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Plant aquaporins (AQPs) facilitate the membrane diffusion of water and small solutes, including hydrogen peroxide (H2O2) and, possibly, cations, essential signalling molecules in many physiological processes. While the determination of the channel activity generally depends on heterologous expression of AQPs in Xenopus oocytes or yeast cells, we established a genetic tool to determine whether they facilitate the diffusion of H2O2 through the plasma membrane in living plant cells. We designed genetic constructs to co‐express the fluorescent H2O2 sensor HyPer and AQPs, with expression controlled by a heat shock‐inducible promoter in Nicotiana tabacum BY‐2 suspension cells. After induction of ZmPIP2;5 AQP expression, a HyPer signal was recorded when the cells were incubated with H2O2, suggesting that ZmPIP2;5 facilitates H2O2 transmembrane diffusion; in contrast, the ZmPIP2;5W85A mutated protein was inactive as a water or H2O2 channel. ZmPIP2;1, ZmPIP2;4 and AtPIP2;1 also facilitated H2O2 diffusion. Incubation with abscisic acid and the elicitor flg22 peptide induced the intracellular H2O2 accumulation in BY‐2 cells expressing ZmPIP2;5. We also monitored cation channel activity of ZmPIP2;5 using a novel fluorescent photo‐switchable Li+ sensor in BY‐2 cells. BY‐2 suspension cells engineered for inducible expression of AQPs as well as HyPer expression and the use of Li+ sensors constitute a powerful toolkit for evaluating the transport activity and the molecular determinants of PIPs in living plant cells. Summary statement: Nicotiana tabacum BY‐2 suspension cells engineered for HyPer expression as well as inducible expression of aquaporins and the use of a Li+ sensor constitute a powerful tool for evaluating hydrogen peroxide and cation transport activity in living plant cells. [ABSTRACT FROM AUTHOR]

Summary: Plasma membrane (PM) intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water and small solutes. The functional importance of the PM organisation of PIPs in the interaction with other cellular structures is not completely understood.We performed a pull‐down assay using maize (Zea mays) suspension cells expressing YFP‐ZmPIP2;5 and validated the protein interactions by yeast split‐ubiquitin and bimolecular fluorescence complementation assays. We expressed interacting proteins tagged with fluorescent proteins in Nicotiana benthamiana leaves and performed water transport assays in oocytes. Finally, a phylogenetic analysis was conducted.The PM‐located ZmPIP2;5 physically interacts with the endoplasmic reticulum (ER) resident ZmVAP27‐1. This interaction requires the ZmVAP27‐1 cytoplasmic major sperm domain. ZmPIP2;5 and ZmVAP27‐1 localise in close vicinity in ER–PM contact sites (EPCSs) and endocytic structures upon exposure to salt stress conditions. This interaction enhances PM water permeability in oocytes. Similarly, the Arabidopsis ZmVAP27‐1 paralogue, AtVAP27‐1, interacts with the AtPIP2;7 aquaporin.Together, these data indicate that the PIP2–VAP27 interaction in EPCSs is evolutionarily conserved, and suggest that VAP27 might stabilise the aquaporins and guide their endocytosis in response to salt stress. [ABSTRACT FROM AUTHOR]

Plasma membrane intrinsic proteins (PIPs) belong to the aquaporin family and facilitate water movement across plasma membranes. Existing data indicate that PIP genes are associated with the abilities of plants to tolerate certain stress conditions. A review of our Glycine soja expressed sequence tag (EST) dataset revealed that abiotic stress stimulated expression of a PIP, herein designated as GsPIP2;1 (GenBank_Accn: FJ825766). To understand the roles of this PIP in stress tolerance, we generated a coding sequence for GsPIP2;1 by in silico elongation and cloned the cDNA by 5′-RACE. Semiquantitative RT-PCR showed that GsPIP2;1 expression was stimulated in G. soja leaves by cold, salt, or dehydration stress, whereas the same stresses suppressed GsPIP2;1 expression in the roots. Transgenic Arabidopsis thaliana plants overexpressing GsPIP2;1 grew normally under unstressed and cold conditions, but exhibited depressed tolerance to salt and dehydration stresses. Moreover, greater changes in water potential were detected in the transgenic A. thaliana shoots, implying that GsPIP2;1 may negatively impact stress tolerance by regulating water potential. These results, deviating from those obtained in previous reports, provide new insights into the relationship between PIPs and abiotic stress tolerance in plants. [ABSTRACT FROM AUTHOR]

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 CO permeability comparable to that of the CO diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes. [ABSTRACT FROM AUTHOR]

