Plant Aquaporins Research Articles

New aspects of plant aquaporin regulation and specificity

meability of these channel proteins for water (Preston et al., 1992). Recent findings concerning the sensitivity of aqua- For a long time, plant physiologists, on the other hand, porins to heavy metal ions and their possible con- have not accepted the requirement of these molecules for sequences to plant physiology are discussed. the simple reason that the majority of plants do not Furthermore, results obtained from heterologous possess organs or sites where comparable tremendous expression of plant aquaporin cRNAs in Xenopus amounts of water are transported through living cells. It oocytes are related to the situation in the plant. An was assumed that the water permeability of a biomemassessment of the significance of water flux regulation brane itself is suYcient for the requirements of plant by modification of the aquaporin protein or change cellular water movement (Fettiplace and Haydon, 1980; of gene activity is given and new data concerning Cullis et al., 1996). Since the counterparts of AQP1 were the selectivity of plasma membrane aquaporins are identified in the plant plasma membrane and tonoplast, presented. this opinion is questioned. To date the existence of aquaporins in virtually all living organisms, including

Plant aquaporins: their molecular biology, biophysics and significance for plant water relations

Over the last decade, considerable advances have occurred in understanding the molecular biology and biophysics of water permeation across plant membranes and tissues. Spurred on by the rapid advances in cloning and functional characterization of a super-family of major intrinsic proteins, some of which function as aquaporins, the biophysics of transport of water and small non-electrolytes across plant membranes is being re-examined based on the proposed function of these membrane-integral proteins in their native membranes. This review focuses on a number of issues that are central to an understanding of aquaporin function: (1) the need to be able to test for water-channel activity in native membranes; (2) the implications of the observed solute/water selectivity of aquaporins; (3) the putative functional roles of aquaporins at the cell, tissue and organ levels in plants; and (4) information that can be obtained from studies of the abundance, diversity and expression patterns of aquaporins. It is clear that to answer many of the critical questions that remain concerning aquaporin function, combined studies using appropriate molecular and biophysical techniques will be required.

Aquaporin Nt-TIPa can account for the high permeability of tobacco cell vacuolar membrane to small neutral solutes.

Members of the major intrinsic protein (MIP) family, described in plants as water-selective channels (aquaporins), can also transport small neutral solutes in other organisms. In the present work, we characterize the permeability of plant vacuolar membrane (tonoplast; TP) and plasma membrane (PM) to non-electrolytes and evaluate the contribution of MIP homologues to such transport. PM and TP vesicles were purified from tobacco suspension cells by free-flow electrophoresis, and membrane permeabilities for a wide range of neutral solutes including urea, polyols of different molecular size, and amino acids were investigated by stopped-flow spectrofluorimetry. For all solutes tested, TP vesicles were found to be more permeable than their PM counterparts, with for instance urea permeabilities from influx experiments of 74.9 +/- 9.6 x 10(-6) and 1.0 +/- 0.3 x 10(-6) cm sec-1, respectively. Glycerol and urea transport in TP vesicles exhibited features of a facilitated diffusion process. This and the high channel-mediated permeability of the same TP vesicles to water suggested a common role for MIP proteins in water and solute transport. A cDNA encoding a novel tonoplast intrinsic protein (TIP) homologue named Nicotiana tabacum TIPa (Nt-TIPa) was isolated from tobacco cells. Immunodetection of Nt-TIPa in purified membrane fractions confirmed that the protein is localized in the TP. Functional expression of Nt-TIPa in Xenopus oocytes showed this protein to be permeable to water and solutes such as urea and glycerol. These features could account for the transport selectivity profile determined in purified TP vesicles. These results support the idea that plant aquaporins have a dual function in water and solute transport. Because Nt-TIPa diverges in sequence from solute permeable aquaporins characterized in other organisms, its identification also provides a novel tool for investigating the molecular determinants of aquaporin transport selectivity.

Characterization of a maize tonoplast aquaporin expressed in zones of cell division and elongation.

