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First published online October 27, 2006; 10.1104/pp.106.088096 Plant Physiology 142:1656-1663 (2006) © 2006 American Society of Plant Biologists Arabidopsis Orthologs of Maize Chloroplast Splicing Factors Promote Splicing of Orthologous and Species-Specific Group II Introns1,[W]Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403
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.
Group II introns are large ribozymes found in bacteria, chloroplasts, and mitochondria that are defined by conserved structural features and patches of sequence similarity (for review, see Michel and Ferat, 1995  ; Pyle, 1996). Mitochondrial and chloroplast genomes in land plants are particularly intron rich, each encoding approximately 20 group II introns (for review, see Barkan, 2004). The chloroplast genomes of vascular and nonvascular land plants have a similar intron complement, with the small differences attributable to lineage-specific intron loss. In contrast, the chloroplasts of green algae have a distinct intron set, and phylogenetic data imply independent intron acquisition in this lineage (for review, see Barkan, 2004).
Although several group II introns have been shown to splice autocatalytically in vivo, self splicing has not been reported for any of the approximately 40 group II introns in land plant organelles. These introns are believed to be degenerate, having lost elements that are necessary to assemble an RNA-based catalytic center. Intron degeneration has been compensated by the recruitment of protein factors that are obligate participants in the splicing reactions (for review, see Lambowitz and Zimmerly, 2004
Despite the intensive use of Arabidopsis (Arabidopsis thaliana) to study chloroplast processes, there is just one report of an Arabidopsis gene that influences the accumulation of a spliced chloroplast RNA: HCF152, a member of the pentatricopeptide repeat family, is required for the accumulation of spliced petB RNA but not for the accumulation of excised petB intron (Meierhoff et al., 2003
T-DNA Insertions in the Arabidopsis Orthologs of crs1, caf1, and caf2 Cause Embryo-Lethal and/or Albino Seedling Phenotypes
We previously predicted the Arabidopsis orthologs of maize crs1, caf1, and caf2 via phylogenetic analysis (Barkan et al., 2007
The plant phenotypes observed in each line are shown in Figure 1 and summarized in Table I. All of the lines except for Atcaf2-1 segregated chlorophyll-deficient seedlings (Fig. 1B), whereas the Atcaf2-1 line segregated defective seeds (Fig. 1C). In those lines that segregated chlorophyll-deficient mutants, genetic linkage was established between the insertion and the chlorophyll-deficient phenotype (Supplemental Fig. S2). Complementation crosses were performed between the two Atcrs1 alleles and between the two Atcaf2 alleles; these crosses yielded chlorophyll-deficient seedlings (Fig. 1B) harboring both mutant alleles (Supplemental Fig. S3). Taken together, these results show that the chlorophyll-deficient phenotypes are caused by the insertions in the targeted genes. Although both Atcrs1 alleles condition chlorophyll-deficient seedlings, their phenotypes are distinct: an insertion in exon 1 (Atcrs1-1) yielded albino seedlings, whereas an insertion in exon 6 (Atcrs1-2) yielded variegated seedlings with green and white patches (Fig. 1B). It seems likely that the Atcrs1-1 phenotype (albino) represents the more severe loss of function as the insertion is in an exon near the beginning of the open reading frame (ORF), whereas the Atcrs1-2 insertion disrupts sequences near the carboxyl terminus of the protein and conditions a milder chlorophyll deficiency. The two insertions in AtCAF2 also condition distinct phenotypes. The siliques resulting from self-pollinating Atcaf2-1/+ plants contained aborted, chlorotic seeds at a frequency of approximately 1:3 aborted to normal seed (Fig. 1C). Furthermore, no homozygous mutant plants were recovered among 20 germinating progeny (data not shown). Together, these results provide strong evidence that Atcaf2-1 conditions an embryo-lethal phenotype. In contrast, Atcaf2-2 conditions slow-growing seedlings with albino cotyledons and pale green leaves (Fig. 1B). The noncomplementing progeny of plants heterozygous for the two alleles (Atcaf2-1/Atcaf2-2) germinated and were similar in appearance to Atcaf2-2 homozygotes. It seems likely that the chlorotic seedling phenotype conditioned by the intronic insertion in Atcaf2-2 represents an incomplete loss of function, whereas the embryo-lethal phenotype conditioned by the Atcaf2-1 insertion, which is in exonic sequences in the middle of the ORF (Fig. 1A), represents the null phenotype.
