WCPSs (AND OTHER MIPs) IN UNICELLULAR ORGANISMS After the discovery of the first WCPS, called AQPZ, in E. coli44 a widespread existence of aquaporins among bacteria was suggested, with roles in measurement of osmotic stress, cell growth and division, or desiccation44, 94. These suggestions have not been confirmed. Although WCPSs have been identified in somebacteria and other unicellular organisms ranging from archaea to eukaryotes, aquaporins are not present in all species of microorganisms. When all sequenced microbial genomes were considered, 153 microbial species apparently devoid of aquaporins were identified, whereas putative aquaporin-encoding genes have been found in only 71 species; in addition, aquaporins seem to be more abundant in eukaryotes (67% of 33 species) than in prokaryotes (26% of 193 species) and are found in only three of the 22 sequenced archaea. Among the fungi, no aquaporins have been reported in the basidiomycetes, whereas they are present in many lower fungi47. The absence of WCPSs in many microorganisms indicates that aquaporins are probably not required for processes that are universally important for microbial survival, including cell growth and division. No clear common phenotypes caused by inactivation of aquaporins are apparent47. The lipid counterpart of the membrane is probably sufficiently permeable to Water, so that, given the high surface area to volume ratio of the microbial cell, a significant role of AQPZ in Water uptake in growth appears unlikely95.
Functional analyses are needed to confirm that all the currently identified putative aquaporins in microorganisms have a function in Watertransport. To date, only the Watertransport functions of AQPZ, of M. marburgensisAQPM and Saccharomyces cerevisiaeAQY1 have been confirmed experimentally47.
The involvement of AQPZ and other microbial aquaporins in osmoregulation is discussed by Kayingo et al.48. Mutants of AQPZ have been used to study the structural basis of aquaporininhibition by mercury96. It was concluded that inhibition of AQPs by mercury is due to the blockage of the pore by a steric mechanism and not by a conformational change of the protein.
On the other hand microorganisms have a high number of GlpFs [reviewed in48]. In fact GlpFs account for the majority of MIPs in microorganisms47. The E. coli GlpF was first identified in 1960s and its permeability to glycerol, urea and glycine was established by 1980 [Heller et al. 1980, cited in71]. However, it was not recognized as a relative of WCPSs until the 1990s, probably because its Water permeability is much lower than that of AQPs71. E. coli GlpF shares a high amino-acidsequence similarity with AQPZ; however, it conducts Water at a rate that is only about one sixth that of AQPZ [Borgnia and Agre, 2001, cited in71].
The GlpF structure67, 68 revealed the aquaporin fold with six membrane-spanning helices and two half helices forming a right-handed bundle surrounding an aqueouschannel (pore), so that GlpF was called the “fraternal twin” of AQP197 The constrictionsite in GlpF (larger than in AQP1 or AQPZ), has a diameter of 3.14 Å, which is large enough to accomodate a glycerol molecule67, and is also more hydrophobic than that in the pore of AQP1. A mutant GlpF, W24F/F200T, reconstituted in liposomes, showed reduced glycerol efflux rates while the Water efflux rate increased68 demonstrating the shifting of GlpF channel properties towards that of aquaporin98.
In regard with WCPSs in archaea the DNA sequence of such a protein, called AQPM, a candidate aquaporin or aquaglyceroporin, has been recognized in the genome of M. marburgensis, a methanogenic thermophilic archaeon99. AQPM was expressed in E. coli, then purified and reconstituted into proteoliposomes, where AQPM behaved like a moderate mercury sensitive Waterchannel and a very poor glyceroltransporter45.
As the MIP superfamily was believed to date back 2.5–3 billion years in evolutionary time57, recognition of an aquaporin in an archaeon suggested an even earlier origin, although it is possible that the gene was transferred horizontally from other microorganisms [Salzberg et al. 2001, cited in45]. From phylogenetic analyses it was suggested45 that eukaryotic members of the MIP family evolved from two basal lineages: AQPZ-like Waterchannels and GlpF-like glycerol facilitators. These divergent lineages may have originated from an AQPM-like sequence, which appears to be intermediate in sequence between the Water-selective aquaporins and the aquaglyceroporins [Zardoya et al., 2002, cited in54].
