Kinetics and regulation of synthesis of the major polypeptides of thylakoid membranes in Chlamydomonas reinhardtii y-1 at elevated temperatures

Abstract

Etiolated cells of Chlamydomonas reinhardtii y-1 exhibit rapid and linear initial kinetics of greening when exposed to light at 38~ The initial rate of chlorophyll accumulation under these conditions is greater than the maximal rate during greening at 25~ Synthesis of the major polypeptides of thylakoid membranes within intact cells was assayed during greening by the incorporation of [3H]leucine and the subsequent electrophoresis of total cellular protein on polyacrylamide gels in the presence of sodium dodecyl sulfate. At 38~ the major membrane polypeptides (about 28,000 and 24,000 daltons in mass) were synthesized at a linear rate after exposure of the cells to light, with no evidence of a lag period. A 1-2 h preincubation in the dark at the higher temperature was necessary to achieve linear initial kinetics. Actinomycin D inhibited synthesis of the membrane polypeptides if added at the beginning of a 2 h dark preincubation, but not when added near the end. These results suggested that transcription of the messenger RNA for the membrane polypeptides occurred during the dark period at 38~ But the major membrane polypeptides were not made by y-1 cells in the dark. The wavelengths of light most effective in eliciting production of the membrane polypeptides were the same as those allowing chlorophyll synthesis. In contrast, wild type cells, which are capable of chlorophyll synthesis in the dark, also make the membrane polypeptides in the dark. The data indicate that at elevated temperatures synthesis of the major thylakoid membrane polypeptides is controlled at a posttranscriptional step, and that this reaction normally proceeds only under conditions which permit reduction of protochlorophyllide.Greening of etiolated plant cells is characterized by the development of photosynthetic competency. The elemental process in the course of this development is the formation of thylakoid membranes within chloroplasts in response to light. Thylakoid membranes, which convert light energy into chemical energy, constitute the major system of membranes within light-grown plant cells. Although these membranes are located within the chloroplast, their formation requires the participation of synthetic systems in the cytoplasmic matrix as well as in the chloroplast. Chlorophyll and 326

In Vitro Processing of Precursors of Thylakoid Membrane Proteins of Chlamydomonas reinhardtii y-1

“…Green cells of C. reinhardtii y-l were obtained by growth at 25°C as previously described (19,20). Yellow label the translated proteins.…”

“…For instance, in vivo labeling of the mature proteins is limited because of the restricted uptake of amino acids by the algal cells (15,19), and also the precursors contain an additional sequence at the N-terminus known as the transit sequence. These problems and their implications have been discussed by Schmidt et al (21) and by Shepherd et al (23).…”

Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chlamydomonas reinhardtii y-1 at 38°C

“…Strain pgl 13 (23) was kindly provided by Dr. A. Boschetti. Green y-l cells, after growth in the light for 1 d, were transferred to the dark and growth was continued for 3 to 4 d at 25°C (3,16). The degreened cells were harvested by centrifugation and suspended in fresh growth medium (15,16) to a density of 1 x 107 cells/mL.…”

“…Green y-l cells, after growth in the light for 1 d, were transferred to the dark and growth was continued for 3 to 4 d at 25°C (3,16). The degreened cells were harvested by centrifugation and suspended in fresh growth medium (15,16) to a density of 1 x 107 cells/mL. For assays of the effects of acetate, degreened cells were suspended in a medium containing 3 mM K2HPO4, 7 mM KH2PO4, 7.5 mM NH4C1, 1.0 mM MgSO4, 0.1 mM CaCl2, and 10 mM NaCl; sodium acetate was added as desired.…”

“…As shown by the response to phenanthro-lines, these cells are fully capable of synthesizing Chlide b in the dark (3)(4)(5). Yet synthesis of LHCPs proceeds at a low rate and no Chl is made until cells are exposed to light (16). As a continuation of this work, we have examined the kinetics of Chl accumulation under conditions that support a maximal rate of greening when degreened cells are exposed to light.…”