Immunoblots were performed to assess the accumulation of the major photosynthetic enzyme complexes in the leaves of those mutants that germinate (Fig. 2
). Representative subunits of each complex were assayed whose abundance reflects that of other closely associated subunits of the same complex: PsaD and PsaF for PSI, PetA for the cytochrome b6f complex, PsbA for PSII, AtpA for the ATP synthase, and RbcL for Rubisco. Whereas the nucleus-encoded chaperone protein Cpn60 accumulated to increased levels in all of the mutants, the levels of all of the assayed core subunits of PSI, PSII, the cytochrome b6f complex, the ATP synthase, and Rubisco were reduced. These protein deficiencies are consistent with underlying global defects in chloroplast gene expression, as each of the affected complexes includes at least one plastid-encoded subunit. The AtCRS1 mutants differ from the AtCAF1 and AtCAF2 mutants in that AtpA is reduced considerably more than the other proteins assayed. This is analogous to what is observed in maize crs1 mutants, which lack the ATP synthase complex due to the failure to splice the atpF RNA (Jenkins et al., 1997
Chloroplast Splicing Defects in AtCRS1, AtCAF1, and AtCAF2 Mutants Mirror Those in Maize To determine whether AtCRS1, AtCAF1, and AtCAF2 function similarly to their maize orthologs in chloroplast group II intron splicing, the metabolism of intron-containing plastid transcripts was analyzed for those mutant alleles that germinated on Suc-containing medium. A poisoned-primer extension (PPE) assay was used for this purpose: an oligonucleotide complementary to exon sequences near a 3' splice junction was used to prime reverse transcription in the presence of a dideoxynucleotide that terminates after different distances on spliced and unspliced RNA templates (see diagram in Fig. 3A ). With this assay, all transcripts for which the intron is spliced are detected as one band, and all those in which the intron is retained are detected as a second band.
Eleven of the 17 group II introns found in both maize and Arabidopsis plastids were examined, including orthologs of all of the introns that require maize CRS1, CAF1, or CAF2. The results show that the splicing of orthologous introns is generally promoted by orthologous splicing factors (Fig. 3B; summarized in Table II ). For example, the ycf3-2 intron was unaffected by mutations in any of these Arabidopsis genes, as in maize. AtCRS1, like maize CRS1, is required for the splicing of the atpF intron but not for the other introns examined. AtCAF2, like maize CAF2, is required for the splicing of the petB, rps12-1, ndhA, ndhB, and ycf3-1 introns, but not for the splicing of the other introns examined. Like its maize counterpart, reduction of AtCAF1 function caused a strong defect in the splicing of the petD and ycf3-1 introns and a distinct defect in rpl16 splicing, but had no impact on the petB, rps12-1, atpF, or ycf3-2 introns. Residual splicing of affected introns in Atcaf1-1 and Atcaf2-2 mutants could be due to the incomplete disruption of gene function by the intron insertions in these alleles.
The role of AtCAF1 in the splicing of the rps16, trnG-UCC, ndhB, and ndhA introns is less clear cut from these data, in that these all show mild splicing defects in Atcaf1-1 mutants. The results for ndhA and ndhB are similar to those in maize, where these introns are strongly dependent on CAF2 and weakly dependent on CAF1 (Ostheimer et al., 2003 ; G. Ostheimer and A. Barkan, unpublished data). The mild ndhA splicing defect in maize caf1 mutants may be an indirect effect, as this intron did not strongly coimmunoprecipitate with CAF1 from chloroplast extract (Schmitz-Linneweber et al., 2005). A more notable difference from the results in maize concerns the rps16 and trnG-UCC introns: in maize, the splicing of these introns is strongly CAF1 dependent and these introns strongly coimmunoprecipitate with CAF1 from stromal extract (Ostheimer et al., 2003; Schmitz-Linneweber et al., 2005). The small effect of the Atcaf1-1 mutation on these introns may reflect a true difference in the intron specificities of maize and Arabidopsis CAF1, redundancy between AtCAF1 and AtCAF2 function with regard to these introns, or simply an incomplete disruption of AtCAF1 gene function by the intron-localized insertion. PPE assays of the ycf3-1 intron in Atcaf1-1 mutants yielded increased amounts of an extension product that is approximately 1 nucleotide (nt) shorter than the product from spliced RNA (marked with an asterisk in Fig. 3). Chain termination at this position corresponds to the predicted branchpoint adenosine, whose 2'OH group forms a branched structure after the first of the two splicing steps; this structure could impede the progress of reverse transcriptase. Thus, AtCAF1 may be necessary specifically for the second of the two trans-esterification reactions involved in group II intron splicing.
