Abstract. The integral membrane proteins of photosystem II (PS II) reaction center complexes are encoded by chloroplast genomes. These proteins are absent from thylakoids of PS II mutants of algae and vascular plants as a result of either chloroplast or nuclear gene mutations. To resolve the molecular basis for the concurrent absence of the PS II polypeptides, protein synthesis rates and mRNA levels were measured in mutants of Chlamydomonas reinhardtii that lack PS II. The analyses show that one nuclear gene product regulates the levels of transcripts from the chloroplast gene encoding the 51-kD chlorophyll a-binding polypeptide (polypeptide 5) but is not involved in the synthesis of other chloroplast mRNAs. Another nuclear product is specifically required for translation of mRNA encoding the 32-34-kD polypeptide, D1. The absence of either D1 or polypeptide 5 does not eliminate the synthesis and thylakoid insertion of two other integral membrane proteins of PS II, the chlorophyll a-binding polypeptide of 46 kD (polypeptide 6) and the 30-kD "Dl-like" protein, D2. However, these two unassembled subunits cannot be properly processed and/or are degraded in the mutants even though they reside in the membrane. In addition, pulse labeling of the nuclear mutants and a chloroplast mutant that does not synthesize D1 mRNA indicates that synthesis of polypeptide 5 and D1 is coordinated at the translational level. A model is presented to explain how absence of one of the two proteins could lead to translational arrest of the other. IN chloroplasts of green algae and higher plants the transfer of electrons from water to plastoquinone in the photosynthetic electron transport chain is accomplished by the photosystem II (PS II) I reaction center complex of thylakoid membranes. PS II consists of at least eight proteins and several protein-bound electron carriers and pigments. Recent interest in PS II structure, function, and biogenesis has focused on five integral membrane proteins encoded in chloroplast DNA and three extrinsic proteins encoded by nuclear genes. The extrinsic proteins are involved in the oxidation of water in the thylakoid lumen and the transfer of electrons to the PS II reaction center (9).The two largest integral membrane proteins of the PS II core complex of Chlamydomonas reinhardtii, polypeptides 5 and 6 (7), possess molecular masses of '~51 and 46 kD, respectively, and bind chlorophyll a (11). The smaller integral membrane proteins considered to be essential components of PS II are cytochrome b559 (3-9 kD), D1 (32-34 kD), and D2 (30 kD) (9). The function of cytochrome b559 is unknown, but it may involve cyclic electron flow around the reaction center (9). D1 participates in binding the QB species of plastoquinone, a secondary electron acceptor of PS II, and herbicides of the urea and triazine classes (30); D1 has also been called the QB protein, the herbicide-binding protein, gions that include putative quinone-binding sites (9, 58). The analysis of PS II mutants provides a valuable approach to understanding...

Levels of mRNA for the chloroplast-encoded elongation factor Tu (tufA) showed a dramatic daily oscillation in the green alga Chlamydomonas reinhardtii, peaking once each day in the early light period. The oscillation of tufA mRNA levels continued in cells shifted to continuous light or continuous dark for at least 2-3 days. Run-off transcription analyses showed that the rate oftufA transcription also peaked early in the light period and, moreover, that this transcriptional oscillation continued in cells shifted to continuous conditions. The half-life of tufA mRNA was estimated at different times and found to vary considerably during a light-dark cycle but not in cells shifted to continuous light. Light-dark patterns of transcription of several other chloroplast-encoded genes were examined and also found to persist in cells shifted to continuous light or dark. These results indicate that a circadian clock controls the transcription of tufA and other chloroplast-encoded genes.

Chloroplast DNA (cpDNA) is under great photooxidative stress, yet its evolution is very conservative compared with nuclear or mitochondrial genomes. It can be expected that DNA repair mechanisms play important roles in cpDNA survival and evolution, but they are poorly understood. To gain insight into how the most severe form of DNA damage, a double-strand break (DSB), is repaired, we have developed an inducible system in Arabidopsis that employs a psbA intron endonuclease from Chlamydomonas, I-CreII, that is targeted to the chloroplast using the rbcS1 transit peptide. In Chlamydomonas, an I-CreII-induced DSB in psbA was repaired, in the absence of the intron, by homologous recombination between repeated sequences (20-60 bp) abundant in that genome; Arabidopsis cpDNA is very repeat poor, however. Phenotypically strong and weak transgenic lines were examined and shown to correlate with I-CreII expression levels. Southern blot hybridizations indicated a substantial loss of DNA at the psbA locus, but not cpDNA as a whole, in the strongly expressing line. PCR analysis identified deletions nested around the I-CreII cleavage site indicative of DSB repair using microhomology (6-12 bp perfect repeats, or 10-16 bp with mismatches) and no homology. These results provide evidence of alternative DSB repair pathways in the Arabidopsis chloroplast that resemble the nuclear, microhomologymediated and nonhomologous end joining pathways, in terms of the homology requirement. Moreover, when taken together with the results from Chlamydomonas, the data suggest an evolutionary relationship may exist between the repeat structure of the genome and the organelle's ability to repair broken chromosomes.DNA repair | evolution | homing endonuclease | I-CreII | plastid DNA C hloroplast DNA (cpDNA) is closely associated with the photosynthetic membranes that harvest radiant energy and strip electrons from water, producing molecular oxygen as a byproduct (1, 2). Highly reactive forms of oxygen are also generated, and despite the presence of detoxifying enzymes, photooxidative damage is a serious problem. Although there has been extensive study of the damage, protection, and repair of photosynthetic membranes, there has been little attention paid to the genetic consequences of photooxidative stress (3). This can be attributed, at least in part, to a grossly incomplete knowledge of how cpDNA is replicated and maintained throughout the life of a plant (3, 4).However, despite the high-stress environment, the evolution of cpDNA is more conservative than nuclear or mitochondrial DNA (5). We can thus infer that DNA repair mechanisms play important roles in protecting cpDNA and are intimately involved in its evolution. And although some processes have been identified (6-11), the panoply of organelle repair mechanisms is not known, nor is it clear which are most important, or how they have coevolved with the genome. Although we expect that some cpDNA repair processes will reflect its prokaryotic ancestry, the consequences of eukaryotic cell integration...

A cloned cpDNA fragment containing a portion of the gene for the 32 -36 kDa thylakoid protein of Chlamydomonas (polypeptide D-l) was isolated. Hybridization probing of RNA from soluble and membrane fractions of Chfamydomonas showed that the mRNA for D-l is bound to thylakoid membranes.Run-off translation of thylakoid-bound polysomes (rough thylakoids) with [35S]-methionine yields polypeptide D-l as the major product. Peptide mapping with S. aureus V-8 protease of D-l synthesized (1) in vivo, (2) in vitro by rough thylakoids and (3) in the reticulocyte lysate directed by non-polyadenylated RNA showed that D-l is synthesized as a precursor in the reticulocyte lysate but as the mature polypeptide by rough thylakoids. ChlamydomonaspsbA gene DNA-RNA hybridization Rough thylakoid In vitro protein synthesis 32 kDa precursor

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