DNA replication in maize leaf protoplasts

Abstract

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of 3H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-a, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytocbemical method. Inclusion of 80 mM Ca 2+ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations: BrdU--5-bromo-2-deoxyuridine; DMSO--dimethyl sulfoxide; n-PG--npropyl gallate; PBS--phosphate-buffered saline

Immunocytochemical study of cell cycle control by cytokinin in cultured soybean cells

“…The immunofluorescence detection of cells in S phase uses a monoclonal antibody against the thymidine analog bromodeoxyuridine (Gratzner 1982) and provides an alternative to autoradiography, which, despite its drawbacks, is still a standard method in cell cycle studies. The application of immunocytochemistry is well established in animal cell cycle work, but it is still relatively new for plant systems (Grosskopf and Kroiher 1988, Levi et al 1987, Pfosser 1989, Wang et al 1989a, 1989b.…”