The Arabidopsis plastid genome encodes three group II introns that are absent in maize: two in the clpP gene and one in the rpoC1 gene. To determine whether the splicing of these introns is dependent on AtCRS1, AtCAF1, or AtCAF2, their splicing was monitored in the corresponding mutant backgrounds. PPE assays showed that the splicing of the clpP-1 intron requires AtCAF1 but not AtCRS1 or AtCAF2, whereas clpP-2 intron splicing was not disrupted in any of these mutant backgrounds (Fig. 4 ; Supplemental Fig. S4A). Splicing of rpoC1 was monitored by RNA gel-blot hybridizations and a ribonuclease protection assay (Fig. 5 ). The RNA gel blots (Fig. 5B) were hybridized with intron-specific (probe 1) and exon-specific (probe 2) probes; the results showed a dramatic increase in the abundance of two unspliced precursors and the near absence of the spliced rpoC1 mRNA in Atcaf1-1 mutants. A low Mr transcript that hybridized specifically to the intron probe (marked with an asterisk in Fig. 5B) may represent excised intron; the ratio of this transcript to the unspliced precursors is also reduced in Atcaf1-1 mutants. The ribonuclease protection assay (Fig. 5C; Supplemental Fig. S4B) likewise revealed a substantial increase in the ratio of unspliced-to-spliced rpoC1 RNAs in Atcaf1 mutants, but not in the other mutants. These results show that AtCAF1, but not AtCRS1 or AtCAF2, is required for the splicing of both the clpP-1 and rpoC1 introns. AtCAF1 is the first protein to be identified that functions in the splicing of these introns.
Proteins that facilitate the splicing of group II introns fall into two general classes: conserved maturases that are usually encoded within the intron whose splicing they facilitate, and proteins of diverse origin that were coopted from the host genome (for review, see Lambowitz and Zimmerly, 2004 ). Little is known about the relative timing of intron acquisition and the recruitment of host-encoded splicing factors, or the coevolution of these proteins with their cognate introns once the relationship had been established. Phylogenetic analyses indicate that most of the group II introns in land plants were acquired prior to the divergence of the charophyte algae, whereas group II introns were acquired independently in more distantly related algal lineages such as the chlorophytes (which include Chlamydomonas; Turmel et al., 2006; for review, see Barkan, 2004). Given the distinct history of intron acquisition in maize and Chlamydomonas plastids, it is perhaps not surprising that there is no similarity among the plastid group II intron splicing factors identified to date in these organisms. Results presented here show that the splicing functions and intron specificities of three group II intron splicing factors in maize are generally conserved with their Arabidopsis orthologs, indicating that these proteins were recruited to function in group II intron splicing prior to the divergence of monocot and dicot plants. The Arabidopsis CAF1 ortholog has an additional role in the splicing of two introns that are found in Arabidopsis but not in maize (rpoC1 and clpP-1), and, to our knowledge, is the first splicing factor to be discovered for these introns. A summary of the intron specificities of CAF1, CAF2, and CRS1 in maize and Arabidopsis is presented in Table II.