The physiologicalrole of aquaporins in archea is not known. It was suggested45 that in some way it should be related with the ability of archaea to withstand exceptional challenges in maintaining Water balance as they thrive in extreme environments including saturated salt solutions, extreme pH and temperatures up to 130°C.
The number of WCPSs (aquaporins plus aquaglyceroporins) in yeasts can range from one to five. Fungal aquaporins have been studied inS. cerevisiae and in C. albicans. There are two aquaporins in S. cerevisiae, called AQY1 and AQY2, and only one in C. albicans. AQY1 and AQY2 are highly similar (88% identical), indicating a recent gene duplication; however, the expression of the two aquaporin genes (AQY1and AQY2) is regulated differently, indicating functional specialization100. The inactivation of AQY1 and AQY2 in S. cerevisiae and deletion of the single aquaporin gene in C. albicans have no conspicuous effects on growth under a variety of conditions (47 and refs. therein). In addition, both S. cerevisiae and C. albicansaquaporin-deletion strains are more resistant to rapid changes in osmolarity compared with wild type. Consequently, a role for aquaporins in microbial osmoregulation (turgor regulation or osmoadaptation) seems improbable, because the Waterchannel activity would aggravate osmotic stress-induced problems rather than counteract them (47, 95).
WCPSs in microorganisms might play a role in Watertransport during natural dehydration processes, for example, spore formation: the Water content of spores is aproximately half of that of vegetative cells100. Such a role has been demonstrated for AQY1 in the formation ofS. cerevisiae spores101. Mutants lacking AQY1show decreased spore viability and this was related to events occuring during spore formation rather than during spore maintenance or germination102. However, subsequent studies102 indicated that S. cerevisiae wine strains lacking AQY1 did not show a decrease in spore fitness or enological aptitude under stressful conditions, limited nitrogen, or increased temperature (heat stress). The physiologicalrole of AQY2 is less clear than for AQY1 [see100 for discussions].
A role for microbial aquaporins in sustaining low-temperature Water permeability, and in this way providing protection of cells against freeze-thaw stress, has been suggested by Tanghe et al. (47, 103). Such a protective effect of aquaporin overexpression has been found in S. cerevisiae, C. albicans, and S. pombe (47 and refs. therein). Some conditions in which WCPSs enhance microbial tolerance against rapid freezing and thus the presence of aquaporins might be advantageous for survival include the following: the liberation and subsequent freeze-thawing of microorganisms by the activities of warm-blooded organisms in frozen environments (breathing, sneezing, flying, stepping and running on frozen ground, contact with trees and other obstacles, foraging on frozen plants, the excretion of feces and urine), large-scale air dispersal of microorganisms associated with dust particles in clouds that can travel between continents, or the “freezing rain”47.
Yeasts have a range of genes that encode aquaglyceroporins, those from S. cerevisiae (Fps1 and Yfl054) being best studied100. Fps1 was in fact one of the first WCPSs discovered104. As discussed in an excellent review by Hohmann105, Fps1 plays a role in the osmoregulation of S. cerevisiae, controlling the intracellular level of glycerol, the compatible osmolyte of proliferating yeastscells.
Fps1-like aquaglyceroporins have been found in other yeasts and a comparison of their structure is discussed by Petterson et al.100. Briefly, the predicted proteins differ in length by as much as 216 residues, as there are several alterations in the NPAmotifs.
S. cerevisiae Yfl054 and Yfl054-like aquaglyceroporins are characterized by an approx. 350 amino-acid long N-terminal extension and an approx. 50 amino-acid long C-terminal extension. The physiologicalrole of these aquaglyceroporins is currently unknown100.
In conclusion, although WCPSs have been found in bacteria, archaea, and unicellular eukaryotes, their absence in many microorganisms appears to indicate that rather than fulfilling a broad role such as osmoregulation, instead WCPSs are involved in specific processes (such as sporulation) or improve freeze tolerance under rapid-freezing conditions; this WCPS function might be important for survival at low temperatures. Future studies are necessary to evaluate other possible physiologicalroles of WCPSs in microorganisms47, 100.
