinsic proteins (PIPs) were co-expressed in various combinations. Permeability increases to glycerol, methylamine and urea were not detected in oocytes expressing PvPIPs. Transport active aquaporins in native plasma membranes of seed coats were demonstrated by measuring rates of osmotic shrinkage of membrane vesicles in the presence and absence of mercuric chloride and silver nitrate. The functional significance of aquaporins in nutrient and water transport in developing seeds is discussed.     

Aquaporins and plant leaf movements

BaCKGROUND: Plant leaf movements can be mediated by specialized motor organs, the pulvini, or can be epinastic (i.e. based on different growth velocities of the adaxial and abaxial halves of the leaf). Both processes are associated with diurnally regulated increases in rates of membrane water transport, which in many cases has been shown to be facilitated by aquaporins. Rhythmic leaf movements are known from many plant species, but few papers deal with the involvement of aquaporins in such movements. SCOPE: Many details of the architecture and function of pulvini were worked out by Ruth Satter and co-workers using Samanea saman as a model organism. More recently a contribution of aquaporins to pulvinar movement in Samanea was demonstrated. Another model plant to study pulvinus-mediated leaf movements is Mimosa pudica. The contribution of both plasma membrane- and tonoplast-localized aquaporins to the seismonastic leaf movements in Mimosa was analysed. In tobacco, as an example of epinastic leaf movement, it was shown that a PIP1 aquaporin family member is an important component of the leaf movement mechanism.     

The aquaporins

Water is the major component of all living cells, and efficient regulation of water homeostasis is essential for many biological processes. The mechanism by which water passes through biological membranes was a matter of debate until the discovery of the aquaporin water channels. Aquaporins are intrinsic membrane proteins characterized by six transmembrane helices that selectively allow water or other small uncharged molecules to pass along the osmotic gradient. In addition, recent observations show that some aquaporins also facilitate the transport of volatile substances, such as carbon dioxide (CO₂) and ammonia (NH₃), across membranes. Aquaporins usually form tetramers, with each monomer defining a single pore. Aquaporin-related proteins are found in all organisms, from archaea to mammals. In both uni- and multicellular organisms, numerous isoforms have been identified that are differentially expressed and modified by post-translational processes, thus allowing fine-tuned tissue-specific osmoregulation. In mammals, aquaporins are involved in multiple physiological processes, including kidney and salivary gland function. They are associated with several clinical disorders, such as kidney dysfunction, loss of vision and brain edema.     

Plant aquaporins: multifunctional water and solute channels with expanding roles

There is strong evidence that aquaporins are central components in plant water relations. Plant species possess more aquaporin genes than species from other kingdoms. According to sequence similarities, four major groups have been identified, which can be further divided into subgroups that may correspond to localization and transport selectivity. They may be involved in compatible solute distribution, gas-transfer (CO2, NH3) as well as in micronutrient uptake (boric acid). Recent advances in determining the structure of some aquaporins gives further details on the mechanism of selectivity. Gating behaviour of aquaporins is poorly understood but evidence is mounting that phosphorylation, pH, pCa and osmotic gradients can affect water channel activity. Aquaporins are enriched in zones of fast cell division and expansion, or in areas where water flow or solute flux density would be expected to be high. This includes biotrophic interfaces between plants and parasites, between plants and symbiotic bacteria or fungi, and between germinating pollen and stigma. On a cellular level aquaporin clusters have been identified in some membranes. There is also a possibility that aquaporins in the endoplasmic reticulum may function in symplasmic transport if water can flow from cell to cell via the desmotubules in plasmodesmata. Functional characterization of aquaporins in the native membrane has raised doubt about the conclusiveness of expression patterns alone and need to be conducted in parallel. The challenge will be to elucidate gating on a molecular level and cellular level and to tie those findings into plant water relations on a macroscopic scale where various flow pathways need to be considered.     

On the pH regulation of plant aquaporins

The majority of plants are unable to evade unfavorable conditions such as flooding, salinity, or drought. Therefore, a fine-tuned water homeostasis appears to be of crucial importance for plant survival, and it was assumed that aquaporins play a significant role in these processes. Regulation of plant aquaporin conductivity was suggested to be achieved by a gating mechanism that involves protein phosphorylation under drought stress conditions and protonation after cytosolic acidification during flooding. The effect of protein phosphorylation or protonation of aquaporins was studied on two plasma membrane intrinsic proteins, NtPIP2;1 and NtAQP1 from tobacco, which were heterologously expressed in yeast. Our results on mutated aquaporins with serine-to-alanine exchange indicate that phosphorylation of the two key serine residues did not affect the pH-dependent modification of water permeability. Protonation on a conserved histidine residue decreased water conductivity of NtPIP2;1. Although cells expressing NtPIP2;1 with a replacement of the histidine by an alanine were found to be pH-insensitive with regard to water permeability, these maintain high water transport rates, similar to those obtained under acidic conditions. The data clearly support the role of histidine at 196 as a component of pH-dependent modification of aquaporin-facilitated water transport. The predictions of combined effects from phosphorylation at conserved serines and histidine protonation were not supported by the results of functional analysis. The obtained results challenge the gating model as a general regulation mechanism for plant plasma membrane aquaporins.     

