Plasma Membrane Aquaporins Research Articles

Effect of maize plasma membrane aquaporin phosphorylation on the channel activity, trafficking and hetero-oligomerisation

Effect of maize plasma membrane aquaporin phosphorylation on the channel activity, trafficking and hetero-oligomerisation

Hydrogen peroxide effects on root hydraulic properties and plasma membrane aquaporin regulation in Phaseolus vulgaris

In the last few years, the role of reactive oxygen species as signaling molecules has emerged, and not only as damage-related roles. Here, we analyzed how root hydraulic properties were modified by different hydrogen peroxide (H2O2) concentrations applied exogenously to the root medium. Two different experimental setups were employed: Phaseolus vulgaris plants growing in hydroponic or in potted soils. In both experimental setups, we found an increase of root hydraulic conductance (L) in response to H2O2 application for the first time. Twenty millimolar was the threshold concentration of H2O2 for observing an effect on L in the soil experiment, while in the hydroponic experiment, a positive effect on L was observed at 0.25 mM H2O2. In the hydroponic experiment, a correlation between increased L and plasma membrane aquaporin amount and their root localization was observed. These findings provide new insights to study how several environmental factors modify L.

Drought Stress-Induced Rma1H1, a RING Membrane-Anchor E3 Ubiquitin Ligase Homolog, Regulates Aquaporin Levels via Ubiquitination in TransgenicArabidopsisPlants

Ubiquitination is involved in a variety of biological processes, but the exact role of ubiquitination in abiotic responses is not clearly understood in higher plants. Here, we investigated Rma1H1, a hot pepper (Capsicum annuum) homolog of a human RING membrane-anchor 1 E3 ubiquitin (Ub) ligase. Bacterially expressed Rma1H1 displayed E3 Ub ligase activity in vitro. Rma1H1 was rapidly induced by various abiotic stresses, including dehydration, and its overexpression in transgenic Arabidopsis thaliana plants conferred strongly enhanced tolerance to drought stress. Colocalization experiments with marker proteins revealed that Rma1H1 resides in the endoplasmic reticulum (ER) membrane. Overexpression of Rma1H1 in Arabidopsis inhibited trafficking of an aquaporin isoform PIP2;1 from the ER to the plasma membrane and reduced PIP2;1 levels in protoplasts and transgenic plants. This Rma1H1-induced reduction of PIP2;1 was inhibited by MG132, an inhibitor of the 26S proteasome. Furthermore, Rma1H1 interacted with PIP2;1 in vitro and ubiquitinated it in vivo. Similar to Rma1H1, Rma1, an Arabidopsis homolog of Rma1H1, localized to the ER, and its overexpression reduced the PIP2;1 protein level and inhibited trafficking of PIP2;1 from the ER to the plasma membrane in protoplasts. In addition, reduced expression of Rma homologs resulted in the increased level of PIP2;1 in protoplasts. We propose that Rma1H1 and Rma1 play a critical role in the downregulation of plasma membrane aquaporin levels by inhibiting aquaporin trafficking to the plasma membrane and subsequent proteasomal degradation as a response to dehydration in transgenic Arabidopsis plants.

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca 2+ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.

An N‐terminal diacidic motif is required for the trafficking of maize aquaporins ZmPIP2;4 and ZmPIP2;5 to the plasma membrane

Maize plasma membrane aquaporins (ZmPIPs, where PIP is the plasma membrane intrinsic protein) fall into two groups, ZmPIP1s and ZmPIP2s, which, when expressed alone in mesophyll protoplasts, are found in different subcellular locations. Whereas ZmPIP1s are retained in the endoplasmic reticulum (ER), ZmPIP2s are found in the plasma membrane (PM). We previously showed that, when co-expressed with ZmPIP2s, ZmPIP1s are relocalized to the PM, and that this relocalization results from the formation of hetero-oligomers between ZmPIP1s and ZmPIP2s. To determine the domains responsible for the ER retention and PM localization, respectively, of ZmPIP1s and ZmPIP2s, truncated and mutated ZmPIPs were generated, together with chimeric proteins created by swapping the N- or C-terminal regions of ZmPIP2s and ZmPIP1s. These mutated proteins were fused to the mYFP and/or mCFP, and the fusion proteins were expressed in maize mesophyll protoplasts, and were then localized by microscopy. This allowed us to identify a diacidic motif, DIE (Asp-Ile-Glu), at position 4-6 of the N-terminus of ZmPIP2;5, that is essential for ER export. This motif was conserved and functional in ZmPIP2;4, but was absent in ZmPIP2;1. In addition, we showed that the N-terminus of ZmPIP2;5 was not sufficient to cause the export of ZmPIP1;2 from the ER. A study of ZmPIP1;2 mutants suggested that the N- and C-termini of this protein are probably not involved in ER retention. Together, these results show that the trafficking of maize PM aquaporins is differentially regulated depending on the isoform, and involves a specific signal and mechanism.

