Induced Changes in Chloroplast Protein Accumulation during Heat Bleaching in Euglena gracilis

Abstract

When growing cultures of light-grown Euglena graciis Z are exposed to slightly elevated temperatures (33°C) there is a time-dependent decrease in chlorophyll (bleaching) and a gradual transformation of chloroplasts into rudimentary plastids. A study was undertaken whose primary objective was to document major changes in polypeptide composition in the stroma and in thylakoids of cells that have been exposed to the bleaching temperature for up to 57 hours. A novel polypeptide of about 60,000 to 63,000 Mr whose function is presently unknown, accumulates in the stroma and in thylakoids in response to growth at the bleaching temperature. The levels of the large and small subunit of ribuolosebisphosphate carboxylase, on the other hand, decrease to very low levels at about 33 hours and remain very low for the duration of the temperature treatment. Of two polypeptides associated with the light-harvesting chlorophyll-protein complex of photosystem II (28,000 and 24,500 Mr) only the level of the smaller polypeptide decreases at the elevated temperature. The levels of 28,000 M, species remain virtually unchanged throughout the temperature treatment period. Changes in chloroplast polypeptide composition were also studied in ceUs that were allowed to recover at room temperature from an initial treatment at 33°C. Bleaching Euglena could provide a useful tool for studying the interaction between the nucleus and chloroplast genetic system that govern the development and maintenance of this vital organelle to plants.Chloroplasts contain their own DNA and the transcription and translation machinery to synthesize the limited number of proteins encoded in the organelle genome (11). The rest of the chloroplast proteins are encoded in the nucleus, translated in the cytoplasm, and imported by the organelle for integration and assembly into the appropriate structural/functional compartment of the chloroplast. Clearly, the development and maintenance of chloroplasts is the result of an intricate interplay between the chloroplast genetic system and the nucleocytoplasmic compartment. Elucidating the molecular basis of this interaction is one of the most fascinating subjects in the field of plant molecular biology.Euglena gracilis provides a convenient system in which to study the nature of the interaction between the nuclear and chloroplast genomes because the alga exhibits a temperature-dependent transformation of chloroplast into rudimentary plastids. When growing cultures of light-grown Euglena are exposed to temperatures of 32 to 34°C there is a time-dependent decrease in the amount of Chl per cell that ultimately leads to the production of irreversibly bleached cultures (26). Interest in this phenomenon, also known as heat-bleaching, dates to the 1950s and was found particularly useful in the generation of colorless strains of Euglena (27). In addition to temperature, ultraviolet light and streptomycin have been used as bleaching agents.Heat bleaching in Euglena appears to target the chloroplast almost exclusively since bleac...

Protein Synthesis during the Initial Phase of the Temperature-Induced Bleaching Response in Euglena gracilis

“…Euglena gracilis Z was grown photoheterotrophically at 23°C in gyratory shakers under cool-white fluorescent lamps as described previously (15). The culture medium was a modified version of Hutner's medium (14) containing 50 ng/ L of vitamin B-12.…”

“…The culture medium was a modified version of Hutner's medium (14) containing 50 ng/ L of vitamin B-12. Temperature-induced bleaching was initiated by transferring the cultures to a gyratory shaker kept at a constant temperature of 33°C ( 15).…”

“…We have recently reported (15) that temperature-induced Chl loss at 33°C occurs in two phases. The initial phase, which involves an increase in total Chl per cell, is followed by a second phase characterized by a steady decline in Chl accumulation per cell that continues for the remainder of the treatment at 33°C.…”

“…The initial phase of the temperature response has been of interest to us since the increase in Chl accumulation per cell suggests a parallel increase in the synthesis of plastid components including proteins. A major change in the profile of stainable polypeptides during this initial phase is in the accumulation of presumably novel polypeptides of 45,000 and 60,000 Mr in the thylakoid (15). ' Total protein synthesis in control and partially bleached cells was determined by blotting 50 ,uL aliquots of cell suspension on filter paper discs 1 h after the addition of the radiolabeled sulfate.…”

“…Radiolabeled cells were harvested by centrifugation and the [35S]plastids isolated on isoosmotic linear gradients of 10 to 50% Percoll underlaid with a cushion of isoosmotic 80% Percoll (14,15). The band of intact plastids was collected from the gradient and washed by dilution with four volumes of ice-cold gradient buffer followed by centrifugation at 5,000 rpm for 3 min (Beckman JA-20 rotor).…”