Membrane water permeability and aquaporin expression increase during growth of maize suspension cultured cells

Aquaporins (AQPs) are water channels that allow cells to rapidly alter their membrane water permeability. A convenient model for studying AQP expression and activity regulation is Black Mexican Sweet (BMS) maize cultured cells. In an attempt to correlate membrane osmotic water permeability coefficient (P_f) with AQP gene expression, we first examined the expression pattern of 33 AQP genes using macro-array hybridization. We detected the expression of 18 different isoforms representing the four AQP subfamilies, i.e. eight plasma membrane (PIP), five tonoplast (TIP), three small basic (SIP) and two NOD26-like (NIP) AQPs. While the expression of most of these genes was constant throughout all growth phases, mRNA levels of ZmPIP1;3 , ZmPIP2;1 , ZmPIP2;2, ZmPIP2;4 and ZmPIP2;6 increased significantly during the logarithmic growth phase and the beginning of the stationary phase. The use of specific anti-ZmPIP antisera showed that the protein expression pattern correlated well with mRNA levels. Cell pressure probe and protoplast swelling measurements were then performed to determine the P_f. Interestingly, we found that the P_f were significantly increased at the end of the logarithmic growth phase and during the steady-state phase compared to the lag phase, demonstrating a positive correlation between AQP abundance in the plasma membrane and the cell P_f.     

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.     

Short-term control of maize cell and root water permeability through plasma membrane aquaporin isoforms

Although it is widely accepted that aquaporins are involved in the regulation of root water uptake, the role of specific isoforms in this process is poorly understood. The mRNA expression and protein level of specific plasma membrane intrinsic proteins (PIPs) were analysed in Zea mays in relation to cell and root hydraulic conductivity. Plants were analysed during the day/night period, under different growth conditions (aeroponics/hydroponics) and in response to short-term osmotic stress applied through polyethylene glycol (PEG). Higher protein levels of ZmPIP1;2, ZmPIP2;1/2;2, ZmPIP2;5 and ZmPIP2;6 during the day coincided with a higher water permeability of root cortex cells during the day compared with night period. Similarly, plants which were grown under aeroponic conditions and which developed a hypodermis ('exodermis') with Casparian bands, effectively forcing more water along a membranous uptake path across roots, showed increased levels of ZmPIP2;5 and ZmPIP1;2 in the rhizodermis and exodermis. When PEG was added to the root medium (2-8 h), expression of PIPs and cell water permeability in roots increased. These data support a role of specific PIP isoforms, in particular ZmPIP1;2 and ZmPIP2;5, in regulating root water uptake and cortex cell hydraulic conductivity in maize.     

Interactions between plasma membrane aquaporins modulate their water channel activity

Plant plasma membrane intrinsic proteins (PIPs) cluster in two evolutionary subgroups, PIP1 and PIP2, with different aquaporin activities when expressed in Xenopus oocytes. Maize ZmPIP1;1 and ZmPIP1;2 do not increase the osmotic water permeability coefficient (Pf), whereas ZmPIP2;1, ZmPIP2;4, and ZmPIP2;5 do. Here, we show that coexpression of the nonfunctional ZmPIP1;2 and the functional ZmPIP2;1, ZmPIP2;4, or ZmPIP2;5 resulted in an increase in Pf that was dependent on the amount of injected ZmPIP1;2 complementary RNA. Confocal analysis of oocytes expressing ZmPIP1;2-green fluorescent protein (GFP) alone or ZmPIP1;2-GFP plus ZmPIP2;5 showed that the amount of ZmPIP1;2-GFP present in the plasma membrane was significantly greater in coexpressing cells. Nickel affinity chromatography purification of ZmPIP2;1 fused to a His tag coeluted with ZmPIP1;2-GFP demonstrated physical interaction and heteromerization of both isoforms. Interestingly, coexpression of ZmPIP1;1 and ZmPIP2;5 did not result in a greater increase in Pf than did the expression of ZmPIP2;5 alone, but coexpression of the ZmPIP1;1 and ZmPIP1;2 isoforms induced a Pf increase, indicating that PIP1 isoform heteromerization is required for both of them to act as functional water channels. Mutational analysis demonstrated the important role of the C-terminal part of loop E in PIP interaction and water channel activity induction. This study has revealed a new mechanism of plant aquaporin regulation that might be important in plant water relations.     

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.     

A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.     

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.     

