David L. Herrin

Chloroplast RNA processing and stability

Primary chloroplast transcripts are processed in a number of ways, including intron splicing, internal cleavage of polycistronic RNAs, and endonucleolytic or exonucleolytic cleavages at the transcript termini. All chloroplast RNAs are also subject to degradation, although a curious feature of many chloroplast mRNAs is their relative longevity. Some of these processes, e.g., psbA splicing and stability of a number of chloroplast mRNAs, are regulated in response to light–dark cycles or nutrient availability. This review highlights recent advances in our understanding of these processes in the model organism Chlamydomonasreinhardtii, focusing on results since the extensive reviews published in 1998 [Herrin DL et al. 1998 (pp. 183–195), Nickelsen Y 1998 (pp. 151–163), Stern DB and Drager RG 1998 (pp. 164–182), in Rochaix JD et al. (eds) The Molecular Biology of Chloroplasts and Mitochondria in Chlamydomonas. Kluwer Academic Publishers, Dordrecht, The Netherlands]. We also allude to studies with other organisms, and to the potential impact of the Chlamydomonas genome project where appropriate.

Renilla luciferase as a vital reporter for chloroplast gene expression in Chlamydomonas

Abstract The use of luciferases as reporters of gene expression in living cells has been extended to the chloroplast genome. We show that the luciferase from the soft coral Renilla reniformis (Rluc) can be successfully expressed in the chloroplast of Chlamydomonas reinhardtii. Expression of the rluc cDNA was driven by the promoter and 5′ untranslated regions of the atpA gene. Western analysis with an anti-Rluc antibody detected a single polypeptide of 38 kDa in the luminescent cells. This is 3 kDa larger than native Rluc, and suggests that translation of the chimeric mRNA begins at the atpA start codon, 29 codons upstream from the rluc start site. We also show that the luminescence of the transformants was sufficient to enable imaging of colonies using a cooled CCD camera.

Processing of a composite large subunit rRNA. Studies with chlamydomonas mutants deficient in maturation of the 23s-like rrna.

Maturation of the chloroplast 23S-like rRNA involves the removal of internal transcribed spacers (ITSs) and, in the case of Chlamydomonas reinhardtii, the splicing of a group I intron (Cr.LSU). Little is known of the cis and trans requirements or of the processing pathway for this essential RNA. Previous work showed that the ribosome-deficient ac20 mutant overaccumulates an unspliced large subunit (LSU) RNA, suggesting that it might be a splicing mutant. To elucidate the molecular basis of the ac20 phenotype, a detailed analysis of the rrn transcripts in ac20 and wild-type cells was performed. The results indicate that processing of the ITSs, particularly ITS-1, is inefficient in ac20 and that ITS processing occurs after splicing. Deletion of the Cr.LSU intron from ac20 also did not alleviate the mutant phenotype. Thus, the primary defect in ac20 is not splicing but most likely is associated with ITS processing. A splicing deficiency was studied by transforming wild-type cells with rrnL genes containing point mutations in the intron core. Heteroplasmic transformants were obtained in most cases, except for P4 helix mutants; these strains grew slowly, were light sensitive, and had an RNA profile indicative of inefficient splicing. Transcript analysis in the P4 mutants also indicated that ITS processing can occur on an unspliced precursor, although with reduced efficiency. These latter results indicate that although there is not an absolutely required order for LSU processing, there does seem to be a preferred order that results in efficient processing in vivo.

Requirement for cytoplasmic protein synthesis during circadian peaks of transcription of chloroplast-encoded genes in Chlamydomonas.

Cycloheximide, an inhibitor of cytoplasmic translation, induced a rapid reduction of 70–80% in levels of mRNA for the chloroplast elongation factor Tu (tufA) in asynchronously growing Chlamydomonas. This effect was shown to be mainly transcriptional, and not restricted to tufA, as transcription of other chloroplast-encoded genes were cycloheximide-sensitive, although not all equally (psbA showed no more than 40% inhibition). Confirmatory evidence that the inhibition of chloroplast transcription was mainly due to blocking cytoplasmic translation was obtained with the cycloheximide-resistant mutant act1, and by using another translation inhibitor, anisomycin. In synchronously growing Chlamydomonas, chloroplast transcription is regulated by the circadian clock, with the daily peak occurring during the early light period. When cycloheximide was added during this period, transcription was inhibited, but not when it was added during the trough period (late light to early dark). Moreover, in synchronized cells switched to continuous light, the drug blocked the scheduled increase in tufA mRNA, but did not remove the pre-existing mRNA. These experiments define two functionally different types of chloroplast transcription in Chlamydomonas, basal (cycloheximide-insensitive) and clock-induced (cycloheximide-sensitive), and indicate that the relative contribution of each type to the overall transcription of a given gene are not identical for all genes. The results also provide evidence for nuclear regulation of chloroplast transcription, thereby obviating the need for an organellar clock, at least for these rhythms.

The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications

Abstract The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1–Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3′ end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11–38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas.

