Basic Proline-Rich Protein-Mediated Microtubules Are Essential for Lobe Growth and Flattened Cell Geometry

Abstract

Complex cell shapes are generated first by breaking symmetry, and subsequent polar growth. Localized bending of anticlinal walls initiates lobe formation in the epidermal pavement cells of cotyledons and leaves, but how the microtubule cytoskeleton mediates local cell growth, and how plant pavement cells benefit from adopting jigsaw puzzle-like shapes, are poorly understood. In Arabidopsis (Arabidopsis thaliana), the basic Pro-rich protein (BPP) microtubule-associated protein family comprises seven members. We analyzed lobe morphogenesis in cotyledon pavement cells of a BPP1;BPP2;BPP5 triple knockout mutant. New image analysis methods (MtCurv and BQuant) showed that anticlinal microtubule bundles were significantly reduced and cortical microtubules that fan out radially across the periclinal wall did not enrich at the convex side of developing lobes. Despite these microtubule defects, new lobes were initiated at the same frequency as in wild-type cells, but they did not expand into well-defined protrusions. Eventually, mutant cells formed nearly polygonal shapes and adopted concentric microtubule patterns. The mutant periclinal cell wall bulged outward. The radius of the calculated inscribed circle of the pavement cells, a proposed proxy for maximal stress in the cell wall, was consistently larger in the mutant cells during cotyledon development, and correlated with an increase in cell height. These bpp mutant phenotypes provide genetic and cell biological evidence that initiation and growth of lobes are distinct morphogenetic processes, and that interdigitated cell geometry effectively suppresses large outward bulging of pavement cells.

Real-time conversion of tissue-scale mechanical forces into an interdigitated growth pattern

“…This exceeds the radius of curvature threshold of 2.5 μm, below which CLASP-dependent stabilization of transfacial microtubules operates 36 . These phenotypes could be explained by a defect in the maintenance of lobe outgrowth rather than lobe initiation, similar to what has been shown for the microtubule-binding protein BPP 30 . A possible effect of the microtubule plus-end binding MOR1 on pavement cell shape has been reported 37 .…”

“…Lobe formation is first detected as a local nanoscale ~300 nm warping 15 that is driven by microtubule and cellulose microfibril-programmed anisotropy in a small patch of the cell wall in an initiating cell. These tiny features have a multiscale influence on morphogenesis because they become stabilized, and slow irreversible growth over a period of days morphs the feature into a well-defined lobe with features at ~10 - 50 μm scales 17,25,30 . This work defines the mechanism, timeline, and type of cell wall patterning that serves as a general model for polarized diffuse growth and provides a foundation to analyze epidermal morphogenesis across wide spatial scales.…”

“…Along the anticlinal wall, the distance from and angles among cell walls at 3-way junctions 51 may also affect stress distributions. Tensile stress is sensitive to the material property gradients in the cell wall and varies inversely with cell wall thickness 30 . Wall stress patterns may also reflect the local growth status, because within a pavement cell, growth rates within a segment often vary by factors of 2 to 7 (Figure 1f and Supplementary Figure 2j), and wall thickness varies by a factor of ~2.5 15 due to imperfect maintenance of wall thickness during cell expansion.…”

“…In pavement cells, longstanding models propose that stable microtubules synthesize cellulose arrays or cell wall thickenings that locally restrict growth and cause lobe outgrowths to appear at adjacent subdomains of the cell 11,22 . In Arabidopsis, the microtubule system is unstable and anticlinal microtubules bundling is not associated with local cell wall thickening 15,30 . An alternative growth-promotion model proposes that localized microtubule arrays pattern cellulose microfibrils to generate a patch of local anisotropic expansion that drives symmetry breaking 13 .…”

Regulation of cytoskeleton‐associated protein activities: Linking cellular signals to plant cytoskeletal function

“…The cytoskeleton maintains cellular architecture, limits membrane protein diffusion, and organizes cellular components (Fletcher and Mullins, 2010; Lv et al, 2017; Ren et al, 2017). Moreover, the cytoskeleton generates the coordinated forces required for many morphogenetic processes during development and acts as a key route for vesicular transport (Wang et al, 2017; Liu et al, 2018; Wong et al, 2019). It physically and biochemically connects cells to the external environment and is therefore considered to function as a sensor of developmental and environmental signals (Nick, 2013; Chen et al, 2016; Hamant et al, 2019).…”