Functional characterization of PIP aquaporins expressed in N2-fixing nodules of Medicago truncatula

CINTIA JOZEFKOWICZ; FLORENCIA SCOCHERA; NICOLAS AYUB; GABRIELA SOTO; GERD PATRICK BIENERT; KARINA ALLEVA

Salto

Congreso; Latin American Crosstalk in Biophysics and Physiology; 2015

Sociedad Argentina de Biofísica y Seccional Biofísica de la Sociedad Uruguaya de Biociencias

Plant aquaporins (AQP) are channel proteins regulating the transmembranetransport of water and small molecules. Here, we aim at studying the roleof AQP expressed in N2-fixing nodules of M truncatula. First, a surveyusing microarray analysis of M. truncatula Gene Expression Atlas(http://mtgea.noble.org/v3/) was conducted, we identified the completecoding sequences of MtAQP which are highly expressed in active nodules.Subsequently, we cloned the coding sequences of the selected AQPbelonging to the PIP (plasma membrane instrinsic protein) subfamily. Weperformed bioinformatic studies and subcloned the MtPIP to functionallycharacterize them in the heterologous yeast expression system S. cerevisiae.We: i- tested their localization by confocal fluorescence microscopy; iiperformedwater transport swelling experiments in spheroplasts using astopped-flow fast kinetics instrument; iii- performed toxicity growth andcomplementation assays in yeast deletion mutants capable to monitor AQPpermeability to hydrogen peroxide, boric acid, ammonia and urea; and iv analyzedthe interaction between PIPs belonging to the PIP1 and PIP2clusters3,4. Results show that both analyzed PIP2 transport water andhydrogen peroxide, and the fluorescent signal derived from GFP:PIP2fusion proteins was mainly detected in the plasma membrane. In contrast,the fluorescent signal pattern observed for GFP:PIP1 was restricted tointracellular structures. Interestingly, upon PIP1-PIP2 co-expression thefluorescent signal of GFP:PIP1 fusion proteins was mainly localized in theplasma membrane. However, PIP1 are not enhancing the membrane waterpermeability or hydrogen peroxide transport upon co-expression with PIP2.These results provide new experimental evidence on the selectivity andfunctionality of legume AQP. for lysis.