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.

Increase in aquaporin activity is involved in leaf succulence of the euhalophyte Suaeda salsa, under salinity

Plasma membrane aquaporins (AQPs) determine, likely among other functions, the water relations of plant cells. We were interested in determining whether AQPs could be regulated in the process of changing leaf succulence, especially during changes in seedlings of a euhalophyte Suaeda salsa (L.), under salinity. Seedlings were treated with 100 mM NaCl for 10 days. The effect of NaCl on hydraulic conductance ( L 0) in roots, succulence degree, osmotic potential, Na + and Cl − content, water permeability of protoplasts, expression of SsPIP, and amount of SsPIP AQP in leaves of S. salsa were determined. The results showed a correlation between leaf succulence and hydraulic conductance in the roots of S. salsa, under salinity. Also, the water permeability of the protoplasts of NaCl-treated leaves was significantly higher than that of controls, and the abundance of transcripts for a plasma-membrane AQP homologous to AtPIP2;7 increased in the presence of NaCl. Immunoblot analyses of PIP AQPs located in plasma membrane-enriched fractions of S. salsa seedlings showed a significant increase under salinity. In conclusion, the increase in salinity-induced AQP activity is by way of an increase in both the transcripts and protein amount, which correlate with the increase in leaf succulence of S. salsa under salinity.

Affinity tags can reduce merohedral twinning of membrane protein crystals

This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.

Rh-PIP2;1, a Rose Aquaporin Gene, Is Involved in Ethylene-Regulated Petal Expansion

Aquaporins are water channel proteins that facilitate the passage of water through biological membranes and play a crucial role in plant growth. We showed that ethylene treatment significantly reduced petal size, inhibited expansion of petal abaxial subepidermal cells, and decreased petal water content in rose (Rosa hybrida 'Samantha'). Here, we report the isolation of a plasma membrane aquaporin (PIP) gene, Rh-PIP2;1, and characterized its potential role in ethylene-inhibited petal expansion. Rh-PIP2;1 is mainly localized on the plasma membrane and belongs to the class 2 subfamily of PIP proteins. We show that Rh-PIP2;1 is an active water channel. The transcripts of Rh-PIP2;1 are highly abundant in petal epidermal cells, especially in the abaxial subepidermal cells. The expression of Rh-PIP2;1 is highly correlated with petal expansion and tightly down-regulated by ethylene. Furthermore, we demonstrate that in Rh-PIP2;1-silenced flowers, petal expansion was greatly inhibited and anatomical features of the petals were similar to those of ethylene-treated flowers. We argue that Rh-PIP2;1 plays an important role in petal cell expansion and that ethylene inhibits petal expansion of roses at least partially by suppressing Rh-PIP2;1 expression.

Maize Plasma Membrane Aquaporins Belonging to the PIP1 and PIP2 Subgroups are in vivo Phosphorylated

Aquaporins are channel proteins that facilitate transmembrane water movement. In this study, we showed that plasma membrane intrinsic proteins (PIPs) from maize shoots are in vitro and in vivo phosphorylated on serine residues by a calcium-dependent kinase associated with the membrane fraction. Mass spectrometry identified phosphorylated peptides corresponding to the C-terminal region of (i) ZmPIP2;1, ZmPIP2;2 and/or ZmPIP2;7; (ii) ZmPIP2;3 and/or ZmPIP2;4; (iii) ZmPIP2;6; together with (iv) a phosphorylated peptide located in the N-terminal region of ZmPIP1;1, ZmPIP1;2, ZmPIP1;3 and/or ZmPIP1;4. The role of phosphorylation in the water channel activity of wild-type and mutant ZmPIP2;1 was studied in Xenopus laevis oocytes. Activation of endogenous protein kinase A increased the osmotic water permeability coefficient of ZmPIP2;1-expressing oocytes, suggesting that phosphorylation activates its channel activity. Mutation of S126 or S203, putative phosphorylated serine residues conserved in all plant PIPs, to alanine decreased ZmPIP2;1 activity by 30-50%, without affecting its targeting to the plasma membrane. Mutation of S285, which is phosphorylated in planta, to alanine or glutamate did not affect the water channel activity. These results indicate that, in oocytes, S126 and S203 play an important role in ZmPIP2;1 activity and that phosphorylation of S285 is not required for its activity.

