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We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

We demonstrate that membranes consisting of certain triblock-copolymers were tight for CO₂. Using a novel approach, we provide evidence for aquaporin facilitated CO₂ diffusion. Plant aquaporins obtained from heterologous expression were inserted into triblock copolymer membranes. These were employed to separate a chamber with a solution maintaining high CO₂ concentrations from one with depleted CO₂ concentrations. CO₂ diffusion was detected by measuring the pH change resulting from membrane CO₂ diffusion from one chamber to the other. An up to 21 fold increase in diffusion rate was determined. Besides the supply of this proof of principle, we could provide additional arguments in favour of protein facilitated CO₂ diffusion to the vivid on-going debate about the principles of membrane gas diffusion in living cells.

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 CO(2)-triggered intracellular acidification was observed. NtPIP2;1, which belongs to the PIP2 subfamily of plant aquaporins, behaved as a true aquaporin but lacked a CO(2)-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 CO(2)-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 CO(2)-related transport processes, a structure built up by the tetramer is the basis of this function.

Photosynthesis is often limited by the rate of CO(2) diffusion from the atmosphere to the chloroplast. The primary resistances for CO(2) diffusion are thought to be at the stomata and at photosynthesizing cells via a combination resulting from resistances of aqueous solution as well as the plasma membrane and both outer and inner chloroplast membranes. In contrast with stomatal resistance, the resistance of biological membranes to gas transport is not widely recognized as a limiting factor for metabolic function. We show that the tobacco (Nicotiana tabacum) plasma membrane and inner chloroplast membranes contain the aquaporin Nt AQP1. RNA interference-mediated decreases in Nt AQP1 expression lowered the CO(2) permeability of the inner chloroplast membrane. In vivo data show that the reduced amount of Nt AQP1 caused a 20% change in CO(2) conductance within leaves. Our discovery of CO(2) aquaporin function in the chloroplast membrane opens new opportunities for mechanistic examination of leaf internal CO(2) conductance regulation.

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.

To study the molecular bases of water transport in olive we characterized cDNAs from Olea europaea cv "Leccino" related to the aquaporin (AQP) gene family. A phylogenetic analysis of the corresponding polypeptides confirmed that they were part of water channel proteins localized in the plasma membrane and in the tonoplast. The full-length sequences were obtained by RACE-PCR and were named OePIP1.1, OePIP2.1 and OeTIP1.1. The OePIP2.1 and OeTIP1.1 encode functional water channel proteins, as indicated by expression assays in Xenopus laevis oocytes. OePIP1.1 and OePIP2.1 expression levels are high in roots and twigs and low in leaves. The highest hybridization signal of OeTIP1.1 was detected in twigs, while in roots and leaves the expression was low. To investigate the effect of abiotic stress on the transcript level of olive AQP genes, olive trees were subjected to drought treatment and the expression levels of the genes were measured by Northern-blot analysis. The transcript levels of each gene diminished strongly in plants submitted to drought stress, when soil moisture, twig water potential and twig hydraulic conductivity progressively decreased. The downregulation of AQP genes may result in reduced membrane water permeability and may limit loss of cellular water during periods of water stress. A possible role for AQPs on shoot embolism repair is discussed.

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.

The formation of ectomycorrhizas, a tight association between fine roots of trees and certain soil fungi, improves plant nutrition in a nutrient-limited environment and may increase plant survival under water stress conditions. To investigate the impact of mycorrhiza formation on plant water uptake, seven genes coding for putative water channel proteins (aquaporins) were isolated from a poplar ectomycorrhizal cDNA library. Four out of the seven genes were preferentially expressed in roots. Mycorrhiza formation resulted in an increased transcript level for three of these genes, two of which are the most prominently expressed aquaporins in roots. When expressed in Xenopus laevis oocytes, the corresponding proteins of both genes were able to transport water. Together, these data indicate, that the water transport capacity of the plasma membrane of root cells is strongly increased in mycorrhized plants. Measurements of the hydraulic conductance of intact root systems revealed an increased water transport capacity of mycorrhized poplar roots. These data, however, also indicate that changes in the properties of the plasma membrane as well as those of the apoplast are responsible for the increased root hydraulic conductance in ectomycorrhizal symbiosis.

