Opposing, Polarity-Driven Nuclear Migrations Underpin Asymmetric Divisions to Pattern Arabidopsis Stomata

Abstract

Multicellular development depends on generating and precisely positioning distinct cell types within tissues. During leaf development, pores in the epidermis called stomata are spaced at least one cell apart for optimal gas exchange. This pattern is primarily driven by iterative asymmetric cell divisions (ACDs) in stomatal progenitors, which generate most of the cells in the tissue. A plasma membrane-associated polarity crescent defined by BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) and BREVIS RADIX family (BRXf) proteins is required for asymmetric divisions and proper stomatal pattern, but the cellular mechanisms that orient ACDs remain unclear. Here, utilizing long-term, quantitative time-lapse microscopy, we identified two oppositely oriented nuclear migrations that precede and succeed ACD during epidermal patterning. The pre- and post-division migrations are dependent on microtubules and actin, respectively, and the polarity crescent is the unifying landmark that is both necessary and sufficient to orient both nuclear migrations. We identified a specific and essential role for MYOXI-I in controlling post-ACD nuclear migration. Loss of MYOXI-I decreases stomatal density, owing to an inability to accurately orient a specific subset of ACDs. Taken together, our analyses revealed successive and polarity-driven nuclear migrations that regulate ACD orientation in the Arabidopsis stomatal lineage.

Keywords: Arabidopsis; MYOXI-I; asymmetric division; cytoskeleton; leaf development; nuclear migration; stomata; tissue patterning.

Figure 1.. Successive nuclear migrations bookend ACD in the stomatal lineage.

A. Schematic of the Arabidopsis stomatal lineage divisions, cell types, and polarity crescents (left). Epidermis of a 3dpg cotyledon (right) from the R2D2 BRXL2::BRXL2-YFP ML1::mCherry-RCI2A line, pseudo-colored to indicate cell types. Scale bar-25μm. B. Percent of asymmetrically dividing cells with nuclei initially positioned proximal or distal to polarizing BRXL2. n-91 cells. C–D. Examples of two classes of nuclear positioning before ACD in R2D2 BRXL2::BRXL2-YFP ML1::mCherry-RCI2A cotyledons. Stills (left) illustrate asymmetrically dividing cells (i) 8.5hr before division, (ii) 0.5hr before division, and (iii) 0.5hr post-ACD. Kymograph (right) generated from the dotted line in (i). The cyan arrowhead indicates the polarity crescent. Scale bars-5μm. E. Stills (left) and kymograph (right) showing the nuclear position during a representative GMC division (same format as in Figure 1C). Scale bars-50μm. F. Stills (left) and kymograph (right) showing the nuclear position during a representative PC division (i) 6hr before division, (ii) 0.5hr before division, and (iii) 0.5hr post-ACD. Scale bars-5μm. G–H. Distribution of the distances from the pre-ACD nuclear center to the polarity crescent, plotted as absolute distance from the distal wall (G) or as a relative distance along the cell axis orthogonal to the division plane (H). n-140 cells. I. Relative nuclear positions before division in the indicated cell types. The ACD data is derived from those plotted in Figures 1G and 1H (see Methods for details). ACD-70 cells, GMC-44 cells, PC-11 cells. Data are represented as mean±standard deviation. J. Stills (left) and kymograph (right) of NM^post in an SLGC. Stills show the cell (i) 0.5hr before ACD, (ii) 0.5hr after ACD, (iii) 4hr after ACD, and (iv) 8hr after ACD. Kymograph (right) generated from the dotted line in (i). The cyan arrowhead indicates the polarity crescent. Scale bars-5μm. K. SLGC nucleus-to-crescent distance during NM^post, quantified from the cell shown in Figure 1J. L. Average SLGC nucleus-to-crescent distance during NM^post. Data are represented as mean±standard deviation. M. Schematic showing the two, oppositely oriented nuclear movements, NM^pre and NM^post, that flank ACD. Nuclear color deepens in later timepoints. See also Figure S1 and Videos S1 and S2.

