Multivariate analysis of leaf tissue morphogenesis
thesisposted on 10.05.2020, 20:49 by Samuel Belteton
Leaf size and shape are strongly influenced by the growth patterns of the epidermal tissue. Pavement cells are the prevalent cell type in the epidermis and during cell expansion they undergo a drastic shape change from a simple polyhedral cells to puzzled-shaped cell. The role of these cell protrusions, more commonly referred to as lobes, remains unknown but their formation has been proposed to help increase the structural integrity of the epidermal tissue. How the symmetry breaking event that initiates a lobe is controlled remains unknown, however pharmacological and genetic disruption of the microtubule system has been shown to interfere not only with lobe initiation but also with lobe expansion. Additionally, the role of microtubules in the pattering of microfibril deposition, the load-bearing structure of the cell wall, makes the microtubule system a good candidate to evaluate its dynamics as a function of shape change. Two main mechanical models for lobe initiation are evaluated here, one where microtubules serve as stable features suppressing local expansion and one where microtubules, similarly to the anisotropic expansion patterning in hypocotyl cells, pro-mote the local anisotropic expansion of the cell resulting in lobe formation. The main method to evaluate these models was through the use of long-term time-lapse image analysis using a plasma-membrane marker for accurate shape change quantification and a microtubule marker to quantify their location, persistence, and density as a function of cell shape change. Using the junctions where three cells come together,cells were sub-divided into segments and the shape of these segments were tracked using a new coordinate system that allowed the detection of new lobes as which can arise from ∼300 deflections. By mapping sub-cellular processes, such as microtubule persistence, to this coordinate system, correlations of microtubule organization and shape change was possible. Additionally, a subset of microtubules bundles that splay across the anticlinal and periclinal walls, perpendicular and parallel to the leaf surface respectively, were identified as marking the location and direction of lobe formation.Disrupting the cell boundary by partially digesting pectin, a main component in the middle lamella, revealed the cell-autonomous morphogenesis mechanism in pavementcells. Under pectinase treatment, cell invaginations were produced and similarly to lobes their initiation was microtubule and cellulose dependent. Lastly, stress prediction using finite-element models, based from live-cell images, co-localized regions of high cell wall stress with both microtubule persistence and shape shape locations in both lobing and invaginated segments. Together, a model of cellular shape change is presented where microtubules translate cell wall stresses to tissue morphogenesis.