Quantitative and dynamic cell polarity tracking in plant cells

Abstract

Quantitative information on the spatiotemporal distribution of polarized proteins is central for understanding cell-fate determination, yet collecting sufficient data for statistical analysis is difficult to accomplish with manual measurements. Here we present POME, a semi-automated pipeline for the quantification of cell polarity, and demonstrate its application to a variety of developmental contexts. POME analysis reveals that during asymmetric cell divisions in the Arabidopsis thaliana stomatal lineage, polarity proteins BASL and BRXL2 are more asynchronous and less mutually dependent than previously thought. While their interaction is important to maintain their polar localization and recruit other effectors to regulate asymmetric cell divisions, BRXL2 polarization precedes that of BASL and can be initiated in BASL's absence. Uncoupling of polarization from BASL activity is also seen in Brachypodium distachyon, where we find that the MAPKKK BdYDA1 is segregated and polarized following asymmetric division. Our results demonstrate that POME is a versatile tool, which by itself or combined with tissue-level studies and advanced microscopy techniques can help uncover new mechanisms of cell polarity.

Tuning of self-renewing capacity in theArabidopsisstomatal lineage by hormonal and nutrition regulation of asymmetric cell divisions

“…The immediate readout of ethylene and glucose antagonism is a shift in population of cells expressing polarized vs. depolarized BRXL2, and the ultimate readout is a change in the size and the cell type composition of the leaf ( Figure 7A). In addition, by developing methods to systematically monitor fluorescent marker dynamics (POME, Gong et al, 2020) and to then track cell divisions and differentiation in the same cells, we uncovered a surprising correlation between the ACD potential of meristemoids and the persistence of polarized BRXL2 in SLGCs ( Figure 7B), suggesting active communication and coordination between these sister cells.…”

“…(B) BRXL2 localization in epidermal cells of 4 dpg Col-0 (top panels) and ctr1 G738R (bottom panels) cotyledons. Gong et al (2020)). (E) Output of polarity measurement (POME) quantification of BRXL2 polarity in 4 dpg Col-0 and ctr1 G738R cotyledons (n=30 cells/genotype, 3 replicates).…”

“…These results suggest a correlation between the degree of BRXL2 polarity and cell identity. Thus, we applied our recently developed polarity measurement tool (POME, Gong et al, 2020) to quantify the distribution of BRXL2-YFP signal at the periphery of each cell. With POME, the pixel intensity of the BRXL2 reporter and an evenly distributed PM reporter are captured along the entire cell circumference, and their relative distributions were used to compute a "polarity index" (Figure1C, Gong et al, 2020).…”

“…Thus, we applied our recently developed polarity measurement tool (POME, Gong et al, 2020) to quantify the distribution of BRXL2-YFP signal at the periphery of each cell. With POME, the pixel intensity of the BRXL2 reporter and an evenly distributed PM reporter are captured along the entire cell circumference, and their relative distributions were used to compute a "polarity index" (Figure1C, Gong et al, 2020). The BRXL2 polarity index (BPI) ranges between 0 and 1, where a BPI close to 0 represents a cell with highly polarized BRXL2, and a BPI of 1 represents a cell with completely depolarized BRXL2.…”

“…From a genetic screen to identify regulators of cell polarity in the stomatal lineage, we found a mutation in CONSTITUTIVE TRIPLE RESPONSE (CTR1), a core component of the ethylene signaling pathway, that caused overall depolarization of BRXL2. To understand the connection between CTR1 and BRXL2 polarity, and how the change of BRXL2 polarity was affecting leaf development, we created long-term tissue-wide lineage tracing methods and employed a recently developed quantitative polarity analysis tool (Gong et al, 2020) to quantify the polarity of individual cells during cell divisions across cell populations. We discovered that ethylene and glucose signaling, respectively involved in environmental and nutritional pathways, antagonistically regulate the balance of asymmetric and symmetric cell divisions in the stomatal lineage.…”