Kinetics of Chlorophyll Accumulation and Formation of Chlorophyll-Protein Complexes during Greening of Chiamydomonas reinhardtii y-1 at 380C'

The initial kinetics of accumulation of chlorophylls (Chi) were analyzed during optimal greening of Chlamydomonas reinhardtii y-1 at 380C. Acetate was required for maximal synthesis of Chi, which occurred at a linear rate when degreened cells were exposed to light. During the first hour Chi a and b accumulated predominantly as geranylgeraniol esters, with lesser amounts of the species with more reduced alcohol side chains. When Chl synthesis was blocked either by treatment with gabaculine or by transfer to the dark, the distribution shifted to the more reduced forms. Similar kinetic pattems indicated that a common pool of chlorophyllides a and b provided substrate for the enzymatic system that performs esterification and reduction of the sidechain for each group of Chi. Chi b was essentially quantitatively integrated into light-harvesting complexes as indicated by energy transfer to Chi a. In the presence of cycloheximide, an inhibitor of cytoplasmic protein synthesis, Chi b did not accumulate and Chi a production was reduced about one-half. The results demonstrate that Chi a/b-protein complexes assemble rapidly during greening and that reduction of the alcohol side chain of the Chi is not required for assembly of these complexes. The rapid and linear greening of yellow cells of Chlamydomonas reinhardtii yl at 38°C offers unique opportunities to examine kinetically light-dependent aspects of chloroplast development. This system, uncomplicated by the lag in greening that occurs in these cells at 25°C (15, 25) can be used to analyze, for example, synthesis of major components of thylakoid membranes such as the light-harvesting Chl a/b-protein complex (LHC23). This complex in higher plants recently was described as an aggregate of three polypeptides (LHCPs) each associated with eight Chl a molecules, seven Chl b molecules, and several carotenoids (7). Degreened C. reinhardtii ycells contain a barely detectable level oftranslatable mRNA for the LHCPs at 25°C (14, 22) but develop a full complement of these mRNAs during a 2-h incubation in the dark at 38°C (14). As shown by the response to phenanthro' This research was supported by National Science Foundation grant DMB 8613585 and Federal Work Study awards to M. A. M. 2 Present address: Department of Biology, Indiana University, Bloomington, IN 47405. 3 Abbreviations: LHC2, light-harvesting complex of PSII; LHCP, light-harvesting Chl a/b-binding apoprotein of LHC2; Chlide, chlorophyllide. lines, these cells are fully capable of synthesizing Chlide b in the dark (3-5). Yet synthesis ofLHCPs 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. This analysis showed that the species that appeared initially were those in which the esterified alcohol is incompletely reduced. Since we found that essentially all the Chl b is associated with light-harvesting complexes, these results indicate that reduction of the alcohol side chain is not essential for assembly of Chl-protein complexes. MATERIALS AND METHODS