In vitro experiments and kinetic models of pollen hydration show that MSL8 is not a simple tension-gated osmoregulator

Pollen, a neighbor-less cell that contains the male gametes, undergoes multiple mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously showed that the pollen-specific mechanosensitive ion channel MscS-Like (MSL)8 is essential for pollen survival during hydration and proposed that it functions as a tension-gated osmoregulator. Here we test this hypothesis with a combination of mathematical modeling and laboratory experiments. Time-lapse imaging revealed that wild-type pollen grains swell and then stabilize in volume rapidly during hydration. msl8 mutant pollen grains, however, continue to expand and eventually burst. We found that a mathematical model wherein MSL8 acts as a simple tension-gated osmoregulator does not replicate this behavior. A better fit was obtained from variations of the model wherein MSL8 inactivation is independent of its membrane tension gating threshold or MSL8 strengthens the cell wall without osmotic regulation. Experimental and computational testing of several perturbations, including hydration in an osmolyte-rich solution, hyper-desiccation of the grains, and MSL8-YFP overexpression, indicated that the Cell Wall Strengthening Model best simulated experimental responses. Finally, expression of a non-conducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that, contrary to our hypothesis and to known MS ion channel function in single-cell systems, MSL8 does not act as a simple membrane tension-gated osmoregulator. Instead, they support a model wherein ion flux through MSL8 is required to alter pollen cell wall properties. These results demonstrate the utility of pollen as a cellular-scale model system and illustrate how mathematical models can correct intuitive hypotheses. ### Competing Interest Statement The authors have declared no competing interest.