This was a nice and fun journey with great colleagues and a supportive PI @robinsonsci.bsky.social, it would not have been possible without the fantastic working environment of @slcuplants.bsky.social 12/12

Since these approaches couldn’t rule out a wounding effect, we’ve decided to use a more gentle approach: tissue folding. Again we observed a change of division orientation after folding 10/12

At this point, we thought that mechanical stress could be the “mysterious factor” orienting stomata division, so we performed mechanical perturbations (stabbing or slicing with a needle), which led to a nice change in division orientations 9/12

Since we’ve found different orientation patterns between the 2 sides of cotyledons we decided to characterize the 2 sides in more detail to try to find the mysterious factor orienting stomata. For this, we used light sheet imaging, dual-view timelapse imaging, full 3D segmentation, and FEM 7/12

We time-lapsed cotyledons during stomata division and analyzed the orientation of division, on the abaxial side we found that the divisions were aligned with the organ axis and not with the cell major axis or growth direction, on the adaxial side the division were more disorganized 6/12

And rather than just looking at a few stomata divisions, we’ve made all epidermal cells turn into stomata using the iMUTE inducible line (Han et al 2018), that’s much funnier 5/12

On the abaxial side, the organ scale alignment is quite obvious in the early stages, but the stomata formed later in development have more random orientations. We’ve also looked at the adaxial side, and interestingly 2 days after germination the new stomata are not aligned anymore 3/12

We first characterized the stomata orientation pattern in Arabidopsis Cotyledons, for this, we’ve manually annotated more than 10000 stomata (that’s a lot of clicking) and mapped their orientation relative to the proximo-distal axis. Here are some of these maps on the abaxial side 2/12

More details on our recent pre-print:
Alignments of stomata have been described in many species, here are 2 examples: Thym from my kitchen, and Leucadendron from a 1935 paper (Smith 1935 on the orientation of stomata)
What is directing these alignments is unknown 1/12
doi.org/10.1101/2024...