Figure 2.. NM^pre and NM^post are oriented by the polarity crescent

A. Schematic of the genotypes used and corresponding polarity protein localizations. B. Stills (left) and kymograph (right) showing NM^pre and NM^post over nine hours in wild-type cells. Stills correspond to the indicated time frames in the kymograph showing the (i) initial, (ii) pre-mitotic, (iii) post-division, and (iv) final nuclear positions. Kymograph (right) generated from the dotted line in (i). The cyan arrowhead indicates the polarity crescent. Scale bars-5μm. C–D. Lower panels as in Figure 2B, with top panels showing broader fields at the start (t=0hr) and end (t=9hr), for basl-2 (C) and MYR-BRX (D). Two representative cells for each genotype are boxed. Scale bars-5μm. E. Schematic illustrating the quantification method (left). Quantification of the nuclear position pre-ACD relative to the membrane opposite the division plane in the indicated genotypes (right). Wild-type-70 cells, basl-2-90 cells, MYR-BRX-128 cells. Data are represented as mean±standard deviation. n.s.-not significant, *** - p<0.001. F. Schematic illustrating the quantification method (left). Quantification of the nuclear position relative to the membrane opposite the division plane five hours post-division in the indicated genotypes (right). Wild-type-64 cells, basl-2-69 cells, MYR-BRX-80 cells. Data are represented as mean±standard deviation. n.s.-not significant, * - p<0.05, ** - p<0.01. G. Representative images of cells immediately before (left) and after (right) ACD in wild-type, basl-2, and MYR-BRX. Yellow dotted lines indicate the dividing cells. Scale bars-10μm. H. Daughter cell size ratios in the indicated genotypes. Wild-type-99 cells, basl-2-83 cells, MYR-BRX-96 cells. Data are represented as mean±standard deviation. n.s.-not significant, See also Figure S2 and Video S3.

Figure 3.. NM^pre and NM^post require distinct cytoskeletal pathways

A–C. Stills (left) and kymographs (right) showing nuclear movement over 8.5hr in cells treated with (A) DMSO, (B) 1μM latrunculin B and (C) 10μM oryzalin. Stills correspond to the indicated time frames in the kymograph showing the (i) initial, (ii) pre-mitotic, (iii) post-division, and (iv) final nuclear positions. Kymograph (right) generated from the dotted line in (i). Two examples are provided for oryzalin treatment (C) to more clearly highlight NM^pre and NM^post. Note the longer time between (ii) and (iii) in (C) due to the requirement for microtubules for mitosis. The cyan arrowheads indicate the polarity crescents. Scale bars-5μm. D. Schematic illustrating the quantification method (left). Quantification of the nuclear position pre-ACD in the indicated treatments, oriented relative to the polarity crescent (right). DMSO-54 cells, latB-77 cells, oryzalin-35 cells. Data are represented as mean±standard deviation. n.s.-not significant, *** - p<0.001. E. Schematic illustrating the quantification method (left). Quantification of the nuclear distance to the polarity crescent in SLGCs five hours after division (right). DMSO-29 cells, latB-34 cells, oryzalin-35 cells. Data are represented as mean±standard deviation. n.s.-not significant, *** - p<0.001. See also Figure S3.

Figure 4.. MYOXI-I regulates NM^post and stomatal patterning

A. Stills (left) and kymograph (right) of NM^post in a wild-type SLGC. Stills show the cell (i) 0.5hr before ACD, (ii) 0.5hr after ACD and (iii) 8hr after ACD. Kymograph (right) generated from the dotted line in (i). The cyan arrowhead indicates the polarity crescent. Scale bars-5μm. B. Stills (left) and kymograph (right) of NM^post in myoxi-i⁴, formatted as in Figure 4A. The cyan arrowhead indicates the polarity crescent. Scale bars-5μm. C. Schematic illustrating the quantification method (left). Quantification of the nuclear distance to the polarity crescent in SLGCs five hours after division (right). Wild-type-31 cells, myoxi-i⁴-33 cells. Data are represented as mean±standard deviation. n.s.-not significant, *** - p<0.001. D. Representative images of 7dpg wild-type and myoxi-i³ cotyledons. Stomata are pseudo-colored purple. Scale bars-25μm. E. Quantification of abaxial stomatal density at 7dpg in wild-type and myoxi-i cotyledons. Wild-type-25 seedlings, myoxi-i³-24 seedlings, myoxi-i⁴-27 seedlings. Data are represented as mean±standard deviation. n.s.-not significant, ** - p<0.01, *** - p<0.001. F. Representative images of correctly and incorrectly oriented spacing divisions, both from myoxi-i⁴. Dotted lines indicate the vectors used to calculate the spacing division angle (θ). Scale bars-10μm. G. Quantification of spacing division angles in wild-type and myoxi-i⁴. Wild-type-88 cells, myoxi-i⁴-81 cells. Kolmogorov-Smirnov test-*- p<0.05. H. Representative images of the abnormal cellular lobing phenotype at 4dpg (top) and 7dpg (below) following incorrect spacing divisions in myoxi-i⁴. Scale bars-10μm. See also Figure S4.