Nuclear mutants of Chlamydomonas reinhardtii defective in the biogenesis of the cytochrome b6f complex

Abstract

The random integration of transforming DNA into the nuclear genome of Chlamydomonas has been employed as an insertional mutagen to generate a collection of photosynthetic mutants that display abnormal steady-state fluorescence levels and an acetate-requiring phenotype. Electron paramagnetic resonance spectroscopy was then used to identify those mutants that specifically lack a functional cytochrome b6f complex. Our analysis of RNA and protein synthesis in five of these mutants reveals four separate phenotypes. One mutant fails to accumulate transcript for cytochrome f, whilst a second displays a severely reduced accumulation of the cytochrome b6 transcript. Two other mutants appear to be affected in the insertion of the haem co-factor into cytochrome b6. The fifth mutant displays no detectable defect in the synthesis of any of the known subunits of the complex. Genetic analysis of the mutants demonstrates that in three cases, the mutant phenotype co-segregates with the introduced DNA. For the mutant affected in the accumulation of the cytochrome f transcript, we have used the introduced DNA as a tag to isolate the wild-type version of the affected gene.

In vivo evidence for 5′→3′ exoribonuclease degradation of an unstable chloroplast mRNA

“…We interpret these results as strong in vivo evidence that in strains containing the mcd1-1 mutation, petD transcripts are degraded by 5Ј→3Ј exoribonuclease activity. Several other non-photosynthetic Chlamydomonas nuclear mutants have been isolated that affect the accumulation of a single chloroplast mRNA (Drapier et al, 1992;Gumpel et al, 1995;Kuchka et al, 1989;Monod et al, 1992;Sieburth et al, 1991). In the case of nac2, the determinant of psbD mRNA stability was localized to the 5Ј UTR of the transcript (Nickelsen et al, 1994).…”

“…Most striking, however, is the role of the nucleus in controlling chloroplast gene expression at the post-transcriptional level. Both genetic (Barkan, 1993;Barkan et al, 1994;Choquet et al, 1988;Gumpel et al, 1995;Jenkins et al, 1997;Kuchka et al, 1989;Levy et al, 1997;Monod et al, 1992;Nickelsen et al, 1994;Rochaix et al, 1989;Stampacchia et al, 1997) and biochemical (Danon and Mayfield, 1991;Gamble and Mullet, 1989;Schuster and Gruissem, 1991) data have established roles for nucleus-encoded proteins in chloroplast mRNA maturation, stability, and translation. Some of these nucleus-encoded factors act on multiple messages, but the majority appear to represent gene-specific factors that are postulated to interact with specific mRNA sequences to carry out their functions.…”

Evidence for regulatory function of nucleus-encoded factors on mRNA stabilization and translation in the chloroplast

“…f to investigate their physiological function in C. reinhardtii. MCA1, required for petA transcript stabilization (13,14) has recently been cloned (C.L., S. Purton, N. J. Gumpel, J.G.-B., F.-A.W., and Y.C., unpublished work; AF330231), while we report here the molecular characterization of TCA1, required for cyt. f translation (15).…”

CytochromefTranslation in Chlamydomonas Chloroplast Is Autoregulated by its Carboxyl-Terminal Domain[W]

“…In the latter view, the CES effector would act as a gene-specific translational activator capable of competitive binding to the 5Ј petA mRNA and to the regulatory motif within the C-terminal domain of cytochrome f. We favor this model because the requirement for nucleus-encoded activators is a common feature of chloroplast gene expression (for recent reviews, see Barkan and Goldschmidt-Clermont, 2000;Zerges, 2000). Among the nuclear factors that control chloroplast gene expression, MCA1 (Gumpel et al, 1995) and TCA1 are required specifically for the expression of petA. MCA1 is involved in the post-transcriptional stabilization of petA mRNA, binds to its 5Ј UTR (S. Purton, Y. Choquet, and D.B.…”