Changes in morphogen kinetics and pollen grain size are potential mechanisms of aberrant pollen aperture patterning in previously observed and novel mutants of Arabidopsis thaliana

Pollen provides an excellent system to study pattern formation at the single-cell level. Pollen surface is covered by the pollen wall exine, whose deposition is excluded from certain surface areas, the apertures, which vary between the species in their numbers, positions, and morphology. What determines aperture patterns is not understood. Arabidopsis thaliana normally develops three apertures, equally spaced along the pollen equator. However, Arabidopsis mutants whose pollen has higher ploidy and larger volume develop four or more apertures. To explore possible mechanisms responsible for aperture patterning, we developed a mathematical model based on the Gierer-Meinhardt system of equations. This model was able to recapitulate aperture patterns observed in the wild-type and higher-ploidy pollen. We then used this model to further explore geometric and kinetic factors that may influence aperture patterns and found that pollen size, as well as certain kinetic parameters, like diffusion and decay of morphogens, could play a role in formation of aperture patterns. In conjunction with mathematical modeling, we also performed a forward genetic screen in Arabidopsis and discovered two mutants with aperture patterns that had not been previously observed in this species but were predicted by our model. The macaron mutant develops a single ring-like aperture, matching the unusual ring-like pattern produced by the model. The doughnut mutant forms two pore-like apertures at the poles of the pollen grain. Further tests on these novel mutants, motivated by the modeling results, suggested the existence of an area of inhibition around apertures that prevents formation of additional apertures in their vicinity. This work demonstrates the ability of the theoretical model to help focus experimental efforts and to provide fundamental insights into an important biological process.Author summary: Pollen is renowned for its ability to form beautiful and complex patterns on its surface. One of the most prominent patterns on the pollen surface is formed by apertures, the regions that lack deposition of the pollen wall exine and develop at precise locations which often vary between the species. How aperture patterns are created is an intriguing and poorly understood question. We developed a mathematical model that aims to explore the mechanisms responsible for the aperture patterning in the pollen of the model plant Arabidopsis. Our model showed that size of the pollen grain could be solely responsible for the increase in aperture number observed in the pollen of some Arabidopsis mutants. Additionally, kinetic parameters, such as diffusion and decay of aperture factors, could also influence aperture number. We coupled our mathematical modeling with a forward genetic screen of a mutagenized population of Arabidopsis. This screen discovered novel mutants with aperture patterns that had been predicted by our mathematical model. Further experiments on these mutants provided additional support to the modeling predictions. These results demonstrate that mathematical modeling could be a powerful tool for understanding the mechanisms responsible for patterning of pollen grains.