WCPSs in protozoa have not only physiological importance, but also relevance for tropical parasitic diseases. A role in sporulation and in spore germination has been suggested for the putative Waterchannel WacA in an amoeba, Dictyostelium discoideum106. The role of Waterchannels in the developmental process of D. discoideum was further investigated by Mitra et al.107, who identified another WCPS called AQPA and sugessted that it is essential to maintain spore dormancy perhaps through the regulation of Water flow.
An AQP gene from Amoeba proteus was recently cloned108. The protein, called ApAQP is a specific Waterchannel present in the membrane of the contractile vacuole (CV) and was implicated in the Watertransfer across the CV membrane. In freshWaterprotozoaWater enters the cell along the osmotic gradient across the plasma membrane since the osmolality of the cytoplasm is higher than that of the environment. The CV periodically repeats a cycle of slow expansion to fill with the fluid from the cytoplasm (diastole) and quick contraction to release the fluid to the cell exterior (systole) [Patterson, 1980, cited in108].
WCPSs from pathogenic protozoan parasites are presented in excellent reviews by Beitz109, 110. Among these parasites are the organisms of the phylum Apicomplexa: Plasmodium species (the causative agents of malaria), Toxoplasma gondii (toxoplasmosis), as well as those of the order Kinetoplastida: Trypanosoma brucei (sleeping sickness), Trypanosoma cruzi (Chagas'disease) andLeishmania species (leishmaniasis). The majority of the Apicomplexa have a single aquaglyceroporin gene, while up to five such genes have been identified in the genomes of the Kinetoplastida. The proteins encoded by these genes appear to be mostly aquaglyceroporins. They have been proposed to playphysiologicalroles in the protection of the parasites from osmotic stress during kidney passages or during transmission between humanand insect as well as in glycerol uptake as a precursor for membranelipid biosynthesis110.
On the other hand the protozoan WCPSs appear to be potentially important for use as a target or entry pathway for chemotherapeutic compounds109.
Inspection of the completed genomes of protozoa provided a major surprise. The Apicomplexan Cryptosporidium parvum does not have any WCPS gene. This is the first identification of a eukaryotic organism that totally lacks WCPSs109.
WCPSs (AND OTHER MIPs) IN PLANTS Since the first identifications of WCPSs in plants42, 43 tremendous progress in the knowledge of plant WCPSs has been achieved, regarding their occurrence, structure, permeability characteristics, regulation, and their physiologicalroles. In addition to WCPSs, plants have MIPs which transport other substances. Plant MIPs are more abundant and show greater diversity than those in bacteria or animals, as MIPs are required for multiple functions (111-117): (a) plants can mediate a large flow of transpiration: soil Water is absorbed by the root, moves radially through living cells to reach the vascular tissues (xylem vessels), by which is transported in shoots. Water is eventually lost in the atmosphere by transpiration, through the stomatal pores of the leaves (on a warm, dry day, the leaf may exchange the equivalent of all its Water content in 20–60 min; (b) plants have to achieve a three-dimensional control of Water exchange in living tissues; (c) this control has to be exerted during all stages of plant growth and development and has to respond to various environmental conditions; (d) plant cells are highly vacuolated and this requires tightly coordinated control of Water and solute transport across the plasma membrane and the intracellular membranes. WCPS-mediated Watertransport seems also to be crucial during leaf and petal movements, reproduction, cell elongation, and seed germination. A role for intracellular WCPSs in plant cell osmoregulation has been proposed (113, 114, 117). In addition, a role of a tobacco aquaporin in CO2 uptake during photosynthesis has been documented90.
It is thus understandable why a high abundance and multitude of MIPs have been identified in plants and it seems that all plants have such proteins. A remarkable multiplicity of MIP isoforms has been identified by genome sequencing: 35 in Arabidopsis thaliana, 33 in rice, 36 in maize116. The plant MIPs are found both in plasma membranes and in intracellular membranes and can be subdivided in four groups (or subfamilies), which to some extent correspond to distinct sub-cellular localizations (113, 115, 116): (a) the tonoplast intrinsicproteins (TIPs) abundantly expressed in the vacuolar membranes; there are various isoforms of TIPs: alpha (seed), gamma (root), and Water-stress induced (Wsi)117; (b) the plasma membraneintrinsicproteins (PIPs), located in plasma membranes, can be subdivided into two phylogenetic subgroups PIP1 and PIP2; (c) the Noduline 26–like intrinsicmembraneproteins (NIPs), that is AQPs that are closed omologues of Gm Nod26, an abundant AQP in the peribacteroid membrane of the symbiotic nitrogen–fixing nodules formed after infection of soybean by Rhizobiaceaebacteria118; (d) the small basic intrinsicproteins (SIPs), first uncovered from genome sequence analysis119, form a class of 1–3 divergent aquaporin homologues and are located in the ER (endoplasmic reticulum) membrane120.
