Arabidopsis orthologs of maize chloroplast splicing factors promote splicing of orthologous and species-specific group II introns

Abstract

Chloroplast genomes in plants and green algae contain numerous group II introns, large ribozymes that splice via the same chemical steps as spliceosome-mediated splicing in the nucleus. Most chloroplast group II introns are degenerate, requiring interaction with nucleus-encoded proteins to splice in vivo. Genetic approaches in maize (Zea mays) and Chlamydomonas reinhardtii have elucidated distinct sets of proteins that assemble with chloroplast group II introns and facilitate splicing. Little information is available, however, concerning these processes in Arabidopsis (Arabidopsis thaliana). To determine whether the paucity of data concerning chloroplast splicing factors in Arabidopsis reflects a fundamental difference between protein-facilitated group II splicing in monocot and dicot plants, we examined the mutant phenotypes associated with T-DNA insertions in Arabidopsis genes encoding orthologs of the maize chloroplast splicing factors CRS1, CAF1, and CAF2 (AtCRS1, AtCAF1, and AtCAF2). We show that the splicing functions and intron specificities of these proteins are largely conserved between maize and Arabidopsis, indicating that these proteins were recruited to promote the splicing of plastid group II introns prior to the divergence of monocot and dicot plants. We show further that AtCAF1 promotes the splicing of two group II introns, rpoC1 and clpP-intron 1, that are found in Arabidopsis but not in maize; AtCAF1 is the first splicing factor described for these introns. Finally, we show that a strong AtCAF2 allele conditions an embryo-lethal phenotype, adding to the body of data suggesting that cell viability is more sensitive to the loss of plastid translation in Arabidopsis than in maize.

Figure 1.

Plant phenotypes associated with T-DNA insertions in AtCRS1 (At5g16180), AtCAF1 (At2g20020), and AtCAF2 (At1g23400). A, Schematic maps of T-DNA insertion sites. Black rectangles are exons and lines are introns. The T-DNA insertion in Atcaf1-2 is within intron 1, 46 nt downstream of that in Atcaf1-1. The sequence flanking each insertion is shown in Supplemental Figure S1. LB, T-DNA left border. B, Seedling phenotypes conditioned by mutant alleles and allele combinations. Where one allele is listed, seedlings are homozygous for the indicated allele. Where two alleles are listed, the plants are the noncomplementing progeny from a cross between plants heterozygous for each allele. Genotypes were confirmed by PCR. Bar=5 mm. C, Segregation of aborted seeds in siliques from a plant heterozygous for Atcaf2-1. The aborted seeds are presumed to be homozygous for the Atcaf2-1 insertion because no homozygous mutants were recovered among the 20 germinating progeny that were genotyped by PCR. The two siliques represent different stages of seed maturation.

Figure 2.

Accumulation of representative subunits of photosynthetic enzyme complexes in AtCRS1, AtCAF1, and AtCAF2 mutants. Duplicate immunoblots of total leaf extract (5 μg protein or the indicated dilution of the wild-type sample) were probed with antibodies to the indicated proteins. The mutant plants were homozygous for the indicated allele. The bottom section shows the filter that had been probed for PsbA stained with Ponceau S, to demonstrate similar protein loading, and to detect the large subunit of Rubisco (RbcL). OE23 and OE33 are luminal proteins found in the oxygen-evolving complex associated with PSII. Bands corresponding to the putative precursor (p) and stromal intermediate (i) forms of OE23 are marked.

Figure 3.

Ratio of spliced to unspliced transcripts from Arabidopsis plastid genes with orthologs in maize. A, Diagram of the PPE assay. The petD intron is used as an example of the strategy. Shaded nts represent exon sequences, and the primer is underlined. Dideoxy CTP is included in the primer extension reaction, such that primer extension terminates at the first guanidine residue encountered in the template RNA (see circled residues). The lengths of the extension products differ for spliced (S) and unspliced (U) transcripts. B, Results of PPE assays for introns whose maize orthologs are influenced by mutations in maize crs1, caf1, or caf2. Mutants were homozygous for the indicated allele. The predicted sizes of the PPE products for spliced (S) and unspliced (U) transcripts are stated in nts, next to each product. Bands corresponding to the radiolabeled primers (P) are marked. Ten micrograms of seedling leaf RNA was analyzed, except in the first lane in each section, which show reactions lacking leaf RNA. The size of the product marked with an asterisk in the ycf3-1 section is consistent with that predicted for chain termination at the branchpoint adenosine. The sizes of the unmarked bands in the petB and rpl16 assays do not correlate with those predicted for termination at the branchpoint adenosines; the origin of these products is unknown.

Figure 4.

PPE assay of clpP intron splicing. The Arabidopsis plastid clpP gene contains two group II introns, clpP-1 and clpP-2. PPE assays were performed as described in Figure 3. U, Unspliced; S, spliced; P, primer. The Atcaf1-2 allele gave results similar to those seen with Atcaf1-1 (Supplemental Fig. S4A).

Figure 5.

Analysis of rpoC1 splicing in AtCRS1, AtCAF1, and AtCAF2 mutants. A, Map of the Arabidopsis plastid rpo locus and the probes used for RNA gel-blot hybridizations. B, RNA gel-blot hybridizations showing rpoC1 transcripts in each mutant line. Three micrograms of seedling leaf RNA was analyzed in each lane. Duplicate blots were probed with intron-specific (probe 1) and exon-specific (probe 2) probes. The asterisk marks a band that may correspond to the excised intron. The positions of RNA size markers are shown to the left. The blot used with probe 1 was stained with methylene blue, and is shown below to illustrate the rRNAs in each sample. C, RNAse protection assay of rpoC1 splicing. The radiolabeled probe spanned the 3′-splice junction, with the indicated number of nts corresponding to intron (111 nt) and exon (321 nt) sequences. Protection of the probe by unspliced or spliced RNAs is predicted to yield 432 nt and 321 nt products, respectively. Three micrograms of total leaf RNA was analyzed. The lane labeled tRNA shows the results when yeast (Saccharomyces cerevisiae) tRNA was substituted for leaf RNA. The positions of DNA size markers are shown to the left. The Atcaf1-2 allele gave results similar to those seen with Atcaf1-1 (Supplemental Fig. S4B).