Plant plasma membrane water channels conduct the signalling molecule H2O2

H(2)O(2) is a relatively long-lived reactive oxygen species that signals between cells and organisms. H(2)O(2) signalling in plants is essential for response to stress, defence against pathogens and the regulation of programmed cell death. Although H(2)O(2) diffusion across membranes is often considered as a passive property of lipid bilayers, native membranes represent significant barriers for H(2)O(2). In the present study we addressed the question of whether channels might facilitate H(2)O(2) conduction across plasma membranes. The expression of several plant plasma membrane aquaporins in yeast, including PIP2;1 from Arabidopsis (where PIP is plasma membrane intrinsic protein), enhanced the toxicity of H(2)O(2) and increased the fluorescence of dye-loaded yeast when exposed to H(2)O(2). The sensitivity of aquaporin-expressing yeast to H(2)O(2) was altered by mutations that alter gating and the selectivity of the aquaporins. The conduction of water, H(2)O(2) and urea was compared, using molecular dynamics simulations based on the crystal structure of SoPIP2;1 from spinach. The calculations identify differences in the conduction between the substrates and reveal channel residues critically involved in H(2)O(2) conduction. The results of the calculations on tetramers and monomers are in agreement with the biochemical data. Taken together, the results strongly suggest that plasma membrane aquaporin pores determine the efficiency of H(2)O(2) signalling between cells. Aquaporins are present in most species and their capacity to facilitate the diffusion of H(2)O(2) may be of physiological significance in many organisms and particularly in communication between different species.

The expression pattern of plasma membrane aquaporins in maize leaf highlights their role in hydraulic regulation

Leaves are key organs for evaporation and photosynthesis and play a crucial role in plant growth and development. In order to function properly, they need to maintain a balanced water content. Water movement through a leaf occurs by a combination of different pathways: water can follow an apoplastic route through the cell wall or a cell-to-cell route via the symplastic and transcellular paths. As aquaporins (AQPs) play an important role in regulating transcellular water flow and CO(2) conductance, studies on AQP mRNA and protein expression in leaves are essential to better understand their role in these physiological processes. Here, we quantified and localized the expression of Zea mays plasma membrane aquaporins (ZmPIPs, plasma membrane intrinsic proteins) in the leaf using quantitative RT-PCR and immunodetection. All ZmPIP genes except ZmPIP2;7 were expressed in leaves. Expression was found to be dependent on the developmental stage of the leaf tissue, with, in general, an increase in expression at the end of the elongation zone and a decrease in mature leaf tissue. These data correlated with the cell water permeability, as determined using a protoplast swelling assay. The diurnal expression of ZmPIPs was also investigated and expression was found to be higher during the first hours of the light period than at night. Immunocytochemical localization of four ZmPIP isoforms indicated that they are involved in leaf radial water movement, in particular in vascular bundles and the mesophyll.

Identification of grapevine aquaporins and expression analysis in developing berries

Aquaporins are membrane water channels that play critical roles in controlling the water content of cells and tissues. In this work, nine full-length cDNAs encoding putative aquaporins were isolated from grape berry cDNA libraries. A phylogenetic analysis conducted with 28 aquaporin genes identified in the grapevine genome and previously characterized aquaporins from Arabidopsis indicates that three cDNAs encode putative tonoplast aquaporins (TIPs) whereas six cDNAs belong to the plasma membrane aquaporin subfamily (PIPs). Specific probes designed on the 3' untranslated regions of each cDNA were used for the preparation of cDNA macroarray filters and in situ hybridization experiments. Macroarray data indicate that expression levels of most TIP and PIP genes depend on grape berry developmental stages and point out to a global decrease of aquaporin gene expression during berry ripening. In young berries, high expression of aquaporin genes was preferentially observed in dividing and elongating cells and in cells involved in water and solutes transport. Taken together, the data provided in this paper indicate that aquaporins are implicated in various physiological aspects of grape berry development.

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.

Characterization of Four Plasma Membrane Aquaporins in Tulip Petals: A Putative Homolog is Regulated by Phosphorylation