We studied aquaporins in maize (Zea mays), an important crop in which numerous studies on plant water relations have been carried out. A maize cDNA, ZmTIP1, was isolated by reverse transcription-coupled PCR using conserved motifs from plant aquaporins. The derived amino acid sequence of ZmTIP1 shows 76% sequence identity with the tonoplast aquaporin gamma-TIP (tonoplast intrinsic protein) from Arabidopsis. Expression of ZmTIP1 in Xenopus laevis oocytes showed that it increased the osmotic water permeability of oocytes 5-fold; this water transport was inhibited by mercuric chloride. A cross-reacting antiserum made against bean alpha-TIP was used for immunocytochemical localization of ZmTIP1. These results indicate that this and/or other aquaporins is abundantly present in the small vacuoles of meristematic cells. Northern analysis demonstrated that ZmTIP1 is expressed in all plant organs. In situ hybridization showed a high ZmTIP1 expression in meristems and zones of cell enlargement: tips of primary and lateral roots, leaf primordia, and male and female inflorescence meristems. The high ZmTIP1 expression in meristems and expanding cells suggests that ZmTIP1 is needed (a) for vacuole biogenesis and (b) to support the rapid influx of water into vacuoles during cell expansion.

Desiccation- and abscisic acid-responsive genes encoding major intrinsic proteins (MIPs) from the resurrection plant Craterostigma plantagineum.

Major intrinsic proteins (MIPs) are a family of channel proteins that are mainly represented by aquaporins in plants. These are divided into TIPs (tonoplast intrinsic proteins) and PIPs (plasma membrane intrinsic proteins) according to their subcellular localization. Homologues to PIPs and TIPs were isolated from the desiccation-tolerant resurrection plant Craterostigma plantagineum by two approaches: firstly, a cDNA library constructed from RNA of dehydrated C. plantagineum leaves was screened with an Arabidopsis thaliana Ath-PIP1b cDNA probe and, secondly, a cDNA library was screened differentially to isolate early drought-induced transcripts. According to sequence homologies the isolated cDNA clones were grouped as follows: Cp-PIPa, Cp-PIPb, Cp-PIPc and Cp-TIP. Cp-PIPa, Cp-PIPc and Cp-TIP transcript accumulation was regulated by dehydration and abscisic acid (ABA). Within the Cp-PIPa group transcripts were regulated either by drought only or by drought and ABA, indicating that ABA-dependent and -independent signal transduction pathways lead to Cp-PIPa expression. Comparison of Cp-PIPa expression in detached leaves and in whole plants suggested the involvement of a signal transmitted in the whole plant in response to drought. Cp-PIPb transcript levels were constitutive in all organs tested. Antibodies raised against a Cp-PIPA protein recognized a polypeptide with an apparent molecular mass of 28 kDa. Using these antibodies it was shown that both Cp-PIPA and Cp-PIPB proteins were localized to the plasma membrane. The role of different members of the MIP group in the dehydration response is discussed.

Mercury-induced Conformational Changes and Identification of Conserved Surface Loops in Plasma Membrane Aquaporins from Higher Plants: TOPOLOGY OF PMIP31 FROM BETA VULGARIS L.

Aquaporins are integral membrane proteins occurring in mammals, plants, and microorganisms, which serve as channels that permit the bidirectional passage of water through cellular membranes. Higher plants contain abundant levels of aquaporins in both the tonoplast and plasma membrane. Aquaporins contain six transmembrane segments with three surface loops located at the apoplastic face of the membrane and two loops at the cytosolic side. In this study, we probed the topology of plasma membrane aquaporins to determine the effects of divalent cations on aquaporin conformation, and to identify structural features that distinguish plasma membrane intrinsic proteins from tonoplast intrinsic proteins. Plasma membrane vesicles from storage tissue of Beta vulgaris L. were subjected to limited proteolysis, and proteolytic fragmentation patterns were detected using affinity-purified antibodies recognizing aquaporins of 31-kDa. In its native membrane-associated state, the 31-aquaporin band, PMIP31, was refractory to proteolysis by trypsin. However, mercuric compounds specifically induced a conformational change resulting in the exposure of a proteolytic cleavage site and formation of a unique 22-kDa proteolytic fragment (p22). N-terminal sequence analysis of p22 established its identity as an aquaporin-derived fragment. Topological studies using sealed right-side-out plasma membrane vesicles established that the proteolytic cleavage site is located at surface loop C, the second apoplastic loop, immediately preceding the sequence Gly-Gly-Gly-Ala-Asn. The Gly-Gly-Gly-Ala-Asn-X-X-X-X-Gly-Tyr motif of loop C and a 14 amino acid motif in apoplastic loop E, Thr-Gly-Ile/Thr-Asn-Pro-Ala-Arg-Ser-Leu/Phe-Gly-Ala-Ala-Ile/Val-Ile/ Val-Phe/Tyr-Asn are completely conserved in all known higher plant aquaporins of plasma membrane origin and are not present in any of the known tonoplast intrinsic proteins. These results demonstrate that the two highly conserved plasma membrane intrinsic protein surface loops are structural features that clearly distinguish plasma membrane from tonoplast aquaporins.

