Mol Gen Genet (1981) 184:460-464 © Springer-Verlag 1981 Protochlorophyllide Photoconversion Mutants of Chlamydomonas reinhardtii Clark Ford, Susan Mitchell, and Wei-yeh Wang Botany Department and Genetics Ph.D. Program, University of Iowa, Iowa City, Iowa 52242, USA Summary. We have developed a procedure for the isolation of Chlamydomonas reinhardtii mutants defective in light-dependent protochlorophyllide reduction (photoconversion), a key step in the biosynthesis of chlorophyll. Mutants were isolated by muta- genizing y-l-4, a temperature-sensitive yellow mutant blocked in the alternative light-independent protochlorophyllide reduc- tion pathway, and screening for colonies which failed to green in the light at the restrictive temperature. Seven mutants were isolated which fail to photoconvert protochlorophyllide in photo- conversion tests. All seven mutants have a single mutation at the pc-1 locus responsible for the defect in photoconversion. pc-1 maps close to y-5 on nuclear linkage group I. The pc-1 mutation is not itself temperature-sensitive because it blocks photoconversion at the permissive temperature when combined with the non-conditional yellow mutations y-5 and y-7. Cells containing the pc-1 mutation alone synthesize about 52% and 36% of the wildtype chlorophyll level in the dark and light, respectively, demonstrating that the light-independent proto- chlorophyllide reduction pathway in C. reinhardtii operates in the light. Introduction The reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide) is a key step in the biosynthesis of chlorophyll. It is closely coupled with the initiation of chlorophyll biosynthesis as well as with the development of functional chloroplasts. In the unicellular green alga Chlamydomonas reinhardtii, two alter- native pathways exist for Pchlide reduction. The first is light- independent, whereas the second is light-dependent. Light-independent Pchlide reduction is catalyzed by an un- known enzyme system. This pathway, which occurs only in some lower plants and algae, allows the synthesis of chlorophyll to proceed in the dark (Bogorad 1976). yellow mutants of C. rein- hardtii are defective in this pathway. When grown in the dark, yellow mutants do not synthesize chlorophyll but instead accu- mulate small amounts of Pchlide (Sager 1961 ; Ford and Wang 1980a, b). In the light, yellow mutants synthesize chlorophyll normally via the alternative, light-dependent Pchlide reduction pathway. Sager (1955) isolated and characterized the first C. reinhardtii yellow mutant, y-1. We have recently characterized six new yellow loci, designated y-5, y-6, y-7, y-8, y-9, and y-lO, and have isolated Offprint requests to." Wei-yeh Wang a temperature-sensitive y-1 allele, designated y-l-4 (Ford and Wang 1980a, b). Because of the number of loci involved in its genetic control, it appears that light-independent Pchlide re- duction either is a process with several reaction steps or involves a complex enzyme with many components. Light-dependent Pchlide reduction, or photoconversion, is catalyzed by a pigment-protein complex termed the Pchlide- holochrome. Holochrome-mediated Pchlide reduction is ubiqui- tous in the plant kingdom and is responsible for the greening (in light) of etiolated plants (Harel 1978). The Pchlide-holo- chrome has been isolated and intensively studied especially in higher plants (for reviews see: Boardman 1966; Bogorad 1976; Harel 1978). Recent biochemical studies have shown that the Pchlide-holochrome is a membrane-bound enzyme which uses NADPH as the hydrogen donor (Griffiths 1978; Oliver and Griffiths 1980; Apel et al. 1980; Beer and Griffiths 1981). Only one mutant blocked in light-dependent Pchlide reduc- tion has previously been studied, the L-6 mutant of Arabidopsis (Robbeln 1956). Since the Pchlide-holochrome can be isolated and studied in vitro, much could be learned from characterizing mutants defective in the photoconversion reaction. In our effort to study the genetic control of Pchlide reduction in C. reinhardtii, we have attempted and succeeded in isolating mutants defective in Pchlide photoconversion. This paper reports the isolation and characterization of seven of these mutants. Materials and Methods Strains. Wildtype and mutant stocks of C. reinhardtii used in this study were derived from strain 137c. Mutants used were the temperature-sensitive y-1 allele y-l-4 (Ford and Wang 1980b), the non-conditional yellow mutations y-5 and y-7 (Ford and Wang 1980a), and the linkage group I marker arg-7. Culture Conditions. Culture conditions were basically those of Ford and Wang (1980a, b). Cells were grown on TAP (Tris Acetate Phosphate) plates for genetic analysis and in HSA (High Salt Acetate) liquid medium for physiological analysis. For ge- netic analysis, cultures were grown at 25° C or at 33° C in the light (1,500 1,600 lux at 25° C, 2,500-3,000 lux at 33° C) or under dim light (about 0.5 lux at both temperatures). Dim light was used interchangeably with darkness for scoring the visible phenotypes of the mutants. All cultures were monitored for puri- ty and recloned when necessary. For photoconversion tests, cells were grown in the dark at 25° C or at 32° C. 32° C was used instead of 33° C in order 0026-8925/81/0184/0460/?-,01.00