Functional characterization of ZmPIP1 aquaporins in maize stomata

Aquaporins are channels facilitating the membrane diffusion of H2O, CO2 and other small solutes. Plasma membrane aquaporins (PIPs) are divided into two groups, PIP1s and PIP2s that show different subcellular localization and activities when expressed alone in Xenopus oocytes or plant cells. Zea mays PIP1s (ZmPIP1s) are retained in the endoplasmic reticulum while ZmPIP2s are found in the plasma membrane. When ZmPIP1s and ZmPIP2s are coexpressed, they assemble as heterotetramers resulting in the colocalization of both proteins in the plasma membrane and an increase in the cell membrane water permeability. PIPs are thought to contribute to the movements of stomata, pores at the leaf surface allowing gas exchange. Therefore, we decided (i) to develop tools to investigate the subcellular regulation of ZmPIP1s in maize stomatal complexes and (ii) to investigate the function of ZmPIP1;1 in maize stomata. We first generated three genetic constructs to express ZmPIP1;1, ZmPIP1;3 and ZmPIP1;6 isoforms fused with the mYFP protein under the control of the respective isoform promoter. These constructs were tested by transient expression in intact maize leaves or leaf mesophyll protoplasts by confocal imaging. Our results showed that the three constructs were functional. Then, parameters such as the stomatal conductance, the transpiration rate and the stomatal density were measured in plants deregulated in ZmPIP1;1. Two ZmPIP1;1 overexpressing lines (ZmPIP1;1 OE) and one knock-down line (zmpip1;1 KD) were compared with their respective wild-type plants. The stomatal conductance (gs) did not vary significantly in well-watered conditions, but the transpiration was higher for the ZmPIP1;1 OE plants. Interestingly, under water deficit conditions, a higher and lower transpiration rate was observed in ZmPIP1;1 OE and zmpip1;1 KD lines, respectively, indicating a role of ZmPIP1;1 in controlling the stomatal behavior in this condition. Further studies are required to determine the exact role of ZmPIP1;1 and to unveil if ZmPIP1;1 is also involved in the abscisic acid signaling pathway acting to close stomata.