Arabidopsis SNAREs SYP61 and SYP121 coordinate the trafficking of plasma membrane aquaporin PIP2;7 to modulate the cell membrane water permeability.

Plant plasma membrane intrinsic proteins (PIPs) are aquaporins that facilitate the passive movement of water and small neutral solutes through biological membranes. Here, we report that post-Golgi trafficking of PIP2;7 in Arabidopsis thaliana involves specific interactions with two syntaxin proteins, namely, the Qc-SNARE SYP61 and the Qa-SNARE SYP121, that the proper delivery of PIP2;7 to the plasma membrane depends on the activity of the two SNAREs, and that the SNAREs colocalize and physically interact. These findings are indicative of an important role for SYP61 and SYP121, possibly forming a SNARE complex. Our data support a model in which direct interactions between specific SNARE proteins and PIP aquaporins modulate their post-Golgi trafficking and thus contribute to the fine-tuning of the water permeability of the plasma membrane.

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.

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).

Identification and characterization of two plasma membrane aquaporins in durum wheat (Triticum turgidum L. subsp. durum) and their role in abiotic stress tolerance.

Plant plasma membrane intrinsic proteins (PIP) cluster in two phylogenetic groups, PIP1 and PIP2 that have different water channel activities when expressed in Xenopus oocytes. PIP2s induce a marked increase of the membrane osmotic water-permeability coefficient (P(f)), whereas PIP1s are generally inactive. Here we report the cloning of two durum wheat (Triticum turgidum L. subsp. durum) cDNAs encoding TdPIP1;1 and TdPIP2;1 belonging to the PIP1 and PIP2 subfamilies, respectively. Contrary to TdPIP1;1, expression of TdPIP2;1 in Xenopus oocytes resulted in an increase in P(f) compared to water-injected oocytes. Co-expression of the non-functional TdPIP1;1 and the functional TdPIP2;1 lead to a significant increase in P(f) compared with oocytes expressing TdPIP2;1 alone. A truncated form of TdPIP2;1, tdpip2;1, missing the first two transmembrane domains, had no water channel activity. Nonetheless, its co-expression with the functional TdPIP2;1 partially inhibits the P(f) and disrupt the activities of plant aquaporins. In contrast to the approach developed in Xenopus oocytes, phenotypic analyses of transgenic tobacco plants expressing TdPIP1;1 or TdPIP2;1 generated a tolerance phenotype towards osmotic and salinity stress. TdPIP1;1 and TdPIP2;1 are differentially regulated in roots and leaves in the salt-tolerant wheat variety when challenged with salt stress and abscisic acid. Confocal microscopy analysis of tobacco roots expressing TdPIP1;1 and TdPIP2;1 fused to the green fluorescent protein showed that the proteins were localized at the plasma membrane.

Maize black Mexican sweet suspension cultured cells are a convenient tool for studying aquaporin activity and regulation.

Aquaporins (AQPs) are channel proteins that facilitate and regulate the permeation of water across biological membranes. Black Mexican sweet suspension cultured cells are a convenient model for studying the regulation of maize AQP expression and activity. Among other advantages, a single cell system allows the contribution of plasma membrane AQPs (PIPs, plasma membrane intrinsic proteins) to the membrane water permeability coefficient (P(f)) to be determined using biophysical measurement methods, such as the cell pressure probe or protoplast swelling assay. We generated a transgenic cell culture line expressing a tagged version of ZmPIP2;6 and used this material to demonstrate that the ZmPIP2;6 and ZmPIP2;1 isoforms physically interact. This kind of interaction could be an additional mechanism for regulating membrane water permeability by acting on the activity and/or trafficking of PIP hetero-oligomers.

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 (Pf < 10 microm/s). However, the presence of a population of cells with a higher Pf (up to 26 microm/s) in mature zone of the root might be correlated with the increased expression of several ZmPIP genes.