Highlights: [•] Significant water amounts are consumed daily for photosynthesis in one chloroplast. [•] Simple diffusion may sustain water supply at steady state photosynthetic rates. [•] Low Ea of water transport indicate the presence of aquaporins in thylakoids. [•] They could contribute to fast regulatory volume changes of the thylakoid lumen. [•] Proteomic evidence of PIPs and TIPs in chloroplasts is worth further investigation. [ABSTRACT FROM AUTHOR]

Calotropis procera, commonly known as 'milkweed', possesses long seed trichomes for seed dispersal and has the ability to survive under harsh conditions such as drought and salinity. Aquaporins are water channel proteins expressed in all land plants, divided into five subfamilies plasma membrane intrinsic proteins (PIPs), tonoplast intrinsic proteins (TIPs), NOD26-like proteins (NIPs), small basic intrinsic proteins (SIPs), and the unfamiliar X intrinsic proteins (XIPs). PIPs constitute the largest group of water channel proteins that are involved in different developmental and regulatory mechanisms including water permeability, cell elongation, and stomata opening. Aquaporins are also involved in abiotic stress tolerance and cell expansion mechanisms, but their role in seed trichomes (fiber cells) has never been investigated. A large number of clones isolated from C. procera fiber cDNA library showed sequence homology to PIPs. Both expressed sequence tags (ESTs) and real-time polymerase chain reaction (PCR) studies revealed that the transcript abundance of this gene family in fiber cells of C. procera is greater than that of cotton. Full-length cDNAs of CpPIP1 and CpPIP2 were isolated from C. procera fiber cDNA library and used for constructing plant expression vectors under constitutive (2×35S) and trichome-specific ( GhLTP3) promoters. Transgenic tobacco plants were developed via Agrobacterium-mediated transformation. The phenotypic characteristics of the plants were observed after confirming the integration of transgene in plants. It was observed that CpPIP2 expression cassette under 2×35S and GhLTP3 promoter enhanced the numbers of stem and leave trichomes. However, 2×35S:: CpPIP2 has a more amplified effect on trichome density and length than GhLTP3:: CpPIP2 and other PIP constructs. These findings imply the role of C. procera PIP aquaporins in fiber cell elongation. The PIPs-derived cell expansion mechanism may be exploited through transgenic approaches for improvement of fiber staple length in cotton and boosting of defense against sucking insects by enhancing plant pubescence. [ABSTRACT FROM AUTHOR]

Membrane intrinsic proteins facilitate movement of small molecules often times functioning as water channels. We have identified two genes from rice which encode proteins with characteristic features of plasma membrane intrinsic proteins (PIP). They possess six membrane-spanning domains, an NPA repeat, overall high sequence homologies and characteristic C- and N-terminal hallmark motifs which allowed assignment of OsPIP1a to the PIP1 subfamily and of OsPIP2a to the PIP2 subfamily. OsPIP1a and OsPIP2a showed similar but not identical expression patterns. The two genes were expressed at higher levels in seedlings than in adult plants and expression in the primary root was regulated by light. In internodes of deepwater rice plants which were induced to grow rapidly by submergence, transcript levels were slightly induced in the intercalary meristem (IM) and slightly reduced in the elongation zone (EZ) after 18 h. In internodes of GA-induced excised stem sections transcript levels transiently declined in the IM and EZ after 1 h and subsequently recovered to elevated levels after 18 h. GA also induced OsPIP expression in non-growing tissue after 18 h. In the IM of submergence-induced stem sections transcript levels remained constitutive. The different growth-promoting treatments showed no direct correlation between growth rate and OsPIP gene expression in dividing or expanding cells. In fact, treatment of excised stem sections with ABA or drought stress induced similar changes in OsPIP expression in the growing zone during the first 6 h as GA did. We conclude that regulation of OsPIP1a and OsPIP2a expression is not primarily controlled by growth. GA-induced growth may however change the water status of cells which in turn results in altered PIP abundance. [ABSTRACT FROM AUTHOR]

To gain insight into the function of plasma membrane intrinsic protein (PIP) genes in apple, two genes, MdPIP1a and MdPIP1b, were isolated. MdPIP1 expression was in accordance with the volume increase during fruit development, which is a loading process of water and solutes. In addition, the expression of MdPIP1 was up‐regulated in the stems by osmotic stress. These results indicate that MdPIP1 may play important roles not only in fruit expansion, but also in maintaining water homeostasis under stress conditions. [ABSTRACT FROM PUBLISHER]