Maize plasma membrane aquaporin ZmPIP2;5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide.

Plant aquaporins play important roles in transmembrane water transport processes, but some also facilitate the diffusion of other small uncharged solutes ranging from gases to metalloids. Recent evidence suggests that the transmembrane movement of hydrogen peroxide, an intra- and intercellular multifunctional signaling and defense compound, can be regulated by aquaporins. We addressed the question whether maize aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily facilitate hydrogen peroxide diffusion using heterologous expression in the yeast Saccharomyces cerevisiae. We showed that ZmPIP proteins belonging to the PIP1 and PIP2 groups were significantly expressed in yeast cells only after codon optimization of their cDNA. In accordance with previous localization studies in oocytes and plants, ZmPIP1;2 was mainly retained in intracellular membranes, while ZmPIP2;5 was localized to the plasma membrane. However, upon co-expression with ZmPIP2;5, ZmPIP1;2 was re-localized to the plasma membrane. Using a non-functional plasma membrane-localized ZmPIP2;5 mutant to deliver ZmPIP1;2 to the plasma membrane, we demonstrated that, in contrast to wild type ZmPIP2;5, ZmPIP1;2 was not permeable to hydrogen peroxide. Our study further highlighted the fact that, when using the yeast system, which is widely employed to study substrates for plant aquaporins and other transporters, although positive transport assay results allow direct conclusions to be drawn regarding solute permeability, negative results require additional control experiments to show that the protein is expressed and localized correctly before concluding on the lack of transport activity.

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.

Role of aquaporins in leaf physiology.

Playing a key role in plant growth and development, leaves need to be continuously supplied with water and carbon dioxide to fulfil their photosynthetic function. On its way through the leaf from the xylem to the stomata, water can either move through cell walls or pass from cell to cell to cross the different tissues. Although both pathways are probably used to some degree, evidence is accumulating that living cells contribute substantially to the overall leaf hydraulic conductance (K(leaf)). Transcellular water flow is facilitated and regulated by water channels in the membranes, named aquaporins (AQPs). This review addresses how AQP expression and activity effectively regulate the leaf water balance in normal conditions and modify the cell membrane water permeability in response to different environmental factors, such as irradiance, temperature, and water supply. The role of AQPs in leaf growth and movement, and in CO(2) transport is also discussed.

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.