FLUORESCENCE ANALYSIS OF PROTOCHLOROPHYLL(IDE) TO CHLOROPHYLL(IDE) CONVERSION IN THE GREEN-ALGA CHLAMYDOMONAS-REINHARDTII MUTANTS

The low-temperature fluorescence spectroscopy allowed to detect three forms of protochlorophyllide in dark-adapted cells of green alga Chlam ydomonas reinhardtii mutant yl (which cannot synthesize chlorophyll - Chl - in the dark), as well as in mutant s48 (which cannot synthesize Chl b). The pigment fluorescence was located at 630, 638 and 656 nm, a nd had excitation maxima at 440 and 444 nm, and a double band at about 425 and 470 nm, respectively. The shape of excitation spectra showed the two former bands belonging to a monomeric pigment while the latter one to the pigment in an aggregated state. The monomeric protochlorop hyllide (PChl) was photochemically active and at room temperature it t ransformed into monomeric chlorophyll(ide). The aggregated PChl was re latively stable and its fluorescence did not change under irradiation. Dark-adapted cells of C. reinhardtii ss107 and vf5 mutants, unable to synthesize light-harvesting complexes (CPLH) or core chlorophyll-prot eins of photosystems 1 and 2, respectively, had the same PChl fluoresc ence. Monomeric PChl only was detected in wild-type cells of C. reinha rdtii in the presence of a substrate for Chl biosynthesis (ALA) or an inhibitor of chloroplast protein formation (CAP). In ALA-treated plant s, the accumulation of PChl was accompanied by accumulation of monomer ic protoporphyrin IX. The latter pigment was the main one detected aft er the alga cultivation at a chilling temperature (8 degrees C). In wi ld-type cells, but not in the s48 or y1 mutants, cultivated in the dar k, fluorescence of monomeric Chi b with the emission maximum at 656 nm was observed. This fluorescence had excitation maximum at 466 nm and seemed to belong to a pigment not bonded to CPLH polypeptides. Hence, in addition to the well-known pathway of Chl formation operating in et iolated higher plants and extensively studied so far, a monomeric path way exists and is responsible for the light-dependent Chl synthesis in cells of C. reinhardtii. This pathway is spectroscopically similar to the recently discovered new pathway of chlorophyll biosynthesis opera ting in light-adapted higher plants.