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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]

In order to improve the understanding of plant water relations under drought stress, the water use behavior of two Fragaria x ananassa Duch. cultivars, contrasting in their drought stress phenotype, is identified. Under drought, stomatal closure is gradual in Figaro. Based on this, we associate Figaro with conservative water use behavior. Contrarily, drought stress causes a sudden and steep decrease in stomatal conductance in Flair, leading to the identification of Flair as a prodigal water use behavior cultivar. Responses to progressive drought on the one hand and an osmotic shock on the other hand are compared between these two cultivars. Tonoplast intrinsic protein mRNA levels are shown to be upregulated under progressive drought in the roots of Figaro only. Otherwise, aquaporin expression upon drought or osmotic stress is similar between both cultivars, i.e. plasma membrane intrinsic proteins are downregulated under progressive drought in leaves and under short term osmotic shock in roots. In response to osmotic shock, root hydraulic conductivity did not change significantly and stomatal closure is equal in both cultivars. De novo abscisic acid biosynthesis is upregulated in the roots of both cultivars under progressive drought. • Figaro is a conservative water use behavior cultivar, Flair a prodigal one. • TIP aquaporins are upregulated in roots of Figaro (conservative) only under drought. • Root ABA biosynthesis is similar between both cultivars. • Root hydraulic conductivity is not different upon osmotic shock. • Root aquaporin expression upon osmotic shock is similar between both cultivars. [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]

Abstract: Background: Hydrogen peroxide (H2O2) is an important signaling compound that has recently been identified as a new substrate for several members of the aquaporin superfamily in various organisms. Evidence is emerging about the physiological significance of aquaporin-facilitated H2O2 diffusion. Scope of review: This review summarizes current knowledge about aquaporin-facilitated H2O2 diffusion across cellular membranes. It focuses on physicochemical and experimental evidence demonstrating the involvement of aquaporins in the transport of this redox signaling compound and discusses the regulation and structural prerequisites of these channels to transmit this signal. It also provides perspectives about the potential importance of aquaporin-facilitated H2O2 diffusion processes and places this knowledge in the context of the current understanding of transmembrane redox signaling processes. Major conclusions: Specific aquaporin isoforms facilitate the passive diffusion of H2O2 across biological membranes and control H2O2 membrane permeability and signaling in living organisms. General significance: Redox signaling is a very important process regulating the physiology of cells and organisms in a similar way to the well-characterized hormonal and calcium signaling pathways. Efficient transmembrane diffusion of H2O2, a key molecule in the redox signaling network, requires aquaporins and makes these channels important players in this signaling process. Channel-mediated membrane transport allows the fine adjustment of H2O2 levels in the cytoplasm, intracellular organelles, the apoplast, and the extracellular space, which are essential for it to function as a signal molecule. This article is part of a Special Issue entitled Aquaporins. [Copyright &y& Elsevier]

Abstract: Seedlings of Citrus sinensis were fertilized with boron (B)-deficient (0μM H3BO3) or -sufficient (10μM H3BO3) nutrient solution for 15weeks. Thereafter, iTRAQ analysis was employed to compare the abundances of proteins from B-deficient and -sufficient roots. In B-deficient roots, 164 up-regulated and 225 down-regulated proteins were identified. These proteins were grouped into the following functional categories: protein metabolism, nucleic acid metabolism, stress responses, carbohydrate and energy metabolism, cell transport, cell wall and cytoskeleton metabolism, biological regulation and signal transduction, and lipid metabolism. The adaptive responses of roots to B-deficiency might include following several aspects: (a) decreasing root respiration; (b) improving the total ability to scavenge reactive oxygen species (ROS); and (c) enhancing cell transport. The differentially expressed proteins identified by iTRAQ are much larger than those detected using 2D gel electrophoresis, and many novel B-deficiency-responsive proteins involved in cell transport, biological regulation and signal transduction, stress responses and other metabolic processes were identified in this work. Our results indicate remarkable metabolic flexibility of citrus roots, which may contribute to the survival of B-deficient plants. This represents the most comprehensive analysis of protein profiles in response to B-deficiency. Biological significance: In this study, we identified many new proteins involved in cell transport, biological regulation and signal transduction, stress responses and other metabolic processes that were not previously known to be associated with root B-deficiency responses. Therefore, our manuscript represents the most comprehensive analysis of protein profiles in response to B-deficiency and provides new information about the plant response to B-deficiency. This article is part of a Special Issue entitled: Translational Plant Proteomics. [Copyright &y& Elsevier]

