CLASP balances two competing cell division plane cues during leaf development

Starting as small, densely packed boxes, leaf mesophyll cells expand to form an intricate mesh of interconnected cells and air spaces, the organization of which dictates the internal surface area of the leaf for light capture and gas exchange during photosynthesis. Despite their importance, little is known about the basic patterns of mesophyll cell division, and how they contribute to cell and intercellular space organization. To address this, we tracked divisions within individual cell lineages in three dimensions over time in Arabidopsis spongy mesophyll. We found that early on, successive cell division planes switch their orientation such that each new cell wall intersects the previous at a right angle, creating a new multi-cell junction (the intersection of three or more cells). These junctions then open to create intercellular spaces. During subsequent enlargement of the spaces, the division planes of the surrounding cells show an increasing tendency to tilt in the direction of their adjacent intercellular spaces. This disrupts the alternating pattern, and by extension, halts the initiation of new multi-cell junctions and intercellular spaces, but allows the expansion of existing spaces. Both division patterns are specified before mitosis by the orientation of interphase cortical microtubules, which gradually narrow to form a preprophase band in the same orientation to establish the future plane of cell division. In the absence of the microtubule-associated protein CLASP, the early alternating division plane and microtubule patterns are compromised, whereas space-oriented divisions are exacerbated. This results in large distortions of the topological relations between cells and intercellular spaces, as well as changes in their relative abundance. Our data reveal the existence of two competing cell division mechanisms that are balanced by CLASP to specify the distribution of cells and intercellular spaces in spongy mesophyll tissue. Division tracking of mesophyll cells shows that initial division plane orientations alternate creating cell walls at right angles, and intercellular spaces. Disruption of this pattern halts initiation of multi-cell junctions and intercellular spaces.