Seedling Lethal1, a pentatricopeptide repeat protein lacking an E/E+ or DYW domain in Arabidopsis, is involved in plastid gene expression and early chloroplast development

Abstract

Chloroplasts are the site of photosynthesis and the biosynthesis of essential metabolites, including amino acids, fatty acids, and secondary metabolites. It is known that many seedling-lethal mutants are impaired in chloroplast function or development, indicating the development of functional chloroplast is essential for plant growth and development. Here, we isolated a novel transfer DNA insertion mutant, dubbed sel1 (for seedling lethal1), that exhibited a pigment-defective and seedling-lethal phenotype with a disrupted pentatricopeptide repeat (PPR) gene. Sequence analysis revealed that SEL1 is a member of the PLS subgroup, which is lacking known E/E(+) or DYW domains at the C terminus, in the PLS subfamily of the PPR protein family containing a putative N-terminal transit peptide and 14 putative PPR or PPR-like motifs. Confocal microscopic analysis showed that the SEL1-green fluorescent protein fusion protein is localized in chloroplasts. Transmission electron microscopic analysis revealed that the sel1 mutant is impaired in the etioplast, as well as in chloroplast development. In sel1 mutants, plastid-encoded proteins involved in photosynthesis were rarely detected due to the lack of the corresponding transcripts. Furthermore, transcript profiles of plastid genes revealed that, in sel1 mutants, the transcript levels of plastid-encoded RNA polymerase-dependent genes were greatly reduced, but those of nuclear-encoded RNA polymerase-dependent genes were increased or not changed. Additionally, the RNA editing of two editing sites of the acetyl-CoA carboxylase beta subunit gene transcripts in the sel1 mutant was compromised, though it is not directly connected with the sel1 mutant phenotype. Our results demonstrate that SEL1 is involved in the regulation of plastid gene expression required for normal chloroplast development.

Figure 1.

Isolation and characterization of the sel1 mutants. A, Pigment-defective and seedling-lethal phenotypes of the sel1 mutants. The wild-type (ecotypes Columbia and Ws-2) and sel1 mutant alleles were grown on MS medium with 1% (w/v) Suc or soil for 4 and 14 d, respectively. Bar = 1 mm. B, Gene structure of SEL1 (At4g18520). The black box represents an exon, and gray boxes represent the 5′ and 3′ untranslated regions. The positions of the T-DNA insertion in sel1-1, sel1-2 (SAIL_793_F11), and sel1-3 (SALK_054374) are represented by triangles. T-DNA is not drawn to scale. C, Northern-blot analysis of the SEL1 gene in wild-type (WT) and sel1 mutant alleles. Twenty micrograms of total RNA was isolated from 7-d-old seedlings grown on MS medium. 18S rRNA was used as a loading control. D, Molecular complementation of the sel1 mutant by pSEL1::SEL1 genomic DNA. The top row shows the 14-d-old wild type, sel1-2, and the complemented line (Comp.). The bottom row shows northern-blot analysis of SEL1 in the wild type, sel1-2, and the complemented line. 18S rRNA was used as a loading control. [See online article for color version of this figure.]

Figure 2.

Sequence analysis of SEL1. A, Predicted motif structure of SEL1 protein. P or P-L-S block motifs are depicted as boxes with letters as previously proposed (Lurin et al., 2004); P, L, and S designate the PPR, PPR-like S, and PPR-like L motif, respectively. B, Amino acid sequence alignment of SEL1. The amino acid sequence of SEL1 was compared with the homologous proteins in other species using ClustalW 2.0 (Larkin et al., 2007). The putative cleavage site of the transit peptide in SEL1 is shown by an arrow. Lines above the sequences show the predicted P- or PPR-like (S and L) motifs. Black boxes indicate amino acid residues that are greater than 80% conserved, and gray boxes indicate amino acids that are greater than 60% conserved. The amino acid sequences are NP_193587.4 for Arabidopsis, XP_002284293.1 for grape, NP_001173562.1 for rice, ACG29369.1 for maize, and XP_001757146.1 for moss.

Figure 3.