We previously identified two high-affinity binding sites for CRS1 within its atpF intron substrate, in intron domains 1 and 4 (Ostersetzer et al., 2005
Relatively little information is available concerning plastid splicing factors in Arabidopsis, with just one candidate described previously: the pentatricopeptide repeat protein HCF152 is required for the accumulation of spliced petB RNA but not for the accumulation of the excised intron (Meierhoff et al., 2003
Maize CAF1 and CAF2 facilitate splicing in complexes with a protein called CRS2, which is related to peptidyl-tRNA hydrolase enzymes and that is necessary for the splicing of CAF-dependent group II introns (Ostheimer et al., 2003
Ortholog Prediction
Arabidopsis (Arabidopsis thaliana) orthologs of CRS1, CAF1, and CAF2 were identified by generating multiple sequence alignments of all members of the CRM domain family in rice (Oryza sativa; 14 proteins) and Arabidopsis (16 proteins), together with maize (Zea mays) CRS1, CAF1, and CAF2. The alignments were used to generate neighbor-joining and parsimony trees; the two methods yielded trees with identical topology and high bootstrap values (Barkan et al., 2007
Seeds from the following T-DNA insertion lines were obtained from the Salk Institute Genomic Analysis Laboratory: SALK_026861 (Atcrs1-1), SALK_027605 (Atcrs1-2), SALK_025042 (Atcaf1-1), SALK_021969 (Atcaf1-2), SALK_049304 (Atcaf2-1), and SALK_08478 (Atcaf2-2). Phenotypically normal seedlings were used as the source of wild-type tissue for phenotypic analyses. Seeds were surface sterilized with 5% (v/v) bleach and 0.1% Tween 20 for 5 min, washed three times in sterilized water, and then sowed on Murashige and Skoog plates (0.3% [w/v] Phytagel [Sigma], 1x Murashige and Skoog salts [Sigma], 1x Gamborg's B5 vitamin [Sigma], and 2% [w/v] Suc, pH 5.8). Plants were grown in a growth chamber at 22°C, 14-h-light/10-h-dark cycles with an irradiance of approximately 200 µE m–2s–1. Plants used for protein and RNA analysis were between 2 and 3 weeks old. Homozygous mutants were seedling or embryo lethal, so mutations were propagated by transferring heterozygous plants to vermiculite 3 weeks after germination, and feeding with a nutrient solution (Liquid Fruit and Bloom, 2-5-1, Down to Earth) until seeds were produced.
Seedlings were frozen in liquid nitrogen and ground to a powder with a toothpick in a 1.5 mL microfuge tube. TPS buffer (100 mM Tris-HCl, pH 9.5, 1 M KCl, and 10 mM EDTA; Thomson and Henry, 1995 T-DNA insertions were confirmed by PCR amplification of either the mutant or wild-type alleles, using the T-DNA left border primer LBa1 (5'-TGGTTCACGTAGTGGGCCATCG-3') and gene-specific primers as follows. Atcrs1-1: At-crs1-1, 5'-GTCCAAAGCTGGTCCACATC-3'; At-crs1-2, 5'-GCAAGTCAGCATCAACGGGA-3'; or atCRS15 (1405), 5'-CTCTCCCATGGCATTTTCCC-3'. Atcrs1-2: At-crs1-3, 5'-AAACCATCGGAGGGAGATGA-3' and At-crs1-4, 5'-TTGGACCCAATTTTGCGTAGC-3'. Atcaf1-1 and Atcaf1-2: AtCAF1-1, 5'-CCTCTGCTCGTTCGATTCCGGGTC-3' and AtCAF1-2, 5'-TAACTGCTCGCAAACATTATCCAT-3'. Atcaf2-1: Atcaf2-1, 5'-GATTCCGGCGTCTCGTATCA-3' and Atcaf2-2, 5'-GGTTCAAGCCCTGGGCAGTC-3'. Atcaf2-2: 1g234-3', 5'-TTGCAATCACTTAAGGTCAATCTCAAATG-3' and 1g234-5', 5'-GTGTTCTGCAGCCAGTGAAGTGCCTT-3'.
