Figure 4

Fluorescence microscopy and flow cytometric analysis of SYTO 42-stained chloroplasts. Brightfield (A) and fluorescence (B) microscopic images of chloroplasts isolated from 14-day-old seedlings after staining with 25 μM SYTO 42. The exposure time in (B) was 0.1 s. (C-E) Flow cytometric analysis of chloroplasts from 14-day-old seedlings treated with and without DNase and stained with the indicated concentrations of SYTO 42. Brightfield (F, H) and fluorescence microscopy (G, I) of chloroplasts from immature leaves of 35-day-old plants after treatment with RNase (F, G) and DNase (H, I) and staining with 25 μM SYTO 42. Insets in (F) and (G) show brightfield and fluorescence images of a chloroplast from a different microscopic field of the same sample. The exposure times were 0.1 s (G) and 0.3 s (I). The number of DNase-treated chloroplasts that did not have visible nucleoids was 8 out of 8. The number of chloroplasts that did not have visible nucleoids was 1 out of 9 after RNase treatment, and 2 out of 19 for untreated controls. (J) Flow cytometric analysis comparing untreated chloroplasts with chloroplasts treated with RNase, DNase, or DNase and RNase from immature leaves of 35-day-old plants. (K) Comparison of unfixed chloroplasts to chloroplasts fixed with 0.8% glutaraldehyde. Differences in cpDNA content (L) and PPoD (M) for chloroplasts isolated from seedlings at 14 and 20 days after imbibition. (N) PCR amplification of a 156-bp fragment of the psb A gene from lysates prepared from chloroplasts that had been previously treated or not treated with DNase. Scale bars are 10 μm. (A, B, L, M (14-day profile)) Chloroplasts were not fixed. (C-K, L, M (20-day profile)) Chloroplasts were fixed in glutaraldehyde. (C-E, J-M) At least 2500 chloroplasts were analyzed for all samples.