RAP,the Sole Octotricopeptide Repeat Protein in Arabidopsis,Is Required for Chloroplast 16S rRNA Maturation

The biogenesis and activity of chloroplasts in both vascular plants and algae depends on an intracellular network of nucleus-encoded, trans-acting factors that control almost all aspects of organellar gene expression. Most of these regulatory factors belong to the helical repeat protein superfamily, which includes tetratricopeptide repeat, pentatricopeptide repeat, and the recently identified octotricopeptide repeat (OPR) proteins. Whereas green algae express many different OPR proteins, only a single orthologous OPR protein is encoded in the genomes of most land plants. Here, we report the characterization of the only OPR protein in Arabidopsis thaliana, RAP, which has previously been implicated in plant pathogen defense. Loss of RAP led to a severe defect in processing of chloroplast 16S rRNA resulting in impaired chloroplast translation and photosynthesis. In vitro RNA binding and RNase protection assays revealed that RAP has an intrinsic and specific RNA binding capacity, and the RAP binding site was mapped to the 5′ region of the 16S rRNA precursor. Nucleoid localization of RAP was shown by transient green fluorescent protein import assays, implicating the nucleoid as the site of chloroplast rRNA processing. Taken together, our data indicate that the single OPR protein in Arabidopsis is important for a basic process of chloroplast biogenesis.     

The Octatricopeptide Repeat Protein Raa8 Is Required for Chloroplast trans Splicing

The mRNA maturation of the tripartite chloroplast psaA gene from the green alga Chlamydomonas reinhardtii depends on various nucleus-encoded factors that participate in trans splicing of two group II introns. Recently, a multiprotein complex was identified that is involved in processing the psaA precursor mRNA. Using coupled tandem affinity purification (TAP) and mass spectrometry analyses with the trans-splicing factor Raa4 as a bait protein, we recently identified a multisubunit ribonucleoprotein (RNP) complex comprising the previously characterized trans-splicing factors Raa1, Raa3, Raa4, and Rat2 plus novel components. Raa1 and Rat2 share a structural motif, an octatricopeptide repeat (OPR), that presumably functions as an RNA interaction module. Two of the novel RNP complex components also exhibit a predicted OPR motif and were therefore considered potential trans-splicing factors. In this study, we selected bacterial artificial chromosome (BAC) clones encoding these OPR proteins and conducted functional complementation assays using previously generated trans-splicing mutants. Our assay revealed that the trans-splicing defect of mutant F19 was restored by a new factor we named RAA8; molecular characterization of complemented strains verified that Raa8 participates in splicing of the first psaA group II intron. Three of six OPR motifs are located in the C-terminal end of Raa8, which was shown to be essential for restoring psaA mRNA trans splicing. Our results support the important role played by OPR proteins in chloroplast RNA metabolism and also demonstrate that combining TAP and mass spectrometry with functional complementation studies represents a vigorous tool for identifying trans-splicing factors.     

Nuclear Mutation Increases Streptomycin and Spectinomycin Sensitivity in Chlamydomonas

A spontaneously arising nuclear mutation, ss-1, has been identified in Chlamydomonas reinhardtii that decreases both streptomycin and spectinomycin resistance levels about 10-fold after its introduction into all wild-type, streptomycin-resistant and spectinomycin-resistant strains examined. The mutations for resistance map to nuclear and uniparentally inherited (chloroplast) loci. In contrast, no modification of erythromycin resistance was detected after introducing ss-1 into wild-type strains or into strains carrying nuclear or uniparentally inherited erythromycin-resistance mutations. We suggest that ss-1 affects the small subunit of the chloroplast ribosome because others have shown that streptomycin and spectinomycin resistance in C. reinhardtii are associated with this subunit, whereas erythromycin resistance is associated with the large subunit. ss-1 shows no linkage with the nuclear locus for streptomycin resistance.     

Extensive methylation of chloroplast DNA by a nuclear gene mutation does not affect chloroplast gene transmission in chlamydomonas

Based on analysis by high pressure liquid chromatography, greater than 35% of the cytosine residues in chloroplast DNA of vegetative cells were found to be methylated constitutively in the nuclear gene mutation (me-1) of Chlamydomonas reinhardtii, which has an otherwise wild-type phenotype. Digestion of chloroplast DNA from vegetative cells and gametes of this mutant with restriction endonucleases Hpa II and Msp I reveals that in the 5′CCGG3′ sequence, CpG is methylated extensively, whereas CpC is only methylated occasionally. Hae III (5′GGCC3′) digestion of the mutant chloroplast DNA also shows extensive methylation of the GpC sequence. In contrast to the results of Sager and colleagues, which show a correlation between methylation of chloroplast DNA and transmission of chloroplast genes in crosses, our results with crosses of the me-1 mutant suggest that extensive chloroplast DNA methylation may be insufficient to account for the pattern of inheritance of chloroplast genes in Chlamydomonas.     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

Atrazine, bromacil, and diuron resistance in chlamydomonas: a single non-mendelian genetic locus controls the structure of the thylakoid binding site

A series of Chlamydomonas reinhardii mutants were selected for resistance to the herbicides atrazine, bromacil, and diuron. Four of these have reduced herbicide binding to the thylakoid membranes and show the non-Mendelian inheritance pattern characteristic of chloroplast genes. These mutants show a variety of selective alterations in binding of the three herbicides. These changes account for the observed patterns of in vivo cross-resistance. Analyses of chloroplast gene recombination indicate that these four mutations are in the same gene. Overall, the results suggest that this gene codes for a protein component of the herbicide binding site. One of the mutants has slow phototrophic growth and altered electron transport as has been observed in atrazine-resistant higher plant varieties, but the others are normal in these respects. The slow growth characteristic of this mutant seems to be the consequence of the same mutation which confers herbicide resistance.