Novel aspects of the regulation of a cDNA (Arf1) from Chlamydomonas with high sequence identity to animal ADP-ribosylation factor 1

ADP-ribosylation factor (ARF) is a highly conserved, low molecular mass (ca. 21 kDa) GTP-binding protein that has been implicated in vesicle trafficking and signal transduction in yeast and mammalian cells. However, little is known of ARF in plant systems. A putative ARF polypeptide was identifed in subcellular fractions of the green alga Chlamydomonas reinhardtii, based on [32P]GTP binding and immunoblot assays. A cDNA clone was isolated from Chlamydomonas (Arf1), which encodes a 20.7 kDa protein with 90% identity to human ARF1. Northern blot analyses showed that levels of Arf1 mRNA are highly regulated during 12 h/12 h light/dark (LD) cycles. A biphasic pattern of expression was observed: a transient peak of Arf1 mRNA occurred at the onset of the light period, which was followed ca. 12 h later by a more prominent peak in the early to mid-dark period. When LD-synchronized cells were shifted to continuous darkness, the dark-specific peak of Arf1 mRNA persisted, indicative of a circadian rhythm. The increase in Arf1 mRNA at the beginning of the light period, however, was shown to be light-dependent, and, moreover, dependent on photosynthesis, since it was prevented by DCMU. We conclude that the biphasic pattern of Arf1 mRNA accumulation during LD cycles is due to regulation by two different factors, light (which requires photosynthesis) and the circadian clock. Thus, these studies identify a novel pattern of expression for a GTP-binding protein gene.

Assessing the relative importance of light and the circadian clock in controlling chloroplast translation in Chlamydomonas reinhardtii

Previous work has shown that transcription of a number of chloroplast-encoded genes, including those for photosynthesis, are under circadian clock control in Chlamydomonas reinhardtii. However, some of these genes encode long-lived mRNAs that are also subject to translational control. Rates of synthesis of the major chloroplast translation products vary dramatically (10–20-fold) during light–dark (LD) cycles, peaking in the light period. To determine whether this pattern reflects circadian clock control, LD-grown cells were shifted to continuous light (LL) and chloroplast protein synthesis monitored by periodic pulse-labeling in the presence of cycloheximide; chloroplast protein synthesis in LD was also examined for comparison. The LD patterns of synthesis of the major polypeptides (including D1, D2, and the large subunit of ribulose-1,5-bisphosphate carboxylase (LS)) were similar to those obtained previously in the absence of cycloheximide. In the LL condition, rates of synthesis of the major chloroplast translation products were high throughout the period examined (∼36 h), fluctuating > 3-fold, although they were generally higher in the subjective light period. LD-grown cells were also shifted to continuous dark (DD) and chloroplast protein synthesis analyzed for ∼24 h starting from the mid-dark period. There was a gradual decline in synthesis of the major proteins during the first subjective light period, which was followed by a very small peak in synthesis around the second subjective dark → light transition. RNA blot analysis showed that the mRNAs for D1, D2 and LS were present at high levels during the period of declining translation. These results indicate that with photoautotrophic growth in LD cycles, the illumination conditions per se are more important than the clock in determining chloroplast translation, but the clock may contribute to this regulation. The advantages of controlling translation by a direct light response and transcription primarily by the circadian clock are discussed. Finally, evidence of translational control of elongation factor Tu synthesis was obtained.

Co-isolation of high-quality DNA and RNA from coenocytic green algae

A protocol is presented for the simultaneous isolation of DNA and RNA from giant-celled green algae. The overall quality of the DNA was examined by the A260/A280 ratio, agarose gel electrophoresis, and restriction enzyme analysis. Denaturing gel electrophoresis and cDNA cloning were used to investigate the quality of the RNA. These assays indicated that both the DNA and RNA isolated by this procedure are of high quality, suitable for further molecular analyses. Since many of these algae are slow growing and therefore only a few grams may be available, the isolation of DNA and RNA from the same plant material has obvious advantages.Abbreviations: Etbr, ethidium bromide.

Self-splicing of the Chlamydomonas chloroplast psbA introns.

We used alpha-32P-GTP labeling of total RNA preparations to identify self-splicing group I introns in Chlamydomonas. Several RNAs become labeled with alpha-32P-GTP, a subset of which is not seen with RNA from a mutant that lacks both copies of the psbA gene. Hybridization of the GTP-labeled RNAs to chloroplast DNA indicates that they originate from the psbA and rrn 23S genes, respectively, the only genes known to contain group I introns in this organism. Introns 1, 2, and 3 of psbA (with flanking exon sequences) were subcloned and transcribed in vitro. The synthetic RNAs were found to self-splice; splicing required Mg2+, GTP, and elevated temperature. In addition, the accuracy of self-splicing was confirmed for introns 1 and 2, and intermediates in the splicing reactions were detected. These results, together with our recent data on the 23S intron, indicate that the ability to self-splice is a general feature of Chlamydomonas group I introns. These findings have significant implications for the mechanism of group I intron splicing and evolution in Chlamydomonas and other chloroplast genomes.

Translational regulation of chloroplast gene expression during the light-dark cell cycle of chlamydomonas: Evidence for control by ATP/energy supply

In Chlamydomonas reinhardtii growing synchronously under a light-dark cycle, the major chloroplast mRNAs are constitutively present but are translated only during the light period. We show that translation of these mRNAs can be induced during the normal dark period by light or by acetate and the induction is blocked by an inhibitor of ATP synthesis, CCCP. Moreover, ATP levels in synchronous cells were found to be 2-5-fold lower during the dark than in the light period; the administration of acetate or light at the mid-dark period increased the ATP level 2-3-fold. These results exclude cell-cycle mediated control and suggest that the regulation of chloroplast translation in light-dark grown Chlamydomonas is mediated, at least in part, by ATP levels.