Pigment mutants of Chlamydomonas with deformed plastids and increased levels of chloroplast nucleic acids

R. E. Wilson 0 H. Swift 0 K.-S. Chiang 0 0 University of Chicago , Illinois 60637 , U.S.A The chloroplast ultrastructure of two Chlamydomonas rcinhardtii pigment variant mutants, U3N and U3A, is strikingly different from that of the wild type. The mutant chloroplast has greatly lowered levels of chlorophyll a and b, and lacks the usual ordered thylakoid membrane structure. The amount of chloroplast ribosomes is increased, but the pyrenoid and surrounding starch grains appear to be unaltered. Our biochemical analyses have shown that, while the properties of chloroplast DNA, ribosomal RNA, and ribosomes in these mutants appeared to be normal, their relative amounts per cell increased markedly when compared to the wild type. In U3N these increases were approximately 60 % for chloroplast DNA and 80 % for chloroplast ribosomes. However, the ratio of chloroplast rDNA genes to total chloroplast DNA remained unchanged as shown by DNA-rRNA hybridization. We propose that (1) The enhanced level of chloroplast ribosomes in these mutants is a direct consequence of the elevated amount of chloroplast DNA. Both of these increases may, in turn, arise from defective mechanism for their control. (2) These mutants grow successfully in the absence of functional photosynthesis, provided an external carbon source is available to them, but functional plastid DNA, ribosomes and protein synthesis may still be a requirement for normal starch metabolism. PIGMENT MUTANTS OF CHLAMYDOMONAS NUCLEIC ACIDS RONALD E. WILSON, HEWSON SWIFT AND KWEN-SHENG CHIANG Department of Biophysics and Theoretical Biology and Department of Biology, University of Chicago, Illinois 60637, U.S.A. SUMMARY In contrast to other unicellular green algae such as Chlorella and Euglena, pigment variants of Chlamydomonas reinhardtii that form stable populations are relatively 1974). Since Chlamydomonas offers a unique possibility for genetic analysis, adequate characterization of such stable mutants should provide a potentially useful tool for dissecting the relative contributions of nuclear and organellar genomes. Parallel studies on the chloroplast ultrastructural and nucleic acid alterations have not yet been documented in any of the stable pigment variant mutants of Chlamydomonas. This paper presents such a study of 2 mutants which exhibit pronounced alterations of pigment constituents (Gross & Dugger, 1969). Both the yellow mutant, U3N, and the white mutant, U3A, resemble the wild type in size, shape, and flagellar length, but their chloroplasts appear much less dense in the light microscope than those of the wild type. U3N, which grows very slowly both in light and in dark, with an organic carbon source, possesses neoxanthin, trollein, violazanthin, lutein, and /?carotene in ratios similar to those found in the wild type; U3A, on the other hand, grows poorly, only in the dark, and is light-sensitive. The unexpected finding of our present study is that, despite the marked alterations of chloroplast ultrastructure, the relative amounts of chloroplast DNA, ribosomes and ribosomal RNA (rRNA) in these mutants all have increased considerably over their counterparts in the wild type. Within the limits of the methods used, the macromolecular properties of mutant nucleic acids appear to be unchanged. While U3 A appeared, during the initial phase of this work, to be the more drastically altered of the 2 mutants, most biochemical work reported here concerns U3N. This is due to an unfortunate loss of our U3A stocks which grew only very poorly under a large variety of conditions evaluated in our laboratory. Requests for U3A strains from several other laboratories, including that of its isolator, were unsuccessful, apparently for the same reason. Because of the rare occurrence of a completely colourless Chlamydomonas mutant, as well as its novel ultrastructural and biochemical characteristics, however, the results of our U3A work are documented here. These data may be of value for comparison not only with any other white mutants which may be isolated in the future, but also with U3N, which was isolated from the same parental unstable mutant, U3, and which exhibited a number of properties similar to U3A. Specific differences also exist between these 2 mutants. Strains and culture conditions Three strains of C. reinhardtii were used: (1) 137C, the wild type, minus mating type, originally obtained from Dr R. P. Levine, (2) U3N, the stable yellow mutant, and (3) U3A, the stable white mutant obtained originally from Dr R. E. Gross. These mutants were derived from a wild type C. reinhardtii minus mating type strain No. 90 of the Indiana Culture Collection (Gross & Dugger, 1969). Wild-type cells were grown in HSM (Sueoka, i960) in 1- or 2-1. Erlenmeyer flasks with cboyn'tcionouoluwshsittier'rifnlugoraensdceanetralatimonpswaitthana imntiexntusirtey ooff 340%0 fCt.Oc2anindleasir.(1Il3luxmiion4atlimonmw~a2)s. pMrouvtaidnetds were grown in the dark in the medium described by Sager & Granick (1954), supplemented with o-i % sodium acetate. Optimum growth in this medium was obtained in 2-1. Erlenmeyer flasks stoppered with a cotton plug, without aeration or stirring. Stationary phase (approx. io6 cells/ml) was attained in about 1 week for U3N and about 15 weeks for U3A. Radioactive labelling and preparation of the S-10 fraction at Baobtohuwtil1d-2tyxpeioa6ndcemllust/amnlt. c[e1l4lCsw]aedreenlianbeel(le5d0/atCtai/pnpirMox)imoarte[l3yH2]axdeinoi5nceel(l2s/5m-8l Cani/dmhMar)vewsteerde added to the media to a final concentration of o-i /tCi/ml and 05/tCi/ml, respectively. Labelled cells were mixed immediately before harvesting and were collected by centrifugation at 6000 g. Subsequent operations were performed on ice. Cells were washed twice with Fig. i. Low-power micrograph of a U3A cell. The nucleus and central nucleolus (n) are normal in appearance, as are the pyrenoid (/>) and its surrounding starch grains. An abnormal membrane whorl surrounds a large vacuole within the plastid at the lower right. Several smaller vacuoles (v), probably autophagic, contain degenerating cell material, x 20000. 25 ml KC1, 25 mM MgCl2, 25 miti Tris-Cl (pH 75), 025 M sucrose (Hoober & Blobel, 1969) supplemented with 10 mM mercaptoethanol. After resuspending in the same buffer, the cells were broken by 2 passages through a French Pressure Cell at 7000 lb/in2 (4'83 x io4 kN m~2). This cell homogenate was centrifuged for 10 min at 10000 g. The supernatant, designated as the S-10 fraction, was either stored at 70 C or used immediately. Sucrose gradient, analysis of ribosomes Linear gradients 0-465 to 101 M in sucrose were used with either buffer A (25 mM KC1, 25 mM MgCl2, 25 mM Tris-Cl, pH 75) or modified buffer A (25 mM KC1, 5 mM MgCl2, 25 mM Tris-HCl, pH 75). 02 ml of the Triton-treated S-10 fraction were layered onto the 12-ml gradient, which was spun for 5 h at 38000 rev/min at 4 CC in an SB-283 rotor of an International Model B60 ultracentrifuge. Forty fracti (...truncated)