Date of Award
Doctor of Philosophy (PhD)
Directional growth in plants is primarily determined by the axis of cell expansion, which is specified by the net orientation of cortical microtubules. Microtubules guide the deposition of cellulose and other cell wall materials. In rapidly elongating cells, transversely oriented microtubules create material anisotropy in the cell wall that prevents radial cell expansion, channeling cell expansion in the longitudinal direction. Mutations perturbing microtubule organization frequently lead to aberrant cell growth in land plants, with some mutations leading to helical growth patterns (called ‘twisted mutants’), often in roots. This phenotype manifests as right-handed or left-handed twisting of cell files along the long axis of plant organs, which correlates with rightward or leftward organ growth, respectively. Helical growth is a common occurrence in the plant kingdom and serves a variety of purposes, but the molecular mechanisms that produce helical growth and define handedness are not well understood. Furthermore, how molecular-level processes propagate across spatial scales to control organ-level growth is undefined. Here, I used the model plant Arabidopsis thaliana as an experimentally tractable system, focusing on the root organ to study the mechanisms underlying helical growth in plants. In this work, I used roots as a model plant organ to investigate the molecular mechanisms that control symmetry maintenance and symmetry breaking in plants. Arabidopsis roots are ideally suited for this work because of their simple, concentric ring-like cellular anatomy and well-defined process of development. I selected two Arabidopsis twisted mutants with opposite chirality to study whether the emergence of right-handed and left-handed helical growth involves conserved or distinct mechanisms. Cortical microtubules are skewed in the right-handed spr1 mutant, which lacks a microtubule plus end-associated protein that regulates polymerization dynamics. In contrast, cortical microtubules tend to be laterally displaced in the left-handed cmu1 mutant, which lacks a protein that contributes to the attachment of cortical microtubules to the plasma membrane. Using a cell-type specific complementation approach, I showed that both SPR1 and CMU1 gene expression in the epidermis alone is sufficient to maintain wild-type-like straight cell files and root growth. In addition, epidermal expression of SPR1 restores both the morphology and skew of the cortical cell file to wild-type-like. By genetically disrupting cell-cell adhesion in the spr1 mutant, I found that a physical connection between epidermal and cortical cells is required for the epidermis to cause organ-level skewed growth. Together, these data demonstrate that the epidermis plays a central role in maintaining straight root growth, suggesting that twisted plant growth in nature could arise by altering microtubule behavior in the epidermis alone and does not require null alleles in all cells. To examine whether cortical microtubule defects in the spr1-3 mutant affect cell growth, I conducted morphometry analysis. I found that while skewed cortical microtubule orientation correlates with asymmetric epidermal cell morphology and growth in the spr1-3 mutant root meristem, cell file twisting is not manifested until the differentiation zone of the root where cell growth slows down and root hairs emerge. Furthermore, I demonstrated that cell file twisting is not sufficient to generate skewed growth at the organ level, which requires that the root is grown on an agar medium, a mechanically heterogeneous environment. Increasing the stiffness of the agar medium caused the spr1-3 and cmu1 mutant roots to grow straight, indicating that mechanical stimuli influence twisted root growth. Despite their important role in root anchorage, root hairs on the epidermis are not required for skewed root growth, nor for reorienting root skewing in response to changes in the mechanical environment. Overall, this work provides new insights into how symmetry breaking affects root mechanoresponse. Spatial heterogeneity in the composition and organization of the plant cell wall affects its mechanics to control cell shape and directional growth. In the last chapter of this work, I describe a new methodology for imaging plant primary cell walls at the nanoscale using atomic force microscopy coupled with infrared spectroscopy (AFM-IR). I contributed to generating a novel sample preparation technique and employed AFM-IR and spectral deconvolution to generate high-resolution spatial maps of the mechanochemical signatures of the Arabidopsis epidermal cell wall. Cross-correlation analysis of the spatial distribution of chemical and mechanical properties suggested that the carbohydrate composition of cell wall junctions correlates with increased local stiffness. In developing this methodology, this chapter provides an essential foundation for applying AFM-IR to understand the complex mechanochemistry of intact plant cell walls at nanometer resolution.
Chair and Committee
Elizabeth Haswell, Dmitri Nusinow, Christopher Topp, Marcus Foston,
Bilkey, Natasha Anriel, "Mechanisms and Consequences of Microtubule-Based Symmetry Breaking in Plant Roots" (2023). Arts & Sciences Electronic Theses and Dissertations. 2832.