Molecular Cloning of a New Aquaporin from Rat Pancreas and Liver

A new water channel (aquaporin-8, gene symbol AQP8) was isolated from rat pancreas and liver by homology cloning. Ribonuclease protection assay showed intense expression of the gene in pancreas and liver, less intense in colon and salivary gland, and negligible in other organs. The full-length cDNA was obtained by ligation of approximately 1.4-kilobase (kb) cDNA isolated from the rat liver cDNA library to approximately 0.5 kb of the 5'-end fragment obtained by the rapid amplification of cDNA ends method. A major transcript of approximately 1.45 kb was demonstrated in liver and colon by Northern blot analysis. Expression of the cRNA in Xenopus oocytes markedly enhanced osmotic water permeability in a mercury-sensitive manner, indicating a water channel function of this molecule. The open reading frame encoded a 263-amino acid protein with a predicted molecular size of 28 kDa. Hydropathy analysis represented six membrane-spanning domains and five connecting loops containing two sites of NPA motif as preserved in other aquaporins. Unlike other mammalian aquaporins, AQP8 has an unusual structure with a long N terminus and a short C terminus, which are found in plant aquaporin, gamma-tonoplast intrinsic protein. By in situ hybridization, AQP8 mRNA expression was assumed in hepatocytes, acinal cells of pancreas and salivary gland, and absorptive colonic epithelial cells. The physiological role(s) of AQP8 remain to be elucidated.

The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group.

Aquaporins, proteins that enhance the permeability of biological membranes to water, are widely distributed in living organisms. They are 26- to 29-kD proteins that belong to the major intrinsic protein (MIP) family of channels. By searching the Arabidopsis thaliana expressed sequence tag database and by using the polymerase chain reaction with oligonucleotides to conserved plant aquaporin domains, we identified 23 expressed Arabidopsis MIP genes. Eight of these had been previously identified as active aquaporins, and two additional ones are now reported to have water-transport activity in Xenopus laevis oocytes. One of these is highly expressed in suspension-cultured cells. On a dendrogram these 23 MIP sequences cluster into three groups: the first group has 11 members and contains the plasma membrane aquaporins, the second group also has 11 members and contains the tonoplast aquaporins, and the third group has only a single member. This MIP protein, provisionally called At-NLM1, is most closely related to the Gm-NOD26 protein that is found in the bacteroid membranes of soybean (Glycine max L.) nodules; At-NLM1 is an active aquaporin when expressed in oocytes. With a semiquantitative slot-blot analysis technique, we determined the expression levels of 22 MIP genes in the various organs. The quantitative polymerase chain reaction was used to determine the effects of various stress treatments on the expression of NLM1.

Expression of an Arabidopsis plasma membrane aquaporin in Dictyostelium results in hypoosmotic sensitivity and developmental abnormalities.