Several processes during sexual reproduction in higher plants involve the movement of water between cells or tissues, such as occurs during dehiscence of the anther and hydration of the pollen grain after it is deposited on a stigma. To get more insight in these processes, a set of putative aquaporins was cloned and it was found that at least 15 are expressed in reproductive organs, which indicates that the control of water flow is important for reproduction. Functional studies in Xenopus laevis oocytes using two of the cDNAs showed that NtPIP2;1 is an efficient aquaporin, whereas NtPIP1;1 is not. Expression studies on RNA and protein levels showed that PIP1 and PIP2 genes are differently expressed in reproductive organs: PIP1 RNA accumulates in the stigma, and PIP1 and PIP2 RNA can be detected in most tissues of the anther.

Aquaporins, found in virtually all living organisms, are membrane-intrinsic proteins that form water-permeable complexes. The mammalian aquaporin AQP1 has also shown CO2 permeability when expressed heterologously in Xenopus oocytes, although whether this is a biochemical curiosity or of physiological significance is a matter of debate. Here we report that, in the same expression system, a CO2 permeability comparable to that of the human AQP1 is observed for the tobacco plasma membrane aquaporin NtAQP1. NtAQP1 facilitates CO2 membrane transport in the homologous plant system at the cellular level, and has a significant function in photosynthesis and in stomatal opening. NtAQP1 overexpression heightens membrane permeability for CO2 and water, and increases leaf growth. The results indicate that NtAQP1-related CO2 permeability is of physiological importance under conditions where the CO2 gradient across a membrane is small, as is the case between the atmosphere and the inside of a plant cell.

Leaf-moving organs, remarkable for the rhythmic volume changes of their motor cells, served as a model system in which to study the regulation of membrane water fluxes. Two plasma membrane intrinsic protein homolog genes, SsAQP1 and SsAQP2, were cloned from these organs and characterized as aquaporins in Xenopus laevis oocytes. Osmotic water permeability (P(f)) was 10 times higher in SsAQP2-expressing oocytes than in SsAQP1-expressing oocytes. SsAQP1 was found to be glycerol permeable, and SsAQP2 was inhibited by 0.5 mM HgCl(2) and by 1 mM phloretin. The aquaporin mRNA levels differed in their spatial distribution in the leaf and were regulated diurnally in phase with leaflet movements. Additionally, SsAQP2 transcription was under circadian control. The P(f) of motor cell protoplasts was regulated diurnally as well: the morning and/or evening P(f) increases were inhibited by 50 microM HgCl(2), by 2 mM cycloheximide, and by 250 microM phloretin to the noon P(f) level. Our results link SsAQP2 to the physiological function of rhythmic cell volume changes.

This paper gives a summary of a project to characterize a tobacco aquaporin. The cDNA and gene, including the 5' upstream region, for the tobacco aquaporin NtAQP1 has been isolated and the encoded protein characterized. The significance of promoter regions for an abscisic acid- and gibberellic acid-induced gene expression could be restricted to a region between -1450 and -1112 upstream of the transcription start point by transient transformation of a bicistronic vector into tobacco protoplasts. NtAQP1 expression in tobacco plants was found to be elevated in flowers, stems and roots. In roots, the protein was detected close to xylem vessels in pitch-like structures. Studies with a NtAQP1-GFP fusion indicated a plasma membrane location. For a functional analysis, the cDNA was expressed in Xenopus oocytes. NtAQP1 was found to be a heavy metal-insensitive aquaporin with additional permeability for glycerol. Mutation of a threonine at position 233 to a cysteine transformed NtAQP1 into a heavy metal-sensitive aquaporin.

A subtractive suppression hybridization technique was used to identify genes that were induced during early phases of the interaction between Cuscuta reflexa, a phanerogamic plant parasite and the incompatible host tomato (Lycopersicon esculentum Mill.). One of the identified genes encodes a new aquaporin (LeAqp2) from tomato. Its function was concluded from the swelling kinetics of LeAqp2-expressing Xenopus laevis oocytes under hypo-osmotic conditions. It was shown that, 6 h after attachment of the plant parasite, the corresponding mRNA accumulated in cells at and adjacent to the attachment site of Cuscuta, while artificial wounding did not modify steady-state LeAqp2- RNA levels. Expression of a close homologue named TRAMP (tomato-ripening-associated protein) was not affected by the plant-plant interaction. Levels of indole-3-acetic acid (IAA) in tomato tissue after infection by Cuscuta have been found to increase at a similar stage of infection. In contrast to the different behavior with respect to infection, IAA induced both LeAqp2 and TRAMP expression. The observed pattern of LeAqp2 expression during the interaction at a stage where cell elongation occurs together with the water-channel activity in the heterologous expression system suggest a function for LeAqp2 during the tomato-Cuscuta interaction.