Four-dimensional quantitative analysis of cell plate development in Arabidopsis using lattice light sheet microscopy identifies robust transition points between growth phases

Abstract

Cell plate formation during cytokinesis entails multiple stages occurring concurrently and requiring orchestrated vesicle delivery, membrane remodelling, and timely deposition of polysaccharides, such as callose. Understanding such a dynamic process requires dissection in time and space; this has been a major hurdle in studying cytokinesis. Using lattice light sheet microscopy (LLSM), we studied cell plate development in four dimensions, through the behavior of yellow fluorescent protein (YFP)-tagged cytokinesis-specific GTPase RABA2a vesicles. We monitored the entire duration of cell plate development, from its first emergence, with the aid of YFP-RABA2a, in both the presence and absence of cytokinetic callose. By developing a robust cytokinetic vesicle volume analysis pipeline, we identified distinct behavioral patterns, allowing the identification of three easily trackable cell plate developmental phases. Notably, the phase transition between phase I and phase II is striking, indicating a switch from membrane accumulation to the recycling of excess membrane material. We interrogated the role of callose using pharmacological inhibition with LLSM and electron microscopy. Loss of callose inhibited the phase transitions, establishing the critical role and timing of the polysaccharide deposition in cell plate expansion and maturation. This study exemplifies the power of combining LLSM with quantitative analysis to decode and untangle such a complex process.

Keywords: 4D imaging; Callose; RABA2a; cell plate; cytokinesis; lattice light sheet microscopy; plant cell division; quantitative image analysis.

Fig. 1.

YFP–RABA2a dynamics at the cell plate. YFP–RABA2a (green) vesicle accumulation and FM4-64- (purple) stained plasma membrane show the transition from vesicle accumulation to mature membrane throughout cell plate development in untreated plants. The accumulation of YFP–RABA2a at the cell plate periphery during maturation with a concurrent increase in the membrane content in the center shows centrifugal growth and maturation. The arrow indicates RABA2a at the cell plate. Data were collected on a Leica SP8 microscope. Scale bar=4 µm. Δt=1 min.

Fig. 2.

Schematic representation of the lattice light sheet data processing pipeline. Schematic representation of the processing workflow for collecting plant cytokinesis events under lattice light sheet microscopy (LLSM). The workflow is separated into four overarching steps: (A) image collection and data organization; (B) data alignment with drift correction and ROI selection; (C) concatenation of each time point to create a 4D stack and bleach correction follow-up processing; (D) image segmentation and quantification. Scale bar=20 µm.

Fig. 3.

Quantitative YFP–RABA2a dynamics at the cell plate. (A) Representative snapshots of a time series showing the transition of YFP–RABA2a (green) in segmented cell plates (purple) during their expansion and maturation. Scale bar=5 μm. (B and C) Volumes of segmented cell plates and their growth rates. (D and E) Bounding surface areas of segmented cell plates and their rates of change. Each line indicates an individually segmented cell plate. Note the cell plate shown in (A) is highlighted in bold in all graphs. Normalized volumes and bounding surfaces , respectively, of developing cell plates were fitted to a polynomial (B and D). Additionally, the polynomials (B and D) were used to derive the rates of change of the volume and bounding surface area (C and E). n=24.

Fig. 4.

YFP–RABA2a cell plate dynamics under Endosidin 7 (ES7) treatment. YFP–RABA2a (green) vesicle accumulation and FM4-64- (purple) stained plasma membrane show cell plate development under ES7 treatment. Note the abnormal pattern and the fragmentation of the cell plate as it transitions during different time points. The pattern of YFP–RABA2a at the cell plate periphery does not expand radially, as seen in the untreated plants. Further, the cell plate cannot follow normal maturation into membranes detectable with FM4-64. Data were collected on a Leica SP8. Scale bar=10 μm. Δt=1.5 min.

Fig. 5.

Quantitative YFP–RABA2a dynamics at the cell plate under ES7 treatment. (A) Representative snapshots of a time series showing the YFP–RABA2a segmented cell plate transition under ES7 treatment. The segmented cell plate is shown in purple from its first emergence, not able to expand radially and flatten. (B–E) Volumes and bounding surfaces of segmented cell plates and their rates of change. Each line indicates an individually segmented cell plate. Note that the cell plate shown in (A) is highlighted in bold in all graphs. Normalized volumes and bounding surface areas of developing cell plates were fitted to a polynomial distribution (B and D). First-degree derivatives present the corresponding rates of change of the polynomial fits for the volume and the bounding surface area, respectively (C and E). Scale bar=10 µm. n=18.

Fig. 6.

Statistical comparison of volume accumulation. YFP–RABA2a normalized volume growth rates, averaged within pre-defined groups and shown with 95% confidence intervals. (A–C) Bins 1–3 represent the rates corresponding to normalized volumes increasing from 0 to 0.33 μm³, 0.33 μm³ to 0.66 μm³, and 0.66 μm³ to 1 μm³, respectively. All bins correspond to the first derivative rates of volume growth. *Indicates P<0.005. (D and E) Bins 4 and 5 represent the rates corresponding to volumes decreasing from 1 μm³ to 0.66 μm³ and from 0.66 μm³ to 0.33 μm³, respectively; bin 6 data are not shown due to the bin spanning over the region in which the end of collection has limited control over noise. All bins correspond to the first derivative rates of volume growth. *Indicates P<0.005. (F) Schematic representation of volume growth grouped in bins for further processing as shown in (A–E).

Fig. 7.

Cell plate diameter expansion under control and Endosidin 7 (ES7) treatment. Cell plate transition based on cell plate diameter expansion under control and ES7 treatment. (A) Control cell plates show a logarithmic expansion pattern, while ES7 treatment causes a more level pattern. Cell plate diameters are normalized to the expected cross-wall length. Note the intersection between control and ES7 treatment, indicating a critical point for cell plate maturation. (B) Volume-to-bounding surface area ratio in control and ES7-treated cell plates. The average of the ratio for cell plates analyzed in Figs 3 and 5 are shown. Control n=23, ES7 n=12. (C) Polynomial fit was applied to cell plate diameters that have been normalized to the corresponding cross-wall. (D) Results of pairwise t-test comparisons along moving averages shown in (C). Analysis shows that all except one time point are statistically different on cell plate expansion when comparing control with the absence of callose via ES7 treatment. Non-treated n=24, treated n=18.

Fig. 8.

Progression of the cell plate in the presence of callose. (A–E) Cell plate development in the presence of callose. (A) An early-stage cell plate before the accumulation of callose. At this stage, only vesicle accumulation by YFP–RABA2a (green) makes up the cell plate. (B–D) Later stage cell plates where callose accumulation stained with aniline blue fluorochrome (magenta) is detectable. Note the transient accumulation of callose in later stages, leading to the maturation of the cell plate during normal cytokinesis. (B) Initial callose deposition overlapping with YFP–RABA2a. (C) As the cell plate maturation continues and the YFP–RABA2a accumulation takes a ‘doughnut shape’ pattern, callose deposition appears in the middle of the cell plate with minimal overlap with YFP–RABA2a at the leading edge. (D) Callose is present throughout the cell plate, while minimal YFP–RABA2a is shown at the discontinuous ring (E). Mature cell plate predominately labeled by callose. Scale bar=5 μm.