Processing and degradation of chloroplast mRNA.

The conversion of genetic information stored in DNA into a protein product proceeds through the obligatory intermediate of messenger RNA. The steady-state level of an mRNA is determined by its relative synthesis and degradation rates, i.e., an interplay between transcriptional regulation and control of RNA stability. When the biological status of an organism requires that a gene product's abundance varies as a function of developmental stage, environmental factors or intracellular signals, increased or decreased RNA stability can be the determining factor. RNA stability and processing have long been known as important regulatory points in chloroplast gene expression. Here we summarize current knowledge and prospects relevant to these processes, emphasizing biochemical data. The extensive literature on nuclear mutations affecting chloroplast RNA metabolism is reviewed in another article in this volume (Barkan and Goldschmidt-Clermont, this issue).

Disruption of the petB-petD intergenic region in tobacco chloroplasts affects petD RNA accumulation and translation.

Translation initiation in chloroplasts is a complex process involving a variety of cis-elements and trans-acting factors. Many chloroplast mRNAs are processed products of polycistronic primary transcripts, but the functional requirement for processing is mostly enigmatic. In tobacco, the petB and petD genes, which encode subunits of the cytochrome b6/f complex, are transcribed from the psbB operon, whose primary transcript is processed into products including di- or tricistronic, but not monocistronic, petB and petD mRNAs. To begin to identify elements important for petB and/or petD translation, we generated tobacco chloroplast transformants by inserting selectable aadA marker cassette in the petB-petD intergenic region. The resulting plants required sucrose for growth, and their phenotypes depended on the orientation of the aadA cassette. When aadA was inserted in the same transcriptional orientation as the psbB operon, petB and petD mRNAs were abundantly produced but aberrant in size, and only 25% of the wild-type amount of the cytochrome b6/f complex accumulated. With the aadA cassette in the opposing orientation, however, very little petD mRNA accumulated, and the cytochrome b6/f complex was undetectable. Polysome analysis suggested that petD mRNAs in both transformants were poorly translated, indicating that the intergenic region contains essential translational elements.

Post-transcriptional defects in tobacco chloroplast mutants lacking the cytochrome b6/f complex.

A variety of post-transcriptional mechanisms govern the synthesis and assembly of photosynthetic protein complexes in chloroplasts. To test whether such mechanisms are conserved between photosynthetic algae and vascular plants, we have interrupted the chloroplast petA, petB and petD genes of tobacco, which encode three subunits of the cytochrome b6/f complex, and compared our results to those previously obtained with Chlamydomonas reinhardtii. As expected, the mutants exhibited high chlorophyll fluorescence, consistent with the loss of a functional cytochrome b6/f complex. Unlike the corresponding mutants of Chlamydomonas, however, cytochrome f was barely detectable in the DeltapetB or DeltapetD mutants. The amounts of petB- and petD-containing mRNAs were reduced in the mutants compared to wild-type plants, but the remaining mRNA was normally associated with polysomes. In contrast, there was a decrease in polysome association of the polycistronic petA mRNA in the DeltapetB and DeltapetD mutants, suggesting that the synthesis of cytochrome f may be decreased in the absence of cytochrome b6 or SUIV. These results are discussed in light of the translational autoregulation model that has been proposed for cytochrome b6/f complex assembly in Chlamydomonas.

The sequence and secondary structure of the 3'-UTR affect 3'-end maturation, RNA accumulation, and translation in tobacco chloroplasts.

RNA maturation and modulation of RNA stability play important roles in chloroplast gene expression. In vitro and in vivo studies have shown that both the 5'- and 3'-untranslated regions (UTRs) contain sequence and structural elements that guide these processes, and interact with specific proteins. We have previously characterized the spinach chloroplast petD 3'-UTR in detail by in vitro approaches. This stem-loop forming sequence is a weak terminator but is required for RNA maturation and also exhibits sequence-specific protein binding. To test petD 3'-UTR function in vivo, tobacco chloroplast transformants were generated containing uidA reporter genes flanked by variants of the petD 3'-UTR, including one which does not form an RNA-protein complex in vitro, and one which lacks a stem-loop structure. Analysis of uidA mRNA indicated that a stable secondary structure is required to accumulate a discrete mRNA, and that changes in the 3'-UTR sequence which affect protein binding in vitro can also affect RNA metabolism in vivo. The 3'-UTR also influenced beta-glucuronidase protein accumulation, but not in proportion to RNA levels. These results raise the possibility that in tobacco chloroplasts, the 3'-UTR may influence translational yield.

Superoxide dismutase in Arabidopsis: an eclectic enzyme family with disparate regulation and protein localization.

A number of environmental stresses can lead to enhanced production of superoxide within plant tissues, and plants are believed to rely on the enzyme superoxide dismutase (SOD) to detoxify this reactive oxygen species. We have identified seven cDNAs and genes for SOD in Arabidopsis. These consist of three CuZnSODs (CSD1, CSD2, and CSD3), three FeSODs (FSD1, FSD2, and FSD3), and one MnSOD (MSD1). The chromosomal location of these seven SOD genes has been established. To study this enzyme family, antibodies were generated against five proteins: CSD1, CSD2, CSD3, FSD1, and MSD1. Using these antisera and nondenaturing-polyacrylamide gel electrophoresis enzyme assays, we identified protein and activity for two CuZnSODs and for FeSOD and MnSOD in Arabidopsis rosette tissue. Additionally, subcellular fractionation studies revealed the presence of CSD2 and FeSOD protein within Arabidopsis chloroplasts. The seven SOD mRNAs and the four proteins identified were differentially regulated in response to various light regimes, ozone fumigation, and ultraviolet-B irradiation. To our knowledge, this is the first report of a large-scale analysis of the regulation of multiple SOD proteins in a plant species.

An AU-rich element in the 3' untranslated region of the spinach chloroplast petD gene participates in sequence-specific RNA-protein complex formation.

In chloroplasts, the 3' untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3'-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3' IR-containing RNA (3' IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b6/f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3' IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3' untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3'-end processing and/or influence the stability of petD mRNA in chloroplasts.

The 41 kDa protein component of the spinach chloroplast petD mRNA 3' stem-loop:protein complex is a nuclear encoded chloroplast RNA-binding protein.

Spinach chloroplast petD gene encodes subunit IV of the cytochrome b6/f complex. Like many chloroplast mRNAs, the spinach petD mRNA contains a 3'UTR stem-loop structure that determines correct 3' processing of the pre-mRNA and stability of the mature mRNA. RNA-protein interactions with this structure may be involved in the regulation of petD mRNA processing and/or stability. In this report, a spinach chloroplast 41 kD protein has been identified as a component of the petD mRNA 3'stem-loop:protein complex. The 41 kD protein has been purified and four internal peptide sequences were obtained. Based on the peptide sequences, a 585 bp fragment was amplified from a spinach cDNA library by PCR. And, a 1404 bp cDNA clone was then isolated by using the initial PCR fragment as probe. Nucleic acid sequence analysis of the cDNA clone identified an ORF encoding a predicted 36 kD mature protein. An apparent N-terminal transit peptide in the coding region and a 3' poly(A) tail exist in the cDNA clone indicated that this chloroplast protein as nuclear encoded. The mature protein was expressed in E. coli, and the expressed product with a molecular mass of 41 kD was purified. A gel mobility shift assay demonstrated that the expressed 41 kD protein interacts with the petD mRNA 3' UTR to form an RNA-protein complex.