Abstract: It is postulated that leaf thermonasty (leaf curling) in rhododendrons under sub-freezing temperatures is caused by water redistribution due to extracellular freezing. We hypothesize that aquaporins (AQPs), the transmembrane water-channels, may be involved in regulating water redistribution and thus leaf curling. Our experimental system includes two Rhododendron species with contrasting leaf curling behavior whereby it was observed in R. catawbiense but not in R. ponticum. We compared leaf movements and the expression of two AQPs, i.e. R. catawbiense/ponticum plasma-membrane intrinsic protein 2 (Rc/RpPIP2;1 and Rc/RpPIP2;2), in the two species under freezing–rewarming and dehydration–rehydration cycles. To determine the relationship between extracellular freezing and leaf-curling, we monitored leaf-curling in R. catawbiense with or without controlled ice-nucleation. Our data indicate that extracellular freezing may be required for leaf curling. Moreover, in both species, PIP2s were up-regulated at temperatures that fell in ice-nucleation temperature range. Such up-regulation could be associated with the bulk-water efflux caused by extracellular freezing. When leaves were frozen beyond the ice-nucleation temperature range, PIP2s were continuously down-regulated in R. catawbiense along with the progressive leaf curling, as also observed for RcPIP2;2 in dehydrated leaves; as leaves uncurled during re-warming/rehydration, RcPIP2 expression was restored. On the other hand, R. ponticum, a non-curling species, exhibited substantial up-regulation of RpPIP2s during freezing/dehydration. Taken together, our data suggest that RcPIP2 down-regulation was associated with leaf curling. Moreover, the contrasting PIP2 expression patterns combined with leaf behavior of R. catawbiense and R. ponticum under these two cycles may reflect different strategies employed by these two species to tolerate/resist cellular dehydration. [Copyright &y& Elsevier]

Abstract: Iron (Fe) homeostasis is essential for life and has been intensively investigated for dicots, while our knowledge for species in the Poaceae is fragmentary. This study presents the first proteome analysis (LC–MS/MS) of plasma membranes isolated from roots of 18-day old maize (Zea mays L.). Plants were grown under low and high Fe conditions in hydroponic culture. In total, 227 proteins were identified in control plants, whereas 204 proteins were identified in Fe deficient plants and 251 proteins in plants grown under high Fe conditions. Proteins were sorted by functional classes, and most of the identified proteins were classified as signaling proteins. A significant number of PM-bound redox proteins could be identified including quinone reductases, heme and copper-containing proteins. Most of these components were constitutive, and others could hint at an involvement of redox signaling and redox homeostasis by change in abundance. Energy metabolism and translation seem to be crucial in Fe homeostasis. The response to Fe deficiency includes proteins involved in development, whereas membrane remodeling and assembly and/or repair of Fe–S clusters is discussed for Fe toxicity. The general stress response appears to involve proteins related to oxidative stress, growth regulation, an increased rigidity and synthesis of cell walls and adaption of nutrient uptake and/or translocation. This article is part of a Special Issue entitled: Plant Proteomics in Europe. [Copyright &y& Elsevier]

Abstract: Jatropha has potential to be an important bio-fuel crop due to such advantages as high seed oil content and the ability to grow well on marginal lands less suited for food crops. Despite its ability to grow on marginal land, Jatropha is still susceptible to high salt and drought stresses, which can significantly reduce plant growth, stomatal conductance, sap-flow rate, and plant sap volume. This study was undertaken to collect basic knowledge of the physiological and molecular aspects of Jatropha response to salt and drought stresses, and to elucidate how Jatropha recovers from stress. From these studies we identified candidate genes that may be useful for the development of Jatropha cultivars that will grow efficiently in arid and barren lands. Of particular interest, two plasma membrane intrinsic proteins were identified: Jatropha plasma membrane intrinsic protein 1 (JcPIP1) and Jatropha plasma membrane intrinsic protein 2 (JcPIP2). The expression levels of JcPIP1 were dramatically increased in roots, stems, and leaves during the recovery from stress, whereas the JcPIP2 gene transcripts levels were induced in roots and stems during the water deficit stress. The protein levels of JcPIP1 and JcPIP2 were consistent with the gene expression patterns. Based on these results, we hypothesized that JcPIP1 plays a role in the recovery events from water stresses, while JcPIP2 is important in early responses to water stress. Virus induced gene silencing technology revealed that both JcPIP1 and JcPIP2 have positive roles in response to water deficit stresses, but have antagonistic functions at the recovery stage. We suggest that both JcPIP1 and JcPIP2 may play important roles in responses to water deficit conditions and both have potential as targets for genetic engineering. [Copyright &y& Elsevier]

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]