Functional characterization of members of plasma membrane intrinsic proteins subfamily and their involvement in metalloids transport in plants

Aquaporins (AQPs) are channel proteins that facilitate the transport of water and various low molecular weight solutes including metalloids. Plant aquaporins have been divided into four major subfamilies: plasma membrane intrinsic proteins (PIPs), NOD26-like intrinsic proteins (NIPs), tonoplast intrinsic proteins (TIPs), and small basic intrinsic proteins (SIPs). Various studies have shown that the transport of metalloids including arsenite, antimonite, silicon and boron in plants is facilitated by members of NIP subfamily. In this study, we provided experimental evidences showing that members of rice PIP subfamily are involved in arsenite and boron permeability. RT-PCR analysis of seven OsPIPs; OsPIP1;2, OsPIP1;3, OsPIP2;4, OsPIP2;5, OsPIP2;6, OsPIP2;7, and OsPIP2;8 showed that these genes were downregulated under arsenite toxicity in shoots and roots. Whereas, these OsPIP genes were deferentially regulated in shoots and highly induced in roots by boron toxicity. Heterologous expression in Xenopus laevis oocytes showed that OsPIP2;4, OsPIP2;6, and OsPIP2;7 significantly increased the transport of arsenite. Expression of OsPIP candidate genes in HD9 yeast strain lacking the metalloids influx and efflux systems resulted in an increased boron sensitivity and accumulation. Overexpression of two OsPIP candidates; OsPIP1;3 and OsPIP2;6 in Arabidopsis yielded enhanced arsenite and boron tolerance with higher biomass and greater root length compared to wild type plants, however there was no difference in arsenic and boron accumulation in long-term uptake assays. Short duration exposure to AsIII resulted in both active influx and efflux of As in shoots and roots, suggesting a bidirectional transport activity of OsPIPs. Whereas, short-term uptake assay of tracer B (10B) in shoots and roots demonstrated increased 10 B influx in transgenic Arabidopsis lines indicating that these OsPIPs are also involved in mediating B transport in plants. We used RNAi approach to knockdown the expression of OsPIP1;3 and OsPIP2;6 in rice. We generated RNAi lines for both genes and qRT-PCR analysis showed a significant decrease in the transcript levels for OsPIP1;3 and OsPIP2;6. These RNAi lines will be the subject of future studies. These OsPIPs genes will be highly useful in developing arsenite and boron tolerant crops for enhanced yield in the areas affected by high As and B toxicity