Protein was extracted from the leaves of 2- to 3-week-old seedlings grown on agar medium, as described previously for maize (Barkan, 1998
RNA was extracted from the leaf tissue of 2- to 3-week-old seedlings grown on agar medium, by using TriZol reagent (GIBCO/BRL) according to the manufacturer's instructions. Phenotypically normal siblings from the same plates were used as the source of wild-type RNA. RNA gel-blot hybridizations and RNAse-protection assays were performed as described previously for maize (Jenkins et al., 1997
Poisoned primer extension reactions were performed with 10 µg of leaf RNA. Antisense primers were designed to obtain extension productions of different sizes from unspliced and spliced RNA templates when performed in the presence of a specific dideoxynucleotide. The primers and dideoxynucleotide used for each intron were as follows: ycf3-1, primer YAAtycf3-R3 5'-AATTTCCTTCAGATTGAGCCGACA-3' ddGTP; ndhA, primer YAAtNdhAR2 5'-ATCAACTGTACTTAAACTGTTAG-3', ddCTP; ndhB, primer YAAtNdhBR2, 5'-AAGCAACGACTGGAGTGGGAGA-3', ddATP; ycf3-2, primer YAAtycf3-1R2, 5'-CTTGTTGAATGGCCTGTTCTCCAC-3', ddCTP; petB, primer YAAtpetB-R2, 5'-ACGTTCTTCGAACCAATCATAAAC-3', ddCTP; petD, primer YAAtpetD-R, 5'-CAGGATCATTCAAATCTGGTTTT-3', ddCTP; atpF, primer YAAtatpF-2, 5'-CTTTCGGTTATCTAATAAATCA-3', ddCTP; clpP-1, primer YAAtclpP-1-R, 5'-ATCTTTCTCGATAAAGTCGGTTG-3', ddCTP; clpP-2, primer YAAtclpP-2-R, 5'-GCGGGTTGATGGATCATTACCC-3', ddCTP; trnG(UCC), primer YAAttrnG[UCC]-R 5'-ATCGTTAGCTTGGAAGGCTAAGGG-3', ddGTP; rpl16, primer YAAtrpl16-R 5'-TGTTGTTTACGAAATCTGGTTCT-3', ddCTP; rps16, primer YAAtrps16-R 5'-TTGCAAGGATTCGATAAACGGCTC-3', ddATP; rps12-1, primer YAAtrps12-1 5'-AAGCAGAGTTTGGTTTTTTGGGGG-3', ddCTP. Primers were 5'-end labeled with [
The following materials are available in the online version of this article.
We thank the Salk Institute Genomic Analysis Laboratory for providing the sequence-indexed Arabidopsis T-DNA insertion mutants, and Harry Roy for providing antibody to Cpn60. We also are grateful to Shingo Kikuchi, Athea Vichas, Jana Prikryl, Mark Carrier, Kenny Watkins, Nigel Walker, and Rosalind Williams-Carrier for technical advice, and to Susan Belcher and Oren Ostersetzer for contributing to the early stages of the AtCRS1 and AtCAF2 work. Received August 28, 2006; accepted September 27, 2006; published October 27, 2006.
1 This work was supported by the National Science Foundation (grant no. DBI–0421799).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Alice Barkan (abarkan{at}molbio.uoregon.edu).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.088096 * Corresponding author; e-mail abarkan{at}molbio.uoregon.edu; fax 541–346–5891.
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pH. It seems likely that the severe ATPase deficiency in AtCRS1 mutants disrupts this targeting pathway by preventing the generation of the necessary pH gradient. 
-32P]ATP and T4 polynucleotide kinase. The RNA was suspended in 4 µL 10 mM Tris-HCl, pH 8.5, 100 mM KCl, combined with 1 µL of labeled primer (50,000–100,000 cpm), heated briefly to 95°C and cooled to 48°C by placing in a 48°C heat block for 10 min. Primer extension was initiated by the addition of 5 µL of a solution containing 5 units AMV reverse transcriptase (Promega) and 10 units of RNAsin Ribonuclease Inhibitor (Promega) in 50 mM Tris-HCl, pH 8.5, 20 mM MgCl2, 20 mM dithiothreitol, 0.5 mM of the appropriate ddNTP, and 0.5 mM of the other three dNTPs. Reactions were incubated at 45°C for 30 min and then stopped by the addition of 12 µL 90% formamide, 20 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.04% bromphenol blue, and 0.04% xylene cyanole and boiling for 2 min. Ten microliters of each reaction was applied to a 15% polyacrylamide gel containing 7 M urea and 1x Tris-borate/EDTA, and electrophoresis was performed at 27 mA with 1x Tris-borate/EDTA buffer. Gels were imaged with a Storm PhosphorImager (Molecular Dynamics). 