Novel types of MIPs have been recently described: a homologue of the bacterial GlpF, acquired by the moss Physcomitrella patens by horizontal gene transfer121, and a fifth class of plant MIPs, which are closely related but yet clearly distinct from PIPs, found in the moss and poplars122.
TIPs promote the transport of Water and small uncharged molecules across the vacuolar membrane (tonoplast). Some TIPs are highly selective AQPs, ensuring a 100-fold higher Water permeability of this membrane compared to plasma membrane122; such TIPs may permit rapid osmotic equilibration between the cytosol and the vacuolar lumen, buffering the osmotic fluctuations of the cytosol, and regulating cell turgor (113, 114, 117). Other TIPs are permeable to Water, urea and glycerol123 and such WCPSs may play a role in equilibrating urea concentrations between the vacuole and the cytoplasm. In addition, the participation of TIPs to transport ammonium (NH) and its gaseous conjugated base (NH3, ammonia) from the cytosol into the vacuole was suggested. NH/NH3 are additional N sources and primary substrates for the synthesis of aminoacids. Consequently, TIPs appear to be involved not only in osmoregulation but may be linked to important metabolic pathways like the urea cycle or aminoacid synthesis113.
PIPs, which form the largest plant MIP subfamily, are the centralpathways for transcellular and intracellular Watertransport (111-117). Although the aminoacid residues at the selectivity filters are similar in PIP1 and PIP2 their permeability and cellular functions are different113. PIP1s could be transporters for small solutes and gases (CO2) in leaves and they need to be activated in roots in order to function as Waterchannels. PIP2s seem to be more efficient Waterchannels than PIP1s, and may provide a major pathway for cellular Watertransport in roots, leaves, reproductive organs and seeds; in addition, CO2transport mediated by PIP2s was proposed (113 and refs. therein).
NIPs are permeable to Water, glycerol, formamide and possibly to gaseous NH3. NIPs are also present in nonleguminous plants, where they have been localized in plasma and intracellular membranes116. NIPs can also be subdivided in two subgroups116. Members of the first subgroup (NIP I) are very comparable to the Gm Nod26 archetype and behave like aquaglyceroporins118. Solute transport mediated by NIPs may play a role in osmoregulation of the peribacteroid space. Members of the NIP II subgroup have a predicted pore of higher size than in the NIP I subgroup and, as a consequence, are permeable to larger solutes such as urea. However, they have a reduced Water permeability for unknown reasons. Recently, very original functions of members of the NIP II subgroup were identified: the uptake of boron by roots124 and the silicon uptake and transport throughout the plant125.
The SIPs subfamily is represented by three members in Arabidopsis (SIP1;1, SIP1;2, and SIP2;1) and in maize and by two members in rice. SIPs have several structural characteristics [reviewed in126]. (a) The first NPAmotifs are changed to NPT, NPC and NPL in SIP1;1, SIP1;2 and SIP2;1, respectively. (b) The N-end is shorter than in other plant MIPs and it is posible that the N-end is related to the intracellular destination. (c) SIPs are relatively rich in basic residues (such as lysine). (d) The loop C between TMD3 and TMD4 is shorter (14–19 residues) than that of PIPs and TIPs (22–26 residues) and this might affect the tertiary structure of SIPs.
All SIPs have been demonstrated to be localized in the ER membrane, although it is not clear which is the ER retention signal [reviewed in126]. Characteristic expression patterns of each SIP has been found; for example SIP1;1 was detected in high amounts in young roots and flower buds, while SIP2;1 was accumulated in young roots and open flowers126. It was suggested that each SIP may playcell-specific roles. However, such roles are not clear. SIP1;1 and SIP2;1 have been demonstrated to have Waterchannel activity, while the transport substrate for SIP2;1 remains to be determined. It is not clear whether SIPs have a role in the specific functions of the ER or in maintaining the tubular/Reticular/sheet shapes of the ER. The nearest homologues of SIPs in mammalians are AQP11 and AQP12, as pointed by Ishibashi49. Further studies are needed to define the physiologicalroles of SIPs in ER membrane.
