S U M M A R YDuring chloroplast development, the large increases in ribulose diphosphate carboxylase (RUDPCase) activity and cytochrome 5 5 2 concentration follow the pattern of chlorophyll synthesis, in that the formation of these two enzymes is inhibited by streptomycin (Sm) and by chloramphenicol (Cm) beyond 12 h of development. Neither enzyme can be detected in W,BUL, a mutant of Euglena in which chloroplasts and chloroplast DNA are undetectable. In contrast, the NADP-linked triose phosphate dehydrogenase (NADP-TPDase), another plastidlocalized enzyme, increases in activity without the 12 h lag normally observed for chlorophyll synthesis; this increase in activity is not inhibited by Sm and Cm, but is inhibited by cycloheximide, an antibiotic which acts on 87 S cytoplasmic ribosomes. NADP-TPDase activity is present at the same level in W,BUL as in the dark-grown wild-type organisms. These data are interpreted to mean that NADP-TPDase is coded in the nuclear DNA, and is translated on 8 7 s cytoplasmic ribosomes. The sensitivity of the increase in cytochrome 552 and RUDPCase activities to Sm and Cm indicates that they are translated, at least in part, on the 68 S ribosomes of the chloroplast. Thus, chloroplast differentiation in Euglena is dependent upon information and synthetic machinery from both the plastid and the rest of the cell. Since total cellular protein does not change significantly during chloroplast development in resting cells, we conclude that protein turnover probably occurs.

Action spectra derived from dose-response curves measured for various processes associated with chloroplast development in Euglena gracilis var. bacillaris are presented. The action spectrum for chlorophyll synthesis during the first 36 hours of continuous illumination of dark-grown resting cells resembles the absorption spectrum of protochlorophyll(ide). The action spectrum for the preillumination phase of potentiation, during which preillumination followed by a dark period brings about lag elimination in chlorophyll synthesis when the cells are subsequently exposed to postilluminating light, shows a high peak in the blue region (at about 433 nm) with a small peak in the yellow-orange region (at about 597 nm); the postillumination phase yields an action spectrum very similar to that obtained for chlorophyll synthesis in continuous light in normal, unpotentiated cells, with peaks at 433 and 631 nm. Alkaline DNase and TPN-linked triose phosphate dehydrogenase, two plastid enzymes which are synthesized outside the chloroplast, yield action spectra which are consistent with protochlorophyll(ide) being the major light receptor. The action spectra which implicate pigments resembling protochlorophyll(ide) holochrome have blue to red peak ratios in the vicinity of 5 : 1 as does the absorption spectrum of the protochlorophyllide holochrome from beans; the action spectrum is not identical with the holochrome spectrum indicating that the Euglena holochrome may differ from the bean pigment in details of its absorption spectrum. The action spectrum for preillumination, shows a ratio of the blue peak to the red effectiveness of about 24: 1. This suggests that preillumination is controlled by a photoreceptor different from the protochlorophyll(ide) holochrome.

We find that cycloheximide completely blocks the light-induced appearance of Euglena chloroplastic aminoacyl-tRNA synthetases in dark-grown cells of Euglerva gracilis var. baciliaris. Streptomycin, on the other hand, has no effect on the light-induction of these organellar enzymes. These observations, together with the finding that an aplastidic mutant (strain WsBUL, which has neither significant plastid structure nor detectable chloroplast DNA) contains low levels of the chloroplastic synthetases, indicate that the chloroplastic synthetases are transcriptional products of nuclear genes and are translated on cytoplasmic ribosomes prior to compartmentalization within the chloroplasts.The chloroplastic aminoacyl-tRNA synthetases (EC 6.1.1.X) of Euglena have been shown to be quantitatively under the control of light (1) in that exposure of dark-grown cells to light results in a marked increase in the level of these organelle-specific enzymes. In the present report, we have taken advantage of this light inducibility as well as of the techniques developed by Schiff et al. (2, 3). These techniques permit the study of chloroplast development in the absence of such complicating factors as cell growth and cell division in order to de-termine the site of translation of chloroplastic synthetases. More specifically, we examined the effect of streptomycin (4-6) and cycloheximide (7), which block protein synthesis on organelle (68S) and cytoplasmic (87S) ribosomes, respectively, on the light induction of chloroplastic synthetases in "resting" or nondividing dark-grown Euglena.We observe that, while cycloheximide completely blocks induction, streptomycin has no effect upon induction of chloroplast synthetases. In addition, we find as indicated previously (1), that low levels of the chloroplast synthetases are present in strain W3BUL (3,(8)(9)(10), a mutant strain that lacks both detectable chloroplast DNA and chloroplast structure. Thus we interpret our results to indicate that the chloroplast-specific aminoacyl-tRNA synthetases are translated on cytoplasmic ribosomes from transcriptional products of the nuclear genome, and are subsequently compartmentalized within the organelle, as has also been suggested for the NADP: triose phosphate dehydrogenase and alkaline DNase of Euglena (11)(12)(13).Abbreviation: SynPh, chloroplast phenylalanyl-tRNA synthetase.

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...

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