Live imaging of microtubule organization, cell expansion, and intercellular space formation in Arabidopsis leaf spongy mesophyll cells

Leaf spongy mesophyll cells form an interconnected network of branched cells and intercellular spaces to maximize the surface area available for light capture and photosynthetic gas exchange. To investigate the morphogenetic events leading to cell separation and branching in Arabidopsis thaliana, we used mesophyll-specific promoters to facilitate imaging of mesophyll cell shape and microtubule (MT) organization over multiple spatiotemporal scales without interference from the overlying epidermal cells. We show that cells enlarge by selective expansion of cell wall regions in contact with intercellular spaces. Cell–cell contacts remain relatively fixed in size, forming the termini of interconnecting branches. Surprisingly, classic schizogeny (de-adhesion of neighboring cells) is relatively infrequent, being related to the local topology of cell junctions during early expansion. Intercellular spaces cue the position of stable MT bundles, which in turn promote efficient dilation of intercellular spaces and cell branching. Our data provide insights into mesophyll morphogenesis and MT organization and lay the groundwork for future investigations.