Localization and Quantification of Plasma Membrane Aquaporin Expression in Maize Primary Root: A Clue to Understanding their Role as Cellular Plumbers

Water movement across root tissues occurs by parallel apoplastic, symplastic, and transcellular pathways that the plant can control to a certain extent. Because water channels or aquaporins (AQPs) play an important role in regulating water flow, studies on AQP mRNA and protein expression in different root tissues are essential. Here, we quantified and localized the expression of Zea mays plasma membrane AQPs (ZmPIPs) in primary root tip using in situ and quantitative RT-PCR and immunodetection approaches. All ZmPIP genes except ZmPIP2;7 were expressed in primary roots. Expression was found to be dependent on the developmental stage of the root, with, in general, an increase in expression towards the elongation and mature zones. Two genes, ZmPIP1;5 and ZmPIP2;5, showed the greatest increase in expression (up to 11- and 17-fold, respectively) in the mature zone, where they accounted for 50% of the total expressed ZmPIPs. The immunocytochemical localization of ZmPIP2;1 and ZmPIP2;5 in the exodermis and endodermis indicated that they are involved in root radial water movement. In addition, we detected a polar localization of ZmPIP2;5 to the external periclinal side of epidermal cells in root apices, suggesting an important role in water uptake from the root surface. Finally, protoplast swelling assays showed that root cells display a variable, but globally low, osmotic water permeability coefficient (P _f < 10 μm/s). However, the presence of a population of cells with a higher P _f (up to 26 μm/s) in mature zone of the root might be correlated with the increased expression of several ZmPIP genes.     

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.     

Aquaporins constitute a large and highly divergent protein family in maize

Aquaporins (AQPs) are an ancient family of channel proteins that transport water and neutral solutes through a pore and are found in all eukaryotes and most prokaryotes. A comparison of the amino acid sequences and phylogenetic analysis of 31 full-length cDNAs of maize (Zea mays) AQPs shows that they comprise four different groups of highly divergent proteins. We have classified them as plasma membrane intinsic proteins (PIPs), tonoplast intrinsic proteins, Nod26-like intrinsic proteins, and small and basic intrinsic proteins. Amino acid sequence identities vary from 16% to 100%, but all sequences share structural motifs and conserved amino acids necessary to stabilize the two loops that form the aqueous pore. Most divergent are the small and basic integral proteins in which the first of the two highly conserved Asn-Pro-Ala motifs of the pore is not conserved, but is represented by alanine-proline-threonine or alanine-proline-serine. We present a model of ZmPIP1-2 based on the three-dimensional structure of mammalian AQP1. Tabulation of the number of times that the AQP sequences are found in a collection of databases that comprises about 470,000 maize cDNAs indicates that a few of the maize AQPs are very highly expressed and many are not abundantly expressed. The phylogenetic analysis supports the interpretation that the divergence of PIPs through gene duplication occurred more recently than the divergence of the members of the other three subfamilies. This study opens the way to analyze the function of the proteins in Xenopus laevis oocytes, determine the tissue specific expression of the genes, recover insertion mutants, and determine the in planta function.     

Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease

Apoptosis is characterized by a conserved series of morphological events beginning with the apoptotic volume decrease (AVD). This study investigated a role for aquaporins (AQPs) during the AVD. Inhibition of AQPs blocked the AVD in ovarian granulosa cells undergoing growth factor withdrawal and blocked downstream apoptotic events such as cell shrinkage, changes in the mitochondrial membrane potential, DNA degradation, and caspase-3 activation. The effects of AQP inhibition on the AVD and DNA degradation were consistent in thymocytes and with two additional apoptotic signals, thapsigargin and C₆-ceramide. Overexpression of AQP-1 in Chinese hamster ovary (CHO-AQP-1) cells enhanced their rate of apoptosis. The AVD is driven by loss of K⁺ from the cell, and we hypothesize that after the AVD, AQPs become inactive, which halts further water loss and allows K⁺ concentrations to decrease to levels necessary for apoptotic enzyme activation. Swelling assays on granulosa cells, thymocytes, and CHO-AQP-1 cells revealed that indeed, the shrunken (apoptotic) subpopulation has very low water permeability compared with the normal-sized (nonapoptotic) subpopulation. In thymocytes, AQP-1 is present and was shown to colocalize with the plasma membrane receptor tumor necrosis factor receptor-1 (TNF-R1) both before and after the AVD, which suggests that this protein is not proteolytically cleaved and remains on the cell membrane. Overall, these data indicate that AQP-mediated water loss is important for the AVD and downstream apoptotic events, that the water permeability of the plasma membrane can control the rate of apoptosis, and that inactivation after the AVD may help create the low K⁺ concentration that is essential in apoptotic cells. Furthermore, inactivation of AQPs after the AVD does not appear to be through degradation or removal from the cell membrane. water movement; major intrinsic protein; channel; enzyme     

Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays

Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO₂ permeability comparable to that of the CO₂ diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes.     

