Live-Cell Imaging of Spongy Mesophyll Morphogenesis and Concomitant Microtubule Organization in Arabidopsis Leaves
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. In spite of their importance for photosynthetic activities, the morphogenesis processes of spongy mesophyll are still poorly understood largely due to the difficulties of internal tissues imaging. To investigate the morphogenetic events leading to cell separation and branching in Arabidopsis thaliana, I 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. By means of live-cell imaging and quantitative images analysis, I show that cells enlarge by selective expansion of cell wall regions in contact with intercellular spaces. Meanwhile the cell–cell contacts remain relatively fixed in size, forming the termini of interconnecting branches. Intercellular spaces provide a strong positional cue for the stable MT bundles, which in turn promote efficient dilation of intercellular spaces and cell branching. My data provide insights into mesophyll morphogenesis and MT organization and lay the groundwork for future investigations. Distinct from its overlying epidermis layer, spongy mesophyll cells not only undergo division and expansion, also separate from their neighbor cells to form the labyrinth of intercellular spaces. Seeking to understand the coordination between cell division, expansion and separation during mesophyll morphogenesis, I carefully examined mesophyll’s cell division pattern. My results revealed a unique alternating division pattern, wherein division planes tend to orient perpendicular to their parental walls. After the absence of CLASP, cell divisions became more or less parallel instead in clasp-1 mutant, which caused severe defects in mesophyll tissue topology and geometry. In short, my results showed that the unique CLASP-dependent alternating cell division pattern programs the distribution pattern of air spaces, which in return affect the subsequent cell division orientations, thus forming a dynamic feedback loop, which is crucial for the normal mesophyll morphogenesis.
live-cell imaging, spongy mesophyll, Arabidopsis
Doctor of Philosophy (Ph.D.)