Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle

Abstract

Plastid genomes (plastomes) are part of the integrated compartmentalised genetic system of photoautotrophic eukaryotes. They are highly redundant and generally dispersed in several regions (nucleoids) within organelles. DNA quantities and number of DNA-containing regions per plastid vary and are developmentally regulated in a way not yet understood. Reliable quantitative data describing these patterns are scarce. We present a protocol to isolate fractions of pure plastids with varying average sizes from leaXets (≤1 mm) and leaves of diVerent developmental stages continuously up to maturity (25 cm) from Beta vulgaris L. (sugar beet) to determine DNA amounts per organelle. The approach is based on plastid puriWcation from homogenates of moderately Wxed tissue by diVerential and isopycnic gradient centrifugations and on application of two diVerent DNA speciWc colorimetric reactions after removing potentially interfering compounds. The sensitive Xuorochrome DAPI (4',6-diamidino-2- phenylindole) was used to estimat numbers and emission intensity of nucleoids per plastid. The amounts determined ranged from 0.15 to 4.9 × 10 -2 pg DNA for plastids of 1→8 'm average diameter, corresponding from approximately a dozen to 330 genome equivalents per organelle and on average four to seven copies per nucleoid. The ratio of plastid/nuclear DNA changed continuously during leaf development from as little as 0.4% to about 20% in fully developed leaves. On the other hand, mesophyll cells of mature leaves diVering in ploidy (di-, tri- and tetraploid) appeared to maintain a relatively constant nuclear genome/plastome ratio, equivalent to about 1,700 copies per C-value.