Drought, salt and wounding stress induce the expression of the plasma membrane intrinsic protein 1 gene in poplar (Populus alba×P. tremula var. glandulosa)

Water uptake across cell membranes is a principal requirement for plant growth at both the cellular and whole-plant levels; water movement through plant membranes is regulated by aquaporins (AQPs) or major intrinsic proteins (MIPs). We examined the expression characteristics of the poplar plasma membrane intrinsic protein 1 gene (PatPIP1), a type of MIP, which was isolated from a suspension cell cDNA library of Populus alba×P. tremula var. glandulosa. Examination of protoplasts expressing the p35S-PatPIP1::sGFP fusion protein revealed that the protein was localized in the plasma membrane. Northern blot analysis revealed that the gene was strongly expressed in poplar roots and leaves. Gene expression was inducible by abiotic factors including drought, salinity, cold temperatures and wounding, and also by plant hormones including gibberellic acid, jasmonic acid and salicylic acid. Since we found that the PatPIP1 gene was strongly expressed in response to mannitol, NaCl, jasmonic acid and wounding, we propose that PatPIP1 plays an essential role in the defense of plants against water stress.     

Coronatine promotes maize water uptake by directly binding to the aquaporin ZmPIP2;5 and enhancing its activity

Water uptake is crucial for crop growth and development and drought stress tolerance. The water channel aquaporins(AQP) play important roles in plant water uptake. Here, we discovered that a jasmonic acid analog, coronatine(COR), enhanced maize(Zea mays) root water uptake capacity under artificial water deficiency conditions. COR treatment induced the expression of the AQP gene Plasma membrane intrinsic protein 2;5(ZmPIP2;5).In vivo and in vitro experiments indicated that COR also directly acts ...     

Upregulation and protein trafficking of aquaporin-2 attenuate cold-induced osmotic damage during cryopreservation

Aquaporins (AQPs) are a recently discovered family of proteins that function as transmembrane water channels. These proteins regulate the delicate osmotic balance across the cell plasma membrane. Given that osmotic damage is the major contributing factor to cell death during freezing, we hypothesized that regulation of AQPs may have an unrealized role in protecting cells from osmotic damage during cryopreservation. Rat kidney inner medullar collecting duct (IMCD) cells were treated with arginine vasopressin (AVP) to increase the amount of AQP2 in the external plasma membrane before freezing in University of Wisconsin solution at -4 degrees C for 24 h. This resulted in a significant increase in cell viability on warming. Conversely, treatment of IMCD cells with AVP and W7 (which inhibits AQP2 protein trafficking to the plasma membrane) before freezing resulted in a 55% decrease in cell viability. These preliminary data indicate that regulation of AQP2 can attenuate cold-induced osmotic damage in rat kidney IMCD cells.     

Biological significance and topological basis of aquaporin-partnering protein-protein interactions

Aquaporins (AQPs) are intramolecular channels essential for transport of H₂O, CO₂, and other small substrates across membranes. Through this function, AQPs can modulate CO₂ uptake and assimilation in plants and regulate water relations and many other physiological processes in all living organisms. To execute their physiological roles, AQPs may experience 3 types of hetero-molecular interaction, between AQPs and their kinases; between AQP isoforms; and between AQPs and other proteins that are neither AQPs nor kinases. Interacting with non-AQP non-kinase proteins may enable AQPs to extend their functions beyond substrate transport, and most fascinatingly, to serve as a gateway control for translocation of virulence effectors from pathogenic bacteria into the cytosol of eukaryotic cells. In this mini review, we will summarize the latter 2 types of interaction and discuss the physiological and/or pathological significance. We will also discuss a research angle to elucidate the structural basis of AQP-partnering protein interactions.     

Analysis of aquaporin-mediated diffusional water permeability by coherent anti-stokes Raman scattering microscopy

Water can pass through biological membranes via two pathways: simple diffusion through the lipid bilayer, or water-selective facilitated diffusion through aquaporins (AQPs). Although AQPs play an important role in osmotic water permeability (P_f), the role of AQPs in diffusional water permeability remains unclear because of the difficulty of measuring diffusional water permeability (P_d). Here, we report an accurate and instantaneous method for measuring the P_d of a single HeLa S3 cell using coherent anti-Stokes Raman scattering (CARS) microscopy with a quick perfusion device for H₂O/D₂O exchange. Ultra-high-speed line-scan CARS images were obtained every 0.488 ms. The average decay time constant of CARS intensities (τ_CARS) for the external solution H₂O/D₂O exchange was 16.1 ms, whereas the intracellular H₂O/D₂O exchange was 100.7 ± 19.6 ms. To evaluate the roles of AQP in diffusional water permeability, AQP4 fused with enhanced green fluorescent protein (AQP4-EGFP) was transiently expressed in HeLa S3 cells. The average τ_CARS for the intracellular H₂O/D₂O exchange in the AQP4-EGFP-HeLa S3 cells was 43.1 ± 15.8 ms. We also assessed the cell volume and the cell surface area to calculate P_d. The average P_d values for the AQP4-EGFP-HeLa S3 cells and the control EGFP-HeLa S3 cells were 2.7 ± 1.0 × 10⁻³ and 8.3 ± 2.6 × 10⁻⁴ cm/s, respectively. AQP4-mediated water diffusion was independent of the temperature but was dependent on the expression level of the protein at the plasma membrane. These results suggest the possibility of using CARS imaging to investigate the hydrodynamics of single mammalian cells as well as the regulation of AQPs.