The mutants isolated also include a large number which achieve resistance by some secondary mechanism. These are all nuclear gene mutations, and represent numerous loci. They also show a variety of patterns of cross-resistance, but the mechanisms behind them have not yet been investigated.

     

The chloroplast gene encoding ribosomal protein S4 in Chlamydomonas reinhardtii spans an inverted repeat — unique sequence junction and can be mutated to suppress a streptomycin dependence mutation in ribosomal protein S12

The ribosomal protein gene rps4 was cloned and sequenced from the chloroplast genome of Chlamydomonas reinhardtii. The N-terminal 213 amino acid residues of the S4 protein are encoded in the single-copy region (SCR) of the genome, while the C-terminal 44 amino acid residues are encoded in the inverted repeat (IR). The deduced 257 amino acid sequence of C. reinhardtii S4 is considerably longer (by 51–59 residues) than S4 proteins of other photosynthetic species and Escherichia coli, due to the presence of two internal insertions and a C-terminal extension. A short conserved C-terminal motif found in all other S4 proteins examined is missing from the C. reinhardtii protein. In E. coli, mutations in the S4 protein suppress the streptomycin-dependent (sd) phenotype of mutations in the S12 protein. Because we have been unable to identify similar S4 mutations among suppressors of an sd mutation in C. reinhardtii S12 obtained using UV mutagenesis, we made site-directed mutations [Arg₆₈ (CGT) to Len (CTG and CTT)] in the wild-type rps4 gene equivalent to an E. coli Gln₅₃ to Len ribosomal ambiguity mutation (ram), which suppresses the sd phenotype and decreases translational accuracy. These mutants were tested for their ability to transform the sd S 12 mutation of C. reinhardtii to streptomycin independence. The streptomycin-independent isolates obtained by biolistic transformation all possessed the original sd mutation in rps12, but none had the expected donor Leu₆₈ mutations in rps4. Instead, six of 15 contained a Gln₇₃ (CAA) to Pro (CCA) mutation five amino acids downstream from the predicted mutant codon, irrespective of rps4 donor DNA. Two others contained six- and ten-amino acid, in-frame insertions at S4 positions 90 and 92 that appear to have been induced by the biolistic process itself. Eight streptomycin-independent isolates analyzed had wild-type rps4 genes and may possess mutations identical to previously isolated suppressors of sd that define at least two additional chloroplast loci. Cloned rps4 genes from streptomycin-independent isolates containing the Gln₇₃ to Pro mutation and the 6-amino acid insertion in r-protein S4 transform the sd strain to streptomycin independence.     

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas.     

Chloroplast suppressors that act on a mitochondrial mutation in Chlamydomonas reinhardtii

We report genetic evidence supporting the existence of suppressor genes in the chloroplast that act on a mitochondrial mutation that impairs heterotrophic growth in the green alga Chlamydomonas reinhardtii. One of these suppressors also acts on a point mutation in the rbcL gene in the chloroplast. These results are consistent with previous data showing that mitochondrial protein synthesis depends on chloroplast protein synthesis in C. reinhardtii. The nature of the interaction between chloroplasts and mitochondria is discussed in light of the requirement for import of tRNAs by plant mitochondria.     

Nuclear factors: Roles related to mitochondrial deafness

Hearing loss (HL) is a common disorder with mitochondrial dysfunction as one of the major causes leading to deafness. Mitochondrial dysfunction may be caused by either mutations in nuclear genes leading to defective nuclear-encoded proteins or mutations in mitochondrial genes leading to defective mitochondrial-encoded products. The specific nuclear genes involved in HL can be classified into two categories depending on whether mitochondrial gene mutations co-exist (modifier genes) or not (deafness-causing genes). TFB1M, MTO1, GTPBP3, and TRMU are modifier genes. A mutation in any of these modifier genes may lead to a deafness phenotype when accompanied by the mitochondrial gene mutation. OPA1, TIMM8A, SMAC/DIABLO, MPV17, PDSS1, BCS1L, SUCLA2, C10ORF2, COX10, PLOG1and RRM2B are deafness-causing genes. A mutation in any of these deafness-causing genes will directly induce variable phenotypic HL.     

Nuclear and chloroplast transformation inChlamydomonas reinhardtii: strategies for genetic manipulation and gene expression

Transformation of the nuclear, chloroplast, and mitochondrial genomes can now be accomplished inChlamydomonas reinhardtii. Many biosynthetic pathways are carried out in the chloroplast, and efforts to manipulate these pathways will require that gene products be directed to this compartment. Chloroplast proteins are encoded in either the chloroplast or nuclear genome. In the latter case they are synthesized in the cytoplasm and imported post-translationally into the chloroplast. Thus, strategies for expressing foreign genes or overexpressing endogenous genes whose products reside in the chloroplast could involve either genome. This paper reviews the present status of transformation methodology for the nuclear and chloroplast genomes inChlamydomonas. Considerations for expressing gene products in the chloroplast are discussed. Experimental evidence for homologous recombination during transformation of the nuclear genome is presented.