Variable amounts of DNA related to the size of chloroplasts III. Biochemical determinations of DNA amounts per organelle |
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Authors: | Uwe Rauwolf Hieronim Golczyk Stephan Greiner Reinhold G Herrmann |
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Institution: | 1. Department für Biologie I, Bereich Botanik, Ludwig-Maximilians-Universit?t München, Menzinger Stra?e 67, 80638, Munich, Germany 2. Department of Plant Cytology and Embryology, Institute of Botany, Jagiellonian University, Grodzka 52, 31-044, Kraków, Poland 3. Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, 14476, Potsdam-Golm, Germany
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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 leaflets (≤1 mm) and leaves of different 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 purification from homogenates of
moderately fixed tissue by differential and isopycnic gradient centrifugations and on application of two different DNA specific
colorimetric reactions after removing potentially interfering compounds. The sensitive fluorochrome DAPI (4′,6-diamidino-2-phenylindole)
was used to estimate 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 μm 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 differing
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. |
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