Studies on Chloroplast Development and Replication in Euglena

Abstract

During chloroplast development in Euglena, the activity of a specific DNase, Euglena alkaline DNase, increases in a manner similar to that of chlorophyll synthesis, but without the lag customarily associated with the early hours of chlorophyll synthesis. The increase in Euglena alkaline DNase activity is not inhibited by chloramphenicol or by streptomycin, but is inhibited by cycloheximide. Euglena alkaline DNase activity is present in a group of aplastidic substrains which contain carotenoids. These results are interpreted to mean that this chloroplast-related DNase is synthesized in the cytoplasm, and that the genetic information for this enzyme is probably nuclear.It is also shown that different bleached substrains exhibit substantial variation, both in total carotenoids and in Euglena and WicBSmL, used in these experiments are described elsewhere (12,30). Agar slants of the bacillaris strain and its three substrains were kindly provided by Dr. Jerome A. Schiff, Brandeis University, Waltham, Massachusetts.E. gracilis, Klebs, Strain Z Pringsheim, from our stock culture was also employed (30). The bleached substrains Y7ZHL and W,,ZHL were obtained by carrying light-grown strain Z through five rounds of culture on Hutner's modified medium (29) at 32 C. The bleached substrains Y,ZSmL and W,5ZSmLwere produced by carrying light-grown cells through five rounds of culture on the same medium, containing 1 mg/ml of streptomycin at 25 C. In both treatments, the fifth culture was plated on agar. Yellow and white colonies appeared on the agar plates, and one colony of each color was isolated and designated as described elsewhere (33). The substrain YZSL was obtained by plating light-grown cells of the Z strain and isolating a rare yellow colony from the plates. None of the Z strain mutants had detctable levels of chlorophyll, and no plastid remnants could be observed by fluorescence microscopy. No reversion to the wild type has been observed over a 2-year period; in addition, the white substrains did not revert to the yellow form during this time period.Growth and Resting Conditions. After growing dark-adapted Euglena to a density of 5 X 10' to 1 X 10' cells/ml, the cells were aseptically transferred to Stern resting medium (37). The cells were kept in the dark for 72 hr and were then exposed to 150 ft-c of white fluorescent light for the specified times. At each time period, cells were harvested, rinsed, sampled for chlorophyll (9), and frozen. Further details of growth and resting conditions are presented in the figure legends.Preparation of Cells for Assays. Frozen cells were thawed and sonicated at the maximum setting on the Branson Biosonik sonicator. The sonication time was 30 sec in the experiment of Figure 3 and 2 min in all other experiments. While 30 sec of sonication was sufficient to release all enzyme activity, sonication periods of 2 min or longer did not appear to affect the specific activities (14). The sonicated suspension was then centrifuged at 30,000g for 30 min, and the supernatant was used as...

Events Surrounding the Early Development of Euglena Chloroplasts: I Cellular Origins of Chloroplast Enzymes in Euglena*

“…The plastid NADP-TPDase, however, is formed even in the presence of Sm and Cm and its appearance is completely inhibited by cycloheximide, indicating that it is not synthesized on plastid ribosomes but on the 8 7 s ribosomes of the cytoplasm. Similar observations and conclusions have been reported for two other enzymes induced during chloroplast development: Euglena alkaline DNase (Egan & Carell, 1972) and the chloroplast phenylalanylt RNA synthetase (Reger, Fairfield, Epler & Barnett, 1970). We surmise, therefore, that enzymes such as NADP-TPDase, Euglena alkaline DNase and chloroplast phenylalanyltRNA synthetase are coded[in nuclear DNA.…”

Events Surrounding the Early Development of Euglena Chloroplasts

“…The successful stabilization of TPN-TPDase, shown in Figure 1, allowed assays to be conducted over a long enough period of time to enable all samples to be assayed without enzyme inactivation. (2,8). Figure 2 presents typical examples of the dose-response curves generated for each of the parameters of Chl synthesis.…”

“…As used here, the substrate was heat-denatured DNA labeled with 3H-thymidine prepared from a thymidineless mutant of Escherichia coli and diluted with heat-denatured calf thymus DNA. Incubation and assay conditions were the same as previously described (3,8), and the reaction was stopped by addition of 0.1 ml of 2 mg/ml of calf thymus DNA and 0.1 ml of 25% trichloroacetic acid. This modified method provided more accurate assay conditions with large numbers of samples, and minimized radioactive contamination problems.…”

“…The procedure for assay of alkaline DNase activity per cell was modified from a previously described method using 'P-DNA (3,8). As used here, the substrate was heat-denatured DNA labeled with 3H-thymidine prepared from a thymidineless mutant of Escherichia coli and diluted with heat-denatured calf thymus DNA.…”

“…It is well established that light induction of chloroplast differentiation results in light-stimulated changes in the plastid and nonplastid compartments (17). Some of these, such as light-induced increases in the activities of the chloroplast TPN-linked triose phosphate dehydrogenase (TPN-TPDase) and alkaline DNase, involve new protein synthesis on cytoplasmic rather than chloroplast ribosomes (2,8). Preliminary studies of the spectral requirements for preillumination leading to elimination of the lag in Chl synthesis and of light-induced 'P incorporation into chloroplast and cytoplasmic ribosomal RNAs suggest that these events involve the participation of PChl(ide) and of a second photoreceptor believed to be sensitive mainly to blue light and to be localized outside the chloroplast (5,6,12).…”