Control of formative cell divisions in the stomatal lineage in Arabidopsis
Final Report Abstract
Work package 1: How is the single formative division of the GMC triggered and restricted? Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue in which other cells transition from proliferation to postmitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We showed that the D-type cyclin, CYCD7;1, is specifically expressed just prior to the symmetric guard cell-forming division, and that it is limiting for this division. Depletion of CYCD7;1 slows down this cell division whereas ectopic expression of CYCD7;1 can trigger cell divisions in GCs. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR1-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell type-specific transcription factors that confine its expression to the appropriate developmental window. CYCD7;1 activity is directly repressed by the lineage specific transcription factor FAMA to ensure a coupling between the cell division which terminates the stomatal lineage, and the formation of terminally fated GCs. This interconnection represents a direct link between cell cycle regulators and developmental decisions. CYCDs are critical for the G1/S transition and commitment to divide and are therefore interesting candidate hubs for the integration of developmental control with the cell cycle machinery. Work package 2: What defines polar domains during asymmetric cell divisions? In this work package we tried to address a fundamental question in biology: how do plant cells set up polar domains to achieve asymmetry necessary for their development? In order to determine which proteins define cellular polarity and set up polar domains in the stomatal lineage, I proposed targeted biotinylation by APEX2 to construct a BASL/BRXL2 interactome consisting of candidate proteins that are proximate to and/or interact with these polarity proteins. Using the original bioID approach to proximity labeling in plants poses a technical problem as the enzyme is optimized to work at 37 degrees °C, whereas plants are typically grown at 22 °C. To overcome this problem, we tested both APEX2 and turboID. APEX2 is based on a plant peroxidase that is naturally active at room temperature and tuboID is based on the original bioID construct where a series of point mutations dramatically increased the enzymatic activity at lower temperatures. In summary, we found that we were able to achieve similar increases in biotinylated protein as observed from visual inspection in immuno-blots with streptavidin to label biotinylated protein. Given that both the approaches show similar activity in plants, tuboID has a couple of advantages making it a more suitable approach for proximity labeling in plants. The most significant advantage of bioID is that it requires biotin, which is readily taken up in plant cells, whereas APEX2 requires biotin-phenol, which we showed only efficiently supplied to the cytoplasm by vacuum infiltration. Vacuum infiltration is a relatively harsh treatment that may cause damage and stress to the cells and the cell that can be reached by this approach may be limited to leaf and superficial cell layers.
Publications
- Lineage- and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells. Development 145, dev160671 (2018)
Weimer A.K., Matos J.L., Sharma N., Patell F., Murray J.A.H., Dewitte W. and Bergmann D.C.
(See online at https://doi.org/10.1242/dev.160671) - A Plant-Specific Polarity Module Establishes Cell Fate Asymmetry in the Arabidopsis Stomatal Lineage
Rowe M.H., Dong J., Weimer A.K. and Bergmann D.C.
(See online at https://doi.org/10.1101/614636)