We suggested previously that temperature-dependent tulip (Tulipa gesneriana) petal movement that is concomitant with water transport is regulated by reversible phosphorylation of an unidentified plasma membrane intrinsic protein (PIP). In this study, four full-length cDNAs of PIPs from tulip petals were identified and cloned. Two PIPs, namely TgPIP1;1 and TgPIP1;2, are members of the PIP1 subfamily, and the remaining two PIPs, namely TgPIP2;1 and TgPIP2;2, belong to the PIP2 subfamily of aquaporins and were named according to the nomenclature of PIP genes in plants. Of these four homologs, only TgPIP2;2 displayed significant water channel activity in the heterologous expression assay using Xenopus laevis oocytes. The water channel activity of this functional isoform was abolished by mercury and was affected by inhibitors of protein kinase and protein phosphatase. Using a site-directed mutagenesis approach to substitute several serine residues with alanine, and assessing water channel activity using the methylotrophic yeast Pichia pastoris expression assay, we showed that Ser35, Ser116 and Ser274 are the putative phosphorylation sites of TgPIP2;2. Real-time reverse transcription-PCR analysis revealed that the transcript levels of TgPIP1;1 and TgPIP1;2 in tulip petals, stems, leaves, bulbs and roots are very low when compared with those of TgPIP2;1 and TgPIP2;2. The transcript level of TgPIP2;1 is negligible in roots, and TgPIP2;2 is ubiquitously expressed in all organs with significant transcript levels. From the data reported herein, we suggest that TgPIP2;2 might be modulated by phosphorylation and dephosphorylation for regulating water channel activity, and may play a role in transcellular water transport in all tulip organs.

Multiple Phosphorylations in the C-terminal Tail of Plant Plasma Membrane Aquaporins: Role in Subcellular Trafficking of AtPIP2;1 in Response to Salt Stress

Aquaporins form a family of water and solute channel proteins and are present in most living organisms. In plants, aquaporins play an important role in the regulation of root water transport in response to abiotic stresses. In this work, we investigated the role of phosphorylation of plasma membrane intrinsic protein (PIP) aquaporins in the Arabidopsis thaliana root by a combination of quantitative mass spectrometry and cellular biology approaches. A novel phosphoproteomics procedure that involves plasma membrane purification, phosphopeptide enrichment with TiO(2) columns, and systematic mass spectrometry sequencing revealed multiple and adjacent phosphorylation sites in the C-terminal tail of several AtPIPs. Six of these sites had not been described previously. The phosphorylation of AtPIP2;1 at two C-terminal sites (Ser(280) and Ser(283)) was monitored by an absolute quantification method and shown to be altered in response to treatments of plants by salt (NaCl) and hydrogen peroxide. The two treatments are known to strongly decrease the water permeability of Arabidopsis roots. To investigate a putative role of Ser(280) and Ser(283) phosphorylation in aquaporin subcellular trafficking, AtPIP2;1 forms mutated at either one of the two sites were fused to the green fluorescent protein and expressed in transgenic plants. Confocal microscopy analysis of these plants revealed that, in resting conditions, phosphorylation of Ser(283) is necessary to target AtPIP2;1 to the plasma membrane. In addition, an NaCl treatment induced an intracellular accumulation of AtPIP2;1 by exerting specific actions onto AtPIP2;1 forms differing in their phosphorylation at Ser(283) to induce their accumulation in distinct intracellular structures. Thus, the present study documents stress-induced quantitative changes in aquaporin phosphorylation and establishes for the first time a link with plant aquaporin subcellular localization.

Characterization and expression of plasma and tonoplast membrane aquaporins in elongating cotton fibers

Cotton fiber (Gossypium hirsutum L. and G. barbadense L.) is a good model for studies of plant cell elongation and cell wall biogenesis. Aquaporins are ancient membrane channel proteins that facilitate the permeation of water across biological membranes. We studied GhPIP1-2, encoding plasma membrane intrinsic protein, and GhgammaTIP1, encoding tonoplast intrinsic protein, during cotton fiber development. The full-length cDNAs of GhPIP1-2 and GhgammaTIP1 were obtained through 5' RACE. The deduced amino acid sequences of GhPIP1-2 and GhgammaTIP1 share high sequence identity with aquaporins from diverse plant species. Phylogenetic analysis of GhPIP1-2 and GhgammaTIP1 with other plant aquaporins showed that GhPIP1-2 belongs to the PIP1 group of the PIP subfamily and GhgammaTIP1 belongs to the gammaTIP group of the TIP subfamily. GhPIP1-2 and GhgammaTIP1 contain three and two introns, respectively. Genomic Southern blot analysis indicated that GhPIP1-2 and GhgammaTIP1 have several copies and multiple homologous genes in allotetraploid cotton. Northern blot analysis with gene-specific probes and real-time PCR demonstrated that GhPIP1-2 and GhgammaTIP1 are predominantly expressed during cotton fiber elongation, with the highest expression levels at 5 days post-anthesis. Moreover, expression patterns of the two genes in G. hirsutum and G. barbadense are similar, whereas the expression levels in G. barbadense are much lower than that in G. hirsutum. The high and preferential expression of GhPIP1-2 and GhgammaTIP1 during fiber cell elongation suggests that they may play important roles in supporting the rapid influx of water into vacuoles during cotton fiber cell expansion.

