William Ortiz

Delayed but Complete Loss of Chloroplast DNA in Heat-Bleaching Cultures of Euglena gracilis

Summary We investigated the effect of incubation at a moderately elevated temperature of 33 °C on the organization of the chloroplast DNA in Euglena gracilis z strain. Our objective was to test whether irreversible loss of chloroplast function is associated with either a complete or partial loss of genetic information in the chloroplast. We show that loss of chloroplast DNA begins at about 60 h and that DNA is undetectable at 100 h. Nevertheless, the number of bleached (white) cells in the culture approaches 80 % before loss of chloroplast DNA can be detected. We conclude that loss of chloroplast DNA is not the root-cause of heat-bleaching but can explain why heat-bleached cells remain permanently white after many months of incubation at the permissive temperature.

Loss of chloroplast transcripts for proteins associated with photosystem II: an early event during heat-bleaching in Euglena gracilis

A shift in the ratio of chlorophyll (Chl) a and Chl b is an early indicator of heat bleaching in Euglena gracilis. This observation prompted us to consider whether or not changes in steady-state levels of chloroplast transcripts and in transcriptional activity could limit the synthesis of Chl a-binding proteins in bleaching plastids. We found that the mature transcripts for CP47 and CP43, the Chl a-binding apoproteins of the proximal antenna of photosystem II, decline sharply very early during bleaching. Our study also shows that transcription of psbB and psbC, the chloroplast genes encoding CP47 and CP43, remains essentially unchanged during the same interval. We conclude that posttranscriptional events, such as mRNA stability, could play a major role in initiating an irreversible loss of chloroplast function in Euglena at a moderately elevated temperature. Lack of these transcripts would eventually impair the assembly of photosystem II in thylakoids.

Cycloheximide delays chlorophyll loss but not the inhibition of plastid protein synthesis during heat-bleaching in Euglena gracilis

Summary Growth at the bleaching temperature of 33 °C induces chlorophyll loss and inhibits chloroplast protein synthesis in photoheterotrophic cultures of Euglena gracilis Z. We now show that brief incubations in the presence of cycloheximide during the early hours of the treatment at the elevated temperature delay the onset of chlorophyll loss. Incubations in the presence of cycloheximide, however, do not bring temporary protection to plastids against the inhibitory effect of temperature on plastid protein synthesis. We conclude that protein synthesis on cytoplasmic ribosomes appears to be required to bring about the loss of chlorophyll at 33 °C. Furthermore, the inability of Euglena gracilis to develop and maintain functional chloroplasts at the elevated temperature can be explained by changes in the ability of plastids to synthesize proteins that are independent from the effect of temperature on chlorophyll accumulation.

Contributions from the plastid and the nucleocytoplasmic compartments to the events associated with heat-bleaching in Euglena gracilis

Summary Growing photoheterotrophic cultures of Euglena gracilis Z show a time-dependent loss of chlorophyll per cell that results in the production of bleached cultures of the alga on prolonged incubation at 33°C. In terms of chlorophyll accumulation, the bleaching process involves an initial rise in chlorophyll accumulation per cell, peaking at about 15 h, followed by a second phase in which chlorophyll levels per cell actually decline. The primary objective of this work was to establish whether loss of chloroplast function at 33°C is associated with a loss of translational activity on chloroplast ribosomes or a loss in the synthesis of chloroplast proteins of nucleocytoplasmic origin, or both. To this effect, pulse-labeling experiments were carried out in vivo on cultures of the alga in the second phase of the temperature response. The results show that synthesis and import of proteins from the nucleocytoplasmic compartment remain high in bleaching plastids during the first 41 h of the temperature treatment. By contrast, protein synthesis inside plastids is gradually lost at the elevated temperature, and only low levels can be detected in 41-h bleached cultures. Consequently, the great majority of the proteins accumulating in 41-h bleached plastids are imported from the nucleocytoplasmic compartment. The loss of translational activity inside the bleaching plastid could explain why Euglena is unable to maintain photosynthetically competent plastids at the elevated temperature.

Factors that affect the synthesis and accumulation of a polypeptide of the light-harvesting chlorophyll-protein complex II in Euglena gracilis

Summary Cultures of photoheterotrophically grown Euglena gracilis Z maintained at 33 ° experience a time-dependent loss of chlorophyll and the preferential loss of the 24,500Mr polypeptide associated with lightharvesting chlorophyll a/b-binding protein the complex of Photosystem II. It is, therefore, important to establish whether temperature or chlorophyll loss is the overriding factor affecting the ability of bleaching plastids to accumulate the 24,500 Mr species. In this study we show that treatment with levulinic acid, an inhibitor of chlorophyll biosynthesis, resulted in a complete loss of the 24,500 Mr species from thylakoids. Light-grown cells exposed to darkness for up to 15 hours, on the other hand, experienced a rapid loss of chlorophyll but little or no reduction in the levels of the 24,500 Mr component. These observations suggest that, in Euglena , loss of chlorophyll and loss of the 24,500 Mr polypeptide from thylakoids are not always directly correlated. We also show that despite the reduction in the levels of the 24,500 Mr species from bleaching thylakoids the polypeptide continues to be synthesized at 33 °C. The failure of bleaching thylakoids to accumulate this polypeptide can be explained by an increased turnover of the newly synthesized polypeptide.

