Oenothera Plastome Mutator-Induced Chloroplast Mutant1

Plastids were isolated from a plastome mutator-induced mutant (pm7) of Oenothera hookeri and were analyzed for various physiological and biochemical attributes. No photosynthetic electron transport activity was detected in the mutant plastids. This is consistent with previous ultrastructural analysis showing the absence of thylakoid membranes in the pm7 plastids and with the observation of aberrant processing and accumulation of chloroplast proteins in the mutant. In comparison to wild type, the mutant tissue lacks chlorophyll, and has significant differences in levels of four fatty acids. The analyses did not reveal any differences in carotenoid levels nor in the synthesis of several chloroplast lipids. The consequences of the altered composition of the chloroplast membrane are discussed in terms of their relation to the aberrant protein processing of the pm7 plastids. The pigment, fatty acid, and lipid measurements were also performed on two distinct nuclear genotypes (A/A and A/C) which differ in their compatibility with the plastid genome (type 1) contained in these lines. In these cases, only chlorophyll concentrations differed significantly. A large number of plastome (or plastid genome) mutants have been generated through the use of the plastome mutator system in the Johansen strain of Oenothera hookeri (15). The mutants thus far accumulated in this collection display various degrees ofchlorosis as well as differing extents ofaberrant chloroplast development as seen at the ultrastructural level (13, 14). Immunological analysis of plastid proteins from one of these plastome mutants, pm7, has provided evidence that indicates the existence of a defect in chloroplast protein processing (EM Johnson, BB Sears, L Schnabelrauch, unpublished data). Proteins affected include one encoded by a chloroplast gene (Cytf) as well as the 16and 23-kD subunits of the oxygen evolving complex of PSII, which are nuclear gene products. The suggestion that the processing of these three proteins in pm7 may be inhibited by a lesion of some shared feature of the proteolytic machinery is supported by the observation that all three proteins appear to be normally ' E. M. J. was supported by a graduate fellowship from the Michigan State University McKnight Photosynthesis training grant and National Science Foundation grant DCB-85002849. Michigan Agricultural Experiment Station Journal No. 13016. 2Current address: Department of Plant Biology, University of California, Berkeley, CA. processed in a putative revertant of pm7 (EM Johnson, BB Sears, L Schnabelrauch, unpublished data). Earlier ultrastructural analysis ofpm7 tissue from leaf-tip culture revealed a striking lack of developed internal membrane structure in the mutant plastids despite the presence of chloroplast ribosomes, plastoglobuli, and a large number of vesicular structures ( 14). It appeared to us that such a mutant with no internal membrane structure would be useful for analysis of thylakoid composition. Specifically, we hoped to determine to what extent individual constituents ofthylakoids accumulated in the absence ofdeveloped internal membranes. Thus, we undertook to characterize pigment, fatty acid, and lipid contents of the mutant. Experiments described in this report were designed to provide a physiological and biochemical characterization of pm7 mutant plastids, including the status of these nonprotein components of the chloroplast and an analysis of the extent of photosynthetic electron transport in the mutant. The results of these experiments are discussed in the context of various possible connections between chloroplast protein processing, accumulation of chloroplast components and the degree of thylakoid development in pm7. Parallel to comparative analyses of pm7 and wild-type plastids, we also examined many of these same characteristics in plastids from Oenothera plants containing two different nuclear backgrounds. These two genotypes, A/A and A/C, differ in their 'compatibility' with the type I plastome (28) as noted by the degree of chlorosis and photosynthetic competence. Hence, analysis ofmembrane composition ofthese two plastome-genome combinations may provide further insight into the phenomenon of 'plastome-genome incompatibility.' MATERIALS AND METHODS