A fruit‐specific plasma membrane aquaporin subtype PIP1;1 is regulated during strawberry (Fragaria × ananassa) fruit ripening

Despite the advances in the physiology of fruit ripening, the role and contribution of water pathways are still barely considered. Our aim was therefore to characterize aquaporins, proteins that render the molecular basis for putative regulatory mechanisms in water transport. We focused our work on strawberry (Fragaria xananassa) fruit, a non-climacteric fruit of special interest because of its forced brief commercial shelf life. A full-length cDNA was isolated with high homology with plasma membrane (PM) intrinsic proteins (named FaPIP1;1), showing a profile with high expression in fruit, less in ovaries and no detection at all in other parts. Its cellular localization was confirmed at the PM. As reported in other plasma membrane intrinsic proteins subtype 1 (PIP1s), when expressing the protein in Xenopus leavis oocytes, FaPIP1;1 shows low water permeability values that only increased when it is coexpressed with a plasma membrane intrinsic protein subtype 2. Northern blotting using total RNA shows that its expression increases during fruit ripening. Moreover, functional characterization of isolated PM vesicles from red stage fruit unequivocally demonstrates the presence of active water channels, i.e. high water permeability values and a low Arrhenius activation energy, both evidences of water transport mediated by proteins. Interestingly, as many ripening-related strawberry genes, the expression pattern of FaPIP1;1 was also repressed by the presence of auxins. We therefore report a fruit specific PIP1 aquaporin with an accumulation pattern tightly associated to auxins and to the ripening process that might be responsible for increasing water permeability at the level of the PM in ripe fruit.

Deactivation of aquaporins decreases internal conductance to CO2 diffusion in tobacco leaves grown under long-term drought.

We compared the diffusion conductance to CO2 from the intercellular air space to the chloroplasts (internal conductance (g i)) between tobacco leaves acclimated to long-term drought (drought-acclimated (DA)) and those grown under sufficient irrigation (well-watered (WW)), and analysed the changes in g i in relation to the leaf anatomical characteristics and a possible CO2 transporter, aquaporin. The g i, which was estimated by combined analyses of CO2 gas exchange with chlorophyll fluorescence, in the DA plants was approximately half of that in the WW plants. The mesophyll and chloroplast surface areas exposing the intercellular air space, which potentially affect g i, were not significantly different between the WW and DA plants. The amounts of plasma membrane aquaporins (PIP), immunochemically determined using radish PIP antibodies, were unrelated to g i. After treatment with HgCl2, an aquaporin inhibitor, the water permeability of the leaf tissues (measured as the weight loss of fully-turgid leaf disks without the abaxial epidermis in 1 m sorbitol) in WW plants decreased with an increase in HgCl2 concentration. The g i in the WW plants decreased to similar levels to the DA plants when the detached leaflets were fed with 0.5 mm HgCl2. In contrast, both water permeability and g i were insensitive to HgCl2 treatments in DA plants. These results suggest that deactivation of aquaporins is responsible for the significant reduction in g i observed in plants growing under long-term drought.

FRET imaging in living maize cells reveals that plasma membrane aquaporins interact to regulate their subcellular localization

Zea mays plasma membrane intrinsic proteins (ZmPIPs) fall into two groups, ZmPIP1s and ZmPIP2s, that exhibit different water channel activities when expressed in Xenopus oocytes. ZmPIP1s are inactive, whereas ZmPIP2s induce a marked increase in the membrane osmotic water permeability coefficient, P(f). We previously showed that, in Xenopus oocytes, ZmPIP1;2 and ZmPIP2;1 interact to increase the cell P(f). Here, we report the localization and interaction of ZmPIP1s and ZmPIP2s in living maize cells. ZmPIPs were fused to monomeric yellow fluorescent protein and/or monomeric cyan fluorescent protein and expressed transiently in maize mesophyll protoplasts. When expressed alone, ZmPIP1 fusion proteins were retained in the endoplasmic reticulum, whereas ZmPIP2s were found in the plasma membrane. Interestingly, when coexpressed with ZmPIP2s, ZmPIP1s were relocalized to the plasma membrane. Using FRET/fluorescence lifetime imaging microscopy, we demonstrated that this relocalization results from interaction between ZmPIP1s and ZmPIP2s. Immunoprecipitation experiments provided additional evidence for the association of ZmPIP1;2 and ZmPIP2;1 in maize roots and suspension cells. These data suggest that PIP1-PIP2 interaction is required for in planta PIP1 trafficking to the plasma membrane to modulate plasma membrane permeability.