Inhibitory effect of hypergravity on photosynthetic carbon dioxide fixation in Euglena gracilis.

Photosynthesis, the conversion of light energy into chemical energy, is a critical biological process, whereby plants synthesize carbohydrates from light, carbon dioxide (CO2) and water. The influence of gravity on this biological process, however, is not well understood. Thus, centrifugation was used to alter the gravity environment of Euglena gracilis grown on nutritive agar plates illuminated with red and blue light emitting diodes. The results showed that hypergravity (up to 10xg) had an inhibitory effect on photosynthetic CO2 fixation. Chlorophyll accumulation per cell was essentially unaffected by treatment; however, Chl a/Chl b ratios decreased in hypergravity when compared to 1xg controls. Photosynthesis in Euglena appears to have limited tolerance for even moderate changes in gravitational acceleration.

Endogenous Substrates of the Euglena Chloroplast Chaperonin 60 at Permissive and Bleaching Temperatures

Summary Cultures of Euglena gracilis grown at the moderately elevated temperature of 33 °C experience an irreversible loss of chlorophyll and chloroplast function known as heat-bleaching. Since molecular chaperones play an important role in protein folding, we considered whether or not chloroplast chaperonin 60 (Cpn60) binds to nascent polypeptides and newly imported polypeptides, its likely targets for folding. In this report, we address the effect of growth temperature on the ability of chloroplast Cpn60 to bind endogenous substrate in vivo using pulse-labeling and co-immunoprecipitation with antibody against Cpn60. Four major polypeptides and about a half-dozen minor polypeptides appear to be the targets of the chaperonin complex at the permissive temperature of 23 °C. Among the principal targets are the large subunit of Rubisco (RbcL) and elongation factor-Tu (EF-Tu). By contrast, only RbcL co-immunoprecipitates with Cpn60 in stromal extracts from partially bleached Euglena but at a much reduced level. Associations between the chaperonin and its target polypeptides resume in a partially bleached culture when returned to the permissive temperature. These observations suggest that chloroplast chaperonin targets only a small, select group of proteins for folding. In addition, the activity of the chaperonin complex in Euglena is sensitive to even a modest increase in growth temperature.

Chaperonin 60 and the stability of polypeptides synthesized in isolated chloroplasts of Euglena gracilis at moderately elevated temperatures

Summary Chloroplast function and continuity is affected when exponential cultures of Euglena gracilis Z are grown at the moderately elevated temperature of 33°C. Since protein instability could be a key factor in the response of chloroplasts to temperature, we carried out pulse-chase experiments with isolated chloroplasts. We have identified a polypeptide of 20.5 ku (kDa) that is degraded quite rapidly when isolated chloroplasts are pulsed at 33°C and then chased at either 23°C or at 33°C. By contrast, most polypeptides synthesized by isolated chloroplasts do not seem to be degraded during the chase period. We believe that the labile nature of the 20.5 ku is exacerbated at 33°C because the amounts of chaperonin 60 detected in Euglena chloroplasts appear to be low and probably insufficient for protection of newly synthesized proteins at elevated temperatures.

Increased Turnover of a Polypeptide Associated with the Lightharvesting Chlorophyll-protein Complex II in Partially Bleached Euglena gracilis

Summary The levels of a 24,500 Mr polypeptide of the light-harvesting chlorophyll-protein complex associated with photosystem II decrease in plastids of growing photoheterotrophic cultures of Euglena gracilis during heat-bleaching at 33 °C. The polypeptide, however, continues to be synthesized, imported and incorporated into thylakoids despite reduced levels of accumulation. The failure of bleaching plastids to maintain normal levels of this polypeptide could be explained by an increase in turnover of the newly synthesized polypeptide at the elevated temperature. In this report, I show that the polypeptide is rapidly degraded when isolated plastids from [ 35 S]labelled partially bleached Euglena are incubated at 33 °C. Pulse-chase experiments carried out in vivo with partially bleached cell cultures essentially confirm the results obtained with isolated plastids. The results presented suggest that post-translational events regulate the levels of the 24,500 Mr polypeptide in bleaching Euglena.

Transcriptional activity in permeabilized chloroplasts ofEuglena

Chloroplasts ofEuglena gracilis rendered permeable with toluene incorporate radiolabeled UTP into cold TCA-precipitable material at higher rates than chloroplast lysates. Incorporation is also linear for approximately 40 minutes. Transcripts produced toluene-treated chloroplasts range from approximately 0.24 to 7.5 kilobases.