SEL1 is localized in the chloroplasts. A, Subcellular localization of SEL1-GFP. Protoplasts were isolated from 35S::GFP and 35S::SEL1:GFP transgenic plants. The fluorescence of GFP and SEL1-GFP fusion protein in protoplasts was observed by confocal laser scanning microscope. Green fluorescence signals, chlorophyll red autofluorescence signals, merged images, and bright-field images are shown. Bar = 5 μm. B, Western-blot analysis of SEL1-FLAG fusion protein in chloroplast subfractions. Chloroplasts from pSEL1::SEL1:FLAG transgenic plants were purified and separated into soluble and membrane fractions by ultracentrifugation. The same proportion of each fraction was separated by SDS-PAGE and transferred to the nitrocellulose membrane. The blot was probed sequentially with antibody to FLAG and specific antibodies to RbcL and D1 as controls for the fractionation of soluble proteins and membrane proteins, respectively. TC, Total chloroplast protein; S, soluble protein; M, membrane protein. [See online article for color version of this figure.]

Figure 4.

Expression patterns of SEL1. GUS expression was analyzed in pSEL1::GUS transgenic plants. A, Three-day-old etiolated seedling. B, Germinating seed. C, Three-day-old seedling. D, Five-day-old seedling. E, Seven-day-old seedling. F, Rosette leaf. G, Flower buds. H, Flower. I, Silique. [See online article for color version of this figure.]

Figure 5.

Ultrastructure of chloroplasts and etioplasts in sel1 mutant. A and B, Chloroplasts are from 7-d-old light-grown cotyledons of the wild type (WT) and sel1 mutant. C and D, Etioplasts are from 7-d-old dark-grown cotyledons of the wild type and sel1 mutant. Prolamellar body and prothylakoid are indicated by arrows and triangles, respectively.

Figure 6.

Analysis of photosynthetic protein complexes in the sel1 mutant. A, Accumulation of representative subunits of photosynthetic protein complexes determined by western-blot analysis with specific antibodies to proteins indicated to the left; RbcL is the large subunit of Rubisco. PsaA and D1 are subunits of PSI and PSII, respectively. AtpB and Cytf (for cytochrome subunit f) are subunits of ATP synthase and cytochrome b₆f complex, respectively. Total proteins (10 μg or the indicated dilution of the wild-type sample) from 7-d-old seedlings were loaded per lane. Actin was used as a loading control. B, Northern-blot analysis for the accumulation of photosynthesis-related transcripts. Five micrograms of total RNA was isolated from 7-d-old seedlings of the wild type (WT) and sel1 mutant and hybridized with the gene-specific probes indicated. 18S rRNA was used as a loading control.

Figure 7.

RNA levels of plastid-encoded genes in sel1 mutant. The transcript abundance of protein-encoding genes and rRNAs of the plastid genome were measured from the wild type (WT) and sel1 mutant by quantitative RT-PCR. The graph shows the log2 ratio of transcript levels in the sel1 mutant compared with levels in the wild type. The genes are sorted according to physical location on the plastid genome. Error bars indicate sd.

Figure 8.

The sel1 mutant is defective in RNA editing of accD transcripts. Sequence analysis for the accD transcripts from the wild type (WT) and sel1 mutant. Nucleotide sequences including the RNA editing sites of accD-1 and accD-2 are shown as chromatograms. Editing sites of accD-1 and accD-2 are indicated by arrows pointing to the corresponding peaks. [See online article for color version of this figure.]

Figure 9.

Chloroplast rRNAs are significantly decreased in the sel1 mutant. A, Northern-blot analysis of chloroplast rRNAs in the wild type (WT) and sel1 mutant. One microgram of total RNA was isolated from 7-d-old seedlings and analyzed by hybridization to probe for the plastid 23S, 16S, 5S, and 4.5S rRNA. 18S rRNA was used as a loading control. B, Polysome analysis of the wild type and sel1 mutant. Total polysome isolation and RNA purification from 7-d-old seedlings were performed as described previously (Kwon and Cho, 2008). The ethidium bromide-stained gel represents equal proportional loading of the 10 gradient fractions.

Figure 10.

Northern-blot analysis of the rpo genes encoding the PEP core subunits in the wild type (WT) and sel1 mutant. A, Schematic representation of chloroplast rpl23 and rpoB operons. rpoA is the last gene of the rpl23 operon. rpoB, rpoC1, and rpoC2 are part of the rpoB operon. The bent lines indicate introns. B, Five micrograms of total RNA from 7-d-old seedlings was analyzed by hybridization to the probe for rpoA, rpoB, rpoC1, and rpoC2. The size marker (M) is a RNA ladder, and ethidium bromide staining is shown as loading control.