The rd28 gene of Arabidopsis thaliana encodes a water channel protein, or aquaporin, of the plasma membrane. A construct in which transcription of the rd28 cDNA is controlled by the Dictyostelium actin15 promoter was transformed into Dictyostelium discoideum cells. Transformants contained RD28 protein in their plasma membranes. When shifted to a low-osmotic-strength buffer, cells expressing rd28 swelled rapidly and burst, indicating that the plant aquaporin allowed rapid water entry in the amoebae. The rate of osmotic lysis was a function of the osmotic pressure of the buffer. We also selected transformants in which the expression of the rd28 cDNA is driven by the promoter of the prespore cotB gene. These transformants accumulated rd28 mRNA uniquely in prespore cells. In low-osmotic-strength buffer, the cotB::rd28 cells aggregated and formed normally proportioned slugs but failed to form normal fruiting bodies. The number of spores was reduced 20-fold, and the stalks of the fruiting bodies were abnormally short. The consequences of expressing RD28 in prespore cells could be partially overcome by increasing the osmolarity of the medium. Under these conditions, the cotB::rd28 cells formed fruiting bodies of more normal appearance, and the number of viable spores increased slightly. Because prespore cells have to shrink and dehydrate to form spores, it was not unexpected that expression of an aquaporin would disrupt this process, but it was surprising to find that stalk differentiation was also affected by expression of rd28 in prespore cells. It appears that osmotic stress on prespore cells alters their ability to signal terminal differentiation in prestalk cells. The results provide independent confirmation that plant aquaporins can function in the cells of other organisms, and that D. discoideum can be used to study the properties of these water channels.

AQUAPORINS AND WATER PERMEABILITY OF PLANT MEMBRANES.

The mechanisms of plant membrane water permeability have remained elusive until the recent discovery in both vacuolar and plasma membranes of a class of water channel proteins named aquaporins. Similar to their animal counterparts, plant aquaporins have six membrane-spanning domains and belong to the MIP superfamily of transmembrane channel proteins. Their very high efficiency and selectivity in transporting water molecules have been mostly characterized using heterologous expression in Xenopus oocytes. However, techniques set up to measure the osmotic water permeability of plant membranes such as transcellular osmosis, pressure probe measurements, or stopped-flow spectrophotometry are now being used to analyze the function of plant aquaporins in their native membranes. Multiple mechanisms, at the transcriptional and posttranslational levels, control the expression and activity of the numerous aquaporin isoforms found in plants. These studies suggest a general role for aquaporins in regulating transmembrane water transport during the growth, development, and stress responses of plants. Future research will investigate the integrated function of aquaporins in long-distance water transport and cellular osmoregulation.

Function and regulation of seed aquaporins

The discovery of water channel proteins named aquaporins has shed new light on the molecular mechanisms of transmembrane water transport in higher plants. As with their animal counterparts, plant aquaporins belong to the large MIP family of transmembrane channels. An increasing number of aquaporins is now being identified on both the vacuolar and plasma membranes of plant cells, but their integrated function remains unclear. Aquaporin α-TIP is specifically expressed in the membrane of protein storage vacuoles in seeds of many plant species. α-TIP was previously shown to undergo phosphorylation in bean seeds. The functional significance of this process was further investigated after heterologous expression of the protein in Xenopus oocytes. Using site-directed mutagenesis of α-TIP and in vitro and in vivo phosphorylation by animal cAMP-dependent protein kinase, it is shown that, in oocytes, direct phosphorylation of α-TIP occurs at three distinct sites and stimulates its water channel activity. In addition to aquaporin phosphorylation, other mechanisms that target aquaporin function are used by living cells to regulate their membrane water permeability. These are the fine control of aquaporin gene expression and, in animal cells only, the regulated trafficking of water channel-containing vesicles. The present work and studies by others on the phosphorylation of nodulin-26, an ion channel protein homologous to α-TIP, provide novel insights into the mechanisms of plant membrane protein regulation. These studies might help identifying and characterizing novel membrane-bound protein kinases and phosphatases. Finally, an integrated function for seed vacuolar aquaporins is discussed. During germination, the rehydration of seed cells, the drastic changes in vacuole morphology, the breakdown and the mobilization of storage products from the vacuole may create osmotic perturbations in the cytoplasm. The fine tuning of TIP aquaporin activity may help control the kinetics and amplitude of osmotic water flows across the tonoplast to achieve proper cytoplasm osmoregulation and control of vacuolar volume.