Maize plasma membrane aquaporin ZmPIP2.docx

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Maize plasma membrane aquaporin ZmPIP2; 5, but not ZmPIP1;2, facilitates transmembrane diffusion of hydrogen peroxide Renz Byron M. Madamba Departmeny of Biological Sciences, Institute of Arts and Sciences, Far Eastern University, Nicanor Reyes Sr., Manila, 1008 Philippines ABSTRACT Aim Aquaporin belong to the major intrinsic protein superfamily and play important roles in facilitating the passive bi-directional diffusion of water and/or a variety of small and non-charged compounds across membranes in organisms. Plant plasma membrane intrinsic proteins (PIPs) cluster in two subgroups, PIP1 and PIP2. Using the yeast Saccharomyces cerevisiae they evaluate the maize aquaporin belonging to the PIP subfamily facilitate hydrogen peroxide diffusion. They demonstrated that, in contrast to wild type ZmPIP2; 5, ZmPIP1; 2 was not permeable to Hydrogen Peroxide. Methods ZmPIP cDNAs were optimized. Their sequences were used to PCR amplify the ZmPIP cDNA and generate ZmPIP constructs. PCR products were directionally sub-cloned into the USER-yeast expression vector. A S. cerevisiae strains 31019b (Δmep1–3) were pre cultured and subjected to Western blotting. Yeast cells expressing the fusion protein with another non-tagged PIP were grown on SD medium. During the exponential growth phase (A600 = 1.3 to 2.0), the fluorescence signal from the expressed fusion protein was observed using an epifluorescence microscope. Results Several Arabidopsis PIP2 facilitates the transmembrane diffusion in H2O2 when expressed in S. cerevisiae but the tested monocots were not sensitive to the concentration of H2O2 . Western blotting showed the presence of strong signals for ZmPIP1;2 and ZmPIP2;5.Expression of ZmPIP2;5 using the ZmPIP2;5OPT vector increased the sensitivity of yeast cells to H2O2. In contrast, yeast cells transformed with an empty vector or the ZmPIP1;2O OPT vector were not sensitive to concentrations of H2O2. These results suggest that ZmPIP2;5, but not ZmPIP1;2, facilitates the transport of H2O2 across yeast membranes. Main conclusion They demonstrated that, in contrast to the wild type ZmPIP2;5, ZmPIP1;2 is not permeable to hydrogen peroxide. Investigation of maize PIP transport properties in S. cerevisiae requires codon optimization of their cDNA to be significantly expressed and verification of their subcellular localization. Moreover, correct ZmPIP1;2 plasma membrane localization in S. cerevisiae relied on the concomitant presence of ZmPIP2;5, a mechanism which is conserved across organisms and must depend on protein features which remain to be identified. Article Bienert, G.P., R.B. Heinen, M.C. Berny and F. Chaumont (2014). Maize plasma membrane aquaporin ZmPIP2;5, not ZmPIP1;2 facilitates transmembrane diffusion of hydrogen peroxide. Biochim. Biophy. Acta 1838: 216-222


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