Extended Data Fig. 5: Phenotypic analysis of the tobacco dpd1 mutant.

a,WT^ptGFP and dpd1^ptGFP plants were grown in the greenhouse. Pictures were taken from 6-week-old plants, 9-week-old plants and fully opened flowers. b, Confocal microscopy images of pollen grains from WT^ptGFP and dpd1^ptGFP plants grown under standard greenhouse conditions. Pollen grains were stained with fluorescein diacetate (FDA) and propidium iodide (PI) to determine pollen viability. This experiment was repeated three times and representative images are shown. Scale bar, 100 μm. c, Quantification of pollen viability. The proportions of viable pollen per biological replicate (imaging session) were modelled using the binomial distribution (Model 5; constructed with n_rep.total = 6; 2,228 pollen grains analysed; see Extended Data Tables 1 and 2). Each replicate consisted of a single harvest of pollen from 2–3 flowers of a single plant, which were stained and imaged in the same session (delimited by dashed lines). During data analysis, it was found that there was significant variation between replicates (represented by replicate parameters with P < 0.05 in Extended Data Table 2). To better represent this variation, the graph shows the proportions of viable pollen in the 5–9 image fields (coloured circles) photographed in each replicate. The mean estimates per genotype are indicated with a black horizontal bar. β_dpd1 represents the effect of the dpd1 genotype on pollen survival (P = 3.00 × 10⁻⁹) compared to the wild type at normal temperature conditions (20–25 °C), expressed as log₁₀ of the fold change (Extended Data Table 2). The parameter estimate was tested by a two-tailed Wald z-test. ***, P < 0.001; α = 0.05. d, Confocal images of WT^ptGFP and dpd1^ptGFP mature pollen stained with DAPI. Images were taken after applying the pollen squash method to release the mature pollen from the anthers. Z-stack overlaid images with maximized projection are displayed (total width of z-slices of WT^ptGFP and dpd1^ptGFP is 2.15 μm and 1.78 μm, respectively). Arrowheads indicate the vegetative nucleus (VN) and the generative nucleus (GN). Arrows indicate organellar DNA released from squashed mature dpd1^ptGFP pollen. This experiment was repeated two times and representative images are shown. Scale bar, 10 μm.