Role of PIP-Aquaporin Phosphorylation in Redox-Dependent Modulation of Osmotic Water Permeability in Plasmalemma from Roots of Pea Seedlings
- 12 Downloads
Investigations were directed to study the mechanism based on redox-dependent modulation of osmotic water permeability in plasmalemma from roots of pea (Pisum sativum L.) seedlings. This mechanism may be associated with oxidation of cysteine residues in PIP-aquaporins or involves redox-dependent phosphorylation of these proteins. Plasmalemma was isolated by partitioning of microsomal membranes in the aqueous polymer two-phase system and using the roots homogenization medium additionally contained SH-reagents, dithiothreitol or diamide, and also phenylarsin oxide, an inhibitor of tyrosine protein phosphatases. Water permeability of plasmalemma was estimated by the kinetics of light scattering changes of membrane vesicles due to their osmotic shrinkage and recorded by the stopped flow method. In order to elucidate whether PIP-aquaporins contain cysteines available for oxidation by SH-reagents, isolated plasmalemma was subjected to PEGylation (reaction, based on binding of 5 kD methoxypolyethylene glycol maleimide with available membrane proteins SH-groups) and further western blot analysis was performed to reveal shift of aquaporins molecular mass by 5 kD after their immunodetection with antibodies. Relative content of PIP-aquaporin phosphorylated forms in plasmalemma was determined using fluorescent labeling proteins in gels with ProQ Diamond after membrane proteins separation by two-dimensional electrophoresis (blue native (BN/PAGE) in first dimension and denaturing (SDS/PAGE) in second dimension). It was established that conservative cysteines in molecules of PIP-aquaporins are available for SH-reagents only under denaturing conditions. This fact excludes the possibility of these cysteines participation in modulation of plasmalemma osmotic water permeability. Comparative analysis of phosphorylated PIP-aquaporins pool with different redox status of membrane proteins and osmotic water permeability coefficients and also the inhibitor analysis data showed that redox-dependent modulation of these proteins activity is due to their phosphorylation with involvement of serine-threonine protein phosphatases of PP2C type and/or thyrosine protein phosphatases.
Keywords:Pisum sativum plasmalemma osmotic water permeability dithiothreitol diamide phenylarsin oxide cysteines PIP-aquaporins 5 kD PEG maleimide BN/SDS/PAGE phosphorylation ProQ Diamond
This work was supported by the Presidium of the Russian Academy of Sciences “Molecular and Cell Biology” Program.
COMPLIANCE WITH ETHICAL STANDARDS
The authors declare that they have no conflict of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.
- 1.Martínez-Ballesta, M.C., Rodríguez-Hernández, M.C., Alcaraz-López, C., Mota-Cadenas, C., Muries, B., and Carvajal, M., Plant hydraulic conductivity: the aquaporins contribution, hydraulic conductivity? in Issues, Determination and Applications, Elango, L., Ed., Rijeka: Intech, 2011, pp. 103–121.Google Scholar
- 4.Aroca, R., Amodeo, G., Fernández-Illescas, S., Herman, E.M., Chaumont, F., and Chrispeels, M.J., The role of aquaporins and membrane damage in chilling and hydrogen peroxide-induced changes in the hydraulic conductance of maize roots, Plant Physiol., 2005, vol. 137, pp. 341–353.CrossRefGoogle Scholar
- 6.Velikanov, G.A., Sibgatullin, T.A., Belova, L.P., and Ionenko, I.F., Membrane water permeability of maize root cells under two levels of oxidative stress, Pro-toplasma, 2015, vol. 252, pp. 1263–1273.Google Scholar
- 11.Santoni, V., Plant aquaporin posttranslational regulation, in Plant Aquaporins: Signaling and Communication in Plants, Chaumont, F. and Tyerman, S., Eds., Berlin: Springer, 2017, pp. 83–106.Google Scholar
- 20.Prak, S., Hem, S., Boudet, J., Viennois, G., Sommerer, N., Rossignol, M., Maurel, C., and Santoni, V., Multiple phosphorylations in the C-terminal tail of plant plasma membrane aquaporins: role in subcellular trafficking of AtPIP;1 in response to salt stress, Mol. Cell Proteomics, 2008, vol. 7, pp. 1019–1030.CrossRefGoogle Scholar
- 24.Garcia-Mata, C., Wang, J., Gajdanowicz, P., Gonzalez, W., Hills, A., Donald, N., Riedelsberger, J., Amtmann, A., Dreyer, I., and Blatt, M.R., A minimal cysteine motif required to activate the SKOR K+ channel of Arabidopsis by the reactive oxygen species H2O2, J. Biol. Chem., 2010, vol. 285, pp. 29286–29294.CrossRefGoogle Scholar