Major intrinsic proteins (MIPs) in plants: a complex gene family with major impacts on plant phenotype

The ubiquitous cell membrane proteins called aquaporins are now firmly established as channel proteins that control the specific transport of water molecules across cell membranes in all living organisms. The aquaporins are thus likely to be of fundamental significance to all facets of plant growth and development affected by plant–water relations. A majority of plant aquaporins have been found to share essential structural features with the human aquaporin and exhibit water-transporting ability in various functional assays, and some have been shown experimentally to be of critical importance to plant survival. Furthermore, substantial evidence is now available from a number of plant species that shows differential gene expression of aquaporins in response to abiotic stresses such as salinity, drought, or cold and clearly establishes the aquaporins as major players in the response of plants to conditions that affect water availability. This review summarizes the function and regulation of these genes to develop a greater understanding of the response of plants to water insufficiency, and particularly, to identify tolerant genotypes of major crop species including wheat and rice and plants that are important in agroforestry. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Aquaporin Tetramer Composition Modifies the Function of Tobacco Aquaporins

Heterologous expression in yeast cells revealed that NtAQP1, a member of the so-called PIP1 aquaporin subfamily, did not display increased water transport activity in comparison with controls. Instead, an increased CO₂-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO₂-related function. Results from split YFP experiments, protein chromatography, and gel electrophoresis indicated that the proteins form heterotetramers when coexpressed in yeast. Tetramer composition had effects on transport activity as demonstrated by analysis of artificial heterotetramers with a defined proportion of NtAQP1 to NtPIP2;1. A single NtPIP2;1 aquaporin in a tetramer was sufficient to significantly increase the water permeability of the respective yeast cells. With regard to CO₂-triggered intracellular acidification, a cooperative effect was observed, where maximum rates were measured when the tetramer consisted of NtAQP1 aquaporins only. The results confirm the model of an aquaporin monomer as a functional unit for water transport and suggest that, for CO₂-related transport processes, a structure built up by the tetramer is the basis of this function.     

Changes in plasma membrane lipids, aquaporins and proton pump of broccoli roots, as an adaptation mechanism to salinity

Salinity stress is known to modify the plasma membrane lipid and protein composition of plant cells. In this work, we determined the effects of salt stress on the lipid composition of broccoli root plasma membrane vesicles and investigated how these changes could affect water transport via aquaporins. Brassica oleracea L. var. Italica plants treated with different levels of NaCl (0, 40 or 80 mM) showed significant differences in sterol and fatty acid levels. Salinity increased linoleic (18:2) and linolenic (18:3) acids and stigmasterol, but decreased palmitoleic (16:1) and oleic (18:1) acids and sitosterol. Also, the unsaturation index increased with salinity. Salinity increased the expression of aquaporins of the PIP1 and PIP2 subfamilies and the activity of the plasma membrane H⁺-ATPase. However, there was no effect of NaCl on water permeability (P_f) values of root plasma membrane vesicles, as determined by stopped-flow light scattering. The counteracting changes in lipid composition and aquaporin expression observed in NaCl-treated plants could allow to maintain the membrane permeability to water and a higher H⁺-ATPase activity, thereby helping to reduce partially the Na⁺ concentration in the cytoplasm of the cell while maintaining water uptake via cell-to-cell pathways. We propose that the modification of lipid composition could affect membrane stability and the abundance or activity of plasma membrane proteins such as aquaporins or H⁺-ATPase. This would provide a mechanism for controlling water permeability and for acclimation to salinity stress.     

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.     

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.     

The zebrafish genome encodes the largest vertebrate repertoire of functional aquaporins with dual paralogy and substrate specificities similar to mammals

Background  

Aquaporins are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. These proteins are vital for maintaining water homeostasis in living organisms. In mammals, thirteen aquaporins (AQP0-12) have been characterized, but in lower vertebrates, such as fish, the diversity, structure and substrate specificity of these membrane channel proteins are largely unknown.     

Aquaporins from pathogenic protozoan parasites: structure, function and potential for chemotherapy

Infectious diseases, caused by protozoa, such as malaria, sleeping sickness, Chagas' disease or leishmaniasis, are a global threat. The increase in the number of affected individuals and the rapid spread of drug-resistant strains call for specific novel strategies to combat human pathogenic parasites. In the search for novel drug targets, transport proteins for nutrients and metabolites of the parasite-host interface are getting into focus. The present review summarizes and discusses the currently available results on protozoan aquaporins. Various genes coding for aquaporin water and solute channels have been identified in the protozoan genomes and they are probable elements of the parasite's cell membrane. Phylogenetic analysis reveals that individual aquaporin genes are of bacterial or plant origin. So far, six protozoan aquaporins have been cloned and functionally characterized. Typically, these are bifunctional channels and pass water at intermediate to high rates as well as uncharged solutes. In the present review, amino acid compositions of the individual pore entries are compared and permeability properties are attributed to specific protein features. Furthermore, possible physiological roles in osmotic protection and metabolism are discussed. Finally, the potential of protozoan aquaporins for use as a target or entry pathway for chemotherapeutic compounds is reviewed.     