There are multiple mechanisms by which plant WCPS activity is changed and regulated. two aspects may be considered: a direct and probably rapid control of the channel activity by gating and a more complex regulation, including changes in gene expression, in trafficking of MIPs, in various physiological conditions or under environmental stresses (92, 113–117, 127). Factors affecting directly the gating include the following: phosphorylation, heterotetramerization, pH, cations, pressure, solute gradients, temperature. In addition, the permeability of WCPSs is influenced by hormones, nutrient stress, plant hormones, attack by pathogens. The phosphorylation sites are located in the N-terminal and C-terminal segments and also in loop B. Calcium dependent protein kinases are involved in phosphorylation that results in the poreopening112, 116. Cytosolic proteins decrease the Water permeability of PIPs (and TIPs). A coordinated inhibition of PIPs and, as a consequence, a general block of root Watertransport during anoxic stress (resulting from soil flooding) was attributed to closure of the channel after cellacidosis128.
In addition to the rapid inhibition of root Water permeability by gating (closure of Waterchannels induced by the acidcytosolic pH) a general downregulation of PIP and TIP genes occurs in response to hypoxia. There are many other changes of MIPs in response to a variable environment. Light116 induces the diurnal regulation of the abundance of TIP in guard cells (controlling the stomatal aperture), or of a PIP2 in the pulvinus, the motor organ controlling the movement of leaves and leaflets in the Mimosaceae. Diurnal variations of root Watertransport (with a twofold to threefold increase during the day) have been observed in many plant species and are matched or slightly preceded by an increase in the abundance of PIP1 and PIP2 transcripts. However, the abundance of the PIP proteins in roots shows a more complex diurnal variation profile, suggesting a role for posttranscriptional regulation129.
The regulation of MIPs under the effects of Water and nutrient stress is a complex issue, because the expression of different MIP genes may be stimulated, reduced, or unchanged under abiotic stress117. In roots of most plant species investigated, drought or salt stresses result in a marked decrease of Water permeability, probably due to the downregulation of Waterchannel activity (downregulation of most WCPStranscripts) during the day; this early response may provide a hydraulic signal to the leaf to trigger stomatal closure, whereas during the night, it may avoid a backflow of Water to the drying soil116. A recovery toward initial transcript abundance occurs over longer term stresses [reviewed in (116-117)].
Adaptation to salinity implies two phases. In the first phase (over the first 24 h of stress) a coordinated transcriptional downregulation and subcellular relocalization of both PIPs and TIPs in roots occur (with sometranscripts downregulated during the first 15–60 min of salt exposure). After 7 days the expression level of transcripts recovered, and even became higher than in plants grown in standard conditions [discussed in (116-117)].
Chilling of plants (i.e., exposure to 4–8°C) induce complex responses involving WCPSs: a marked decrease in root Watertransport (associated with a twofold to fourfold decrease in the abundance of most PIP transcripts), closure of petals (for instance in tulip), associated with decreased phosphorylation of the putative WCPS [reviewed in116].
Biotic interactions of plants with soil microorganisms are important in plant Water relations and tolerance to environmental stress. NIPs behave like aquaglyceroporins and when phosphorylated their Water and solute permeability increased, while gas permeability decreased. Phosphorylation of Gm Nod26 (by calcium-signaling pathways) was enhanced in response to osmotic stress (both drought and salt stress).
In conclusion, WCPSs (and other MIPs) play a key role in plant Water relations. Novel functions and regulation mechanisms of plant WCPSs have been recently uncovered. Besides Watersome plant MIPs can transportphysiologicalimportant molecules such as neutral solutes (urea, boric acid, silicic acid) or gases (NH3, CO2). Thus plant MIPs are involved in many great functions of plants, including nutrient aquisition, carbon fixation, cell signaling, and stress responses (111, 117, 123).