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

Pollen, a neighbor-less cell containing the male gametes, undergoes mechanical challenges during plant sexual reproduction, including desiccation and rehydration. It was previously shown 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 they 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 inactivates 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, the expression of a nonconducting MSL8 variant did not complement the msl8 overexpansion phenotype. These data indicate that contrary to our hypothesis and to the current understanding of MS ion channel function in bacteria, 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.

Interactions between a mechanosensitive channel and cell wall integrity signaling influence pollen germination in Arabidopsis thaliana.

Cells employ multiple systems to maintain cellular integrity, including mechanosensitive ion channels and the cell wall integrity (CWI) pathway. Here, we use pollen as a model system to ask how these different mechanisms are interconnected at the cellular level. MscS-Like 8 (MSL8) is a mechanosensitive channel required to protect Arabidopsis thaliana pollen from osmotic challenges during in vitro rehydration, germination, and tube growth. New CRISPR/Cas9 and artificial miRNA-generated msl8 alleles produced unexpected pollen phenotypes, including the ability to germinate a tube after bursting, dramatic defects in cell wall structure, and disorganized callose deposition at the germination site. We document complex genetic interactions between MSL8 and two previously established components of the CWI pathway, MARIS and ANXUR1/2. Overexpression of MARISR240C-FP suppressed the bursting, germination, and callose deposition phenotypes of msl8 mutant pollen. Null msl8 alleles suppressed the internalized callose structures observed in MARISR240C-FP lines. Similarly, MSL8-YFP overexpression suppressed bursting in the anxur1/2 mutant background, while anxur1/2 alleles reduced the strong rings of callose around ungerminated pollen grains in MSL8-YFP overexpressors. These data show that mechanosensitive ion channels modulate callose deposition in pollen and provide evidence that cell wall and membrane surveillance systems coordinate in a complex manner to maintain cell integrity.