Nucleus–Cytosol Interactions—A Source of Stoichiometric Error in Flow Cytometric Estimation of Nuclear DNA Content in Plants

Variation of the genome size estimate with environmental conditions in Drosophila melanogaster

“…Fluctuations in genome size estimates of the kind observed in our experiments can thus simply be explained in terms of changes in the topology of chromatin, which modifies the accessibility of DNA to fluorochromes, especially to propidium iodide, which is often used in flow cytometry, as shown in various experiments (23,26,30,31). Changes in DNA accessibility after a temperature treatment have been indeed reported for ethanolfixed cells of rat thymocytes (27).…”

“…Because the environment in which an organism grows seems to influence DNA content (6), it is concluded that genome size variation is an adaptation to changing environment. With the increasing use of flow cytometry based on intercalating fluorochromes, some of the correlations observed between genome size estimates and environmental factors may be artifacts, because DNA accessibility to the fluorochrome depends on chromatin structure, particularly its sensitivity to decondensation (23). Some slight fluctuations between individuals within populations and between populations may thus result, at least in part, from this kind of artifact, whereas differences between species are more likely really to involve natural selection and correspond to changes in genome size due to a change in the amount of DNA.…”

“…We must therefore be cautious when using flow cytometry to estimate DNA content, because many endogenous or exogenous factors, as well as the conditions to which the nuclei were submitted before being introduced into the cytometer (e.g., temperature, and pH of buffers [23]), may greatly influence the genome size estimate. Hence, before assigning a genome size value to a species, we need measurements for more than one population.…”

“…A correlation between estimates obtained using different methods is not in itself sufficient, however, to eliminate the possibility of an artifact, because DNA accessibility depends on complex interactions between intercalating or nonintercalating fluorochromes and DNA and various proteins. Variation in DNA accessibility due to the state of condensation of the chromatin has been reported in plants (21,23), during spermatogenesis (24) and in frozen and thawed human spermatozoa (25), in mouse thymocyte nuclei (26), in ethanol-fixed cells (27), in dividing and stationary Euglena cells (28), during differentiation of Friend leukemia cells (29), and in DNAse I treated HeLa nuclei (30,31). The exact impact of external environmental conditions on the genome size estimate therefore needs to be determined before a protocol can be defined that would permit reliable and comparable genome size estimates between individuals and populations within a given experiment and between different experiments.…”

Regeneration of Allotriploid <i>Coffea</i> Plants from Tissue Culture: Resolving the Propagation Problems Promoted by Irregular Meiosis

“…In several woody plants secondary metabolites can promote pseudo-intraspecific variations in the nuclear genome size by affecting the PI fluorescence (Price et al 2000, Noirot et al 2000. Particularly in Coffea species, caffeine and chlorogenic acids are known to interfere in the accessibility of the dye to the DNA, which may lead to stoichiometric errors in nuclear 2C value measurements by FCM (Noirot et al 2003).…”