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. 2022 Dec 20;11:e79913. doi: 10.7554/eLife.79913

Figure 2. BdPAN1 and BdPOLAR display opposite, reciprocal polarisation in subsidiary mother cells (SMCs).

(A, B) BdPAN1p:BdPAN1-YFP reporter expression in stage 1 stomatal files (A) and during SMC stage 3 (B). Images of propidium iodide (PI)-stained cell outlines (left), and fluorescence intensity of YFP channel (right) (C, D). BdPOLARp:BdPOLAR-mVenus reporter expression in stage 1 stomatal files (C) and during SMC stage 3 (D). Images of PI-stained cell outlines (left), and fluorescence intensity of mVenus channel (right). The absence of BdPOLAR at the guard mother cell (GMC)/SMC interface is indicated with white arrowheads. The single white arrow indicates a newly formed subsidiary cell; note that BdPOLAR-mVenus dissociated from the SMC distal polarity domain. (E) Manually traced, normalised fluorescence intensity at the SMC plasma membrane of BdPAN1-YFP lines (n=13) and BdPOLAR-mVenus lines (n=8). Average fluorescence intensity with standard error is plotted as a function of the ‘unfolded’ SMC starting with the GMC/SMC interface as indicated by the model. Note that the signal gets noisy towards the end as SMCs are differently sized. Only SMCs with a length/width ratio (LWR) >0.8 for BdPAN1-YFP and LWR >0.9 for BdPOLAR-mVenus were included. (F) BdPOLARp:3xNLS-eGFP reporter expression during stomatal development. Overlay images show BdPOLAR transcriptional signals (in yellow) and PI-stained cell outlines in magenta. The stomatal files are indicated with a grey asterisk. GMCs are indicated with blue asterisks. Developmental stages are indicated. Confocal images shown are single focal planes midway from top to bottom. Scale bar, 10 µm.

Figure 2—source data 1. Quantification of fluorescence intensity of cell outlines of SMCs expressing BdPOLAR-mVenus or BdPAN1-YFP.
elife-79913-fig2-data1.xlsx (201.1KB, xlsx)

Figure 2.

Figure 2—figure supplement 1. BdPAN1 and BdPOLAR expression throughout stomatal development in Brachypodium distachyon.

Figure 2—figure supplement 1.

(A)BdPAN1p:BdPAN1-YFP reporter expression during stomatal development. Fluorescence intensity images of YFP channel only (upper) and images of PI-stained cell outlines only (bottom). (B) BdPOLARp:BdPOLAR-mVenus reporter expression during stomatal development. Fluorescence intensity images of mVenus channel only (upper) and images of propidium iodide (PI)-stained cell outlines only (bottom). Note that the laser was adjusted for different stages to visualise the signal and expression level between stages cannot be quantitatively compared. The stomatal files are indicated with grey asterisks. Guard mother cells (GMCs) are indicated with blue asterisks. Stomatal stages are indicated. (C) Simplified model of the spatiotemporal dynamics of the two polarity domains. Note that the proximal BdPAN1 domain forms earlier and is stable post-mitotically, whereas BdPOLAR quickly dissociates from the distal domain after subsidiary cell division. Confocal images shown are single focal planes midway from top to bottom. Scale bar, 10 µm.
Figure 2—figure supplement 2. BdPAN1, BdPOLAR, and BdTAN1 reporter expression in representative subsidiary mother cells (SMCs).

Figure 2—figure supplement 2.

(A–C) Different SMCs expressing either BdPAN1-YFP (A), BdPOLAR-mVenus (B), or BdTAN1-mCitrine (C). Depicted are SMCs of which circumference was manually traced to measure fluorescence intensity for Figure 2E and/or Figure 4I. Note that only SMCs that did not divide yet were measured. For Figure 2E, only SMCs with a length/width ratio (LWR) >0.8 for BdPAN1-YFP and LWR >0.9 for BdPOLAR-mVenus were included. Names of images correspond to names in the supplementary dataset 1. Confocal images shown are single focal planes midway from top to bottom. Scale bar, 5 µm.