Investigating the Role of Arabidopsis Plasma Membrane Intrinsic Protein AtPIP2;1 in Seed Germination

Abstract

Soil salinity can cause osmotic stress during seed germination by limiting water uptake and causing NaCl accumulation. Exposure to salinity stress during seed imbibition can alter germination percentage, slow the germination rate and seedling growth. Subsets of membrane intrinsic proteins called aquaporins contribute to salinity stress tolerance in plants, and some isoforms of these proteins could have roles in helping seeds to germinate in saline conditions. Aquaporins have multiple roles in many physiological processes, such as solute transport, hydraulic conductance, signalling, development and adjusting to changes in the environment. Recent studies have revealed that some plant aquaporins are candidates for being involved in both water and Na+ transport. For example, Arabidopsis thaliana Plasma Membrane Intrinsic (PIP) proteins AtPIP2;1 and AtPIP2;2 transported both water and univalent cations (Na+ and K+) when tested in heterologous systems. To explore whether AtPIP2;1 influenced Na+ transport in plants, Atpip2;1 loss of function mutants and wild type Columbia-0 seed were germinated on media containing 50 mM NaCl. The wild type Arabidopsis seed appeared to germinate earlier than the seed from the Atpip2;1 mutant lines. This preliminary observation led to the hypothesis that there may be a link between the function of AtPIP2;1 and factors that influence seed germination percentage in Arabidopsis. To test this hypothesis, wild type Arabidopsis, two Atpip2;1 mutant lines, a Atpip2;1*Atpip2;2 mutant line, and two transgenic lines overexpressing AtPIP2;1 (35S::AtPIP2;1) and a null control line were propagated in controlled conditions and seed was harvested from each of these seven genotypes. The seed was used to test whether the seven genotypes differed in germination percentage and seed area, weight and Na+ and K+ content when imbibed in control or saline treatment. This research aims to investigate the role of an aquaporin protein, AtPIP2;1, in seeds in the model plant Arabidopsis thaliana. A series of exploratory experiments were carried to explore possible roles for this protein in seed germination, with a particular focus in germination under salt stress. In saline conditions, seed from Atpip2;1 mutant lines and lines overexpressing AtPIP2;1 germinated slower than wild type and null control lines, respectively. Fifty hours after sowing, the seedlings of one mutant Atpip2;1 line weighed more than wild type, and seedlings of both 35S::AtPIP2;1 overexpression lines weighed less than null controls. When imbibed in saline conditions, Atpip2;1 mutant line seed contained more K+ than wild type seed. Whereas seed from lines overexpressing AtPIP2;1 contained more K+ than null line seed when imbibed for 30 h in water then transferred to a solution containing 75 mM NaCl from 30 to 50 h after sowing, but not when imbibed in the saline treatment from 0 to 50 h. In conclusion, the potential for these observations to be a consequence of AtPIP2;1 transport of water, ions or hormones, or linked to protein-protein interactions and signaling roles are discussed. Options for future experiments targeted at distinguishing which aspects of AtPIP2;1 functions are important in contributing to optimal seed germination in saline treatment are considered.