In silico study of human aquaporin AQP11 and AQP12 channels

AQP11 and AQP12 are the most distantly related paralogs of the aquaporin family in human. They share indeed a low sequence similarity with other aquaporins and exhibit a modified N‐terminal NPA signature motif. Furthermore, they have an anomalous subcellular localization. The AQP11 and AQP12 biological role remains to be fully clarified and their ability to allow transport of water is still debated. We have built accurate 3D‐models for AQP11 and AQP12 and comprehensively compared their sequence and structure to other known aquaporins. In order to investigate whether they appear compatible or not with water permeability, we especially focused on the amino acid composition and electrostatics of their channels, keeping the structure of the low‐water efficiency AQP0 as a reference system. Our analysis points out a possible alternative ar/R site and shows that these aquaporins feature unique residues at key pore‐lining positions that make the shape, composition and electrostatics of their channel peculiar. Such residues can represent pivotal hints to study and explain the AQP11 and AQP12 biological and molecular function.     

Insight into factors directing high production of eukaryotic membrane proteins; production of 13 human AQPs in Pichia pastoris

Membrane proteins are key players in all living cells. To achieve a better understanding of membrane protein function, significant amounts of purified protein are required for functional and structural analyses. Overproduction of eukaryotic membrane proteins, in particular, is thus an essential yet non-trivial task. Hence, improved understanding of factors which direct a high production of eukaryotic membrane proteins is desirable. In this study we have compared the overproduction of all human aquaporins in the eukaryotic host Pichia pastoris. We report quantitated production levels of each homologue and the extent of their membrane localization. Our results show that the protein production levels vary substantially, even between highly homologous aquaporins. A correlation between the extents of membrane insertion with protein function also emerged, with a higher extent of membrane insertion for pure water transporters compared to aquaporin family members with other substrate specificity. Nevertheless, the nucleic acid sequence of the second codon appears to play an important role in overproduction. Constructs containing guanine at the first position of this codon (being part of the mammalian Kozak sequence) are generally produced at a higher level, which is confirmed for hAQP8. In addition, mimicking the yeast consensus sequence (ATGTCT) apparently has a negative influence on the production level, as shown for hAQP1. Moreover, by mutational analysis we show that the yield of hAQP4 can be heavily improved by directing the protein folding pathway as well as stabilizing the aquaporin tetramer.     

Plant aquaporins: Roles in plant physiology

Background

Aquaporins are membrane channels that facilitate the transport of water and small neutral molecules across biological membranes of most living organisms.

Scope of review

Here, we present comprehensive insights made on plant aquaporins in recent years, pointing to their molecular and physiological specificities with respect to animal or microbial counterparts.

Major conclusions

In plants, aquaporins occur as multiple isoforms reflecting a high diversity of cellular localizations and various physiological substrates in addition to water. Of particular relevance for plants is the transport by aquaporins of dissolved gases such as carbon dioxide or metalloids such as boric or silicic acid. The mechanisms that determine the gating and subcellular localization of plant aquaporins are extensively studied. They allow aquaporin regulation in response to multiple environmental and hormonal stimuli. Thus, aquaporins play key roles in hydraulic regulation and nutrient transport in roots and leaves. They contribute to several plant growth and developmental processes such as seed germination or emergence of lateral roots.

General significance

Plants with genetically altered aquaporin functions are now tested for their ability to improve plant resistance to stresses. This article is part of a Special Issue entitled Aquaporins.     

Identification of the family of aquaporin genes and their expression in upland cotton (<Emphasis Type="Italic">Gossypium hirsutum</Emphasis>L.)

Background  

Cotton (Gossypium spp.) is produced in over 30 countries and represents the most important natural fiber in the world. One of the primary factors affecting both the quantity and quality of cotton production is water. A major facilitator of water movement through cell membranes of cotton and other plants are the aquaporin proteins. Aquaporin proteins are present as diverse forms in plants, where they function as transport systems for water and other small molecules. The plant aquaporins belong to the large major intrinsic protein (MIP) family. In higher plants, they consist of five subfamilies including plasma membrane intrinsic proteins (PIP), tonoplast intrinsic proteins (TIP), NOD26-like intrinsic proteins (NIP), small basic intrinsic proteins (SIP), and the recently discovered X intrinsic proteins (XIP). Although a great deal is known about aquaporins in plants, very little is known in cotton.