The Pentatricopeptide Repeat Protein SOT5/EMB2279 Is Required for Plastid rpl2 and trnK Intron Splicing

Photosynthetic characterization of sot5 mutants. A, Analysis of F_v/F_m of 30-d-old wild-type (WT) and sot5 plants. The graph at right shows quantified F_v/F_m of the mature and young leaves shown at left (Chl fluorescence image). The data represent means ± sd (n = 6). Asterisks indicate a significant difference between the wild type and sot5 (Student’s t test, **, P < 0.05). B, Nonphotochemical quenching (NPQ) measurement of mature and young leaves from 30-d-old wild-type and sot5 plants. The data represent means ± sd (n = 6). C, Immunoblot analysis of photosynthetic proteins accumulated in mature and young leaves from 30-d-old wild-type and sot5 plants. Coomassie Brilliant Blue (CBB) staining is shown to check the difference in sample loading. D, BN-PAGE analysis of photosynthetic complexes in thylakoid membranes isolated from 30-d-old wild-type and sot5 plants. Each lane was loaded with 6 μg of Chl.

Leaf morphology and adaxial-abaxial polarity are altered in sot5. A, Morphology and cross section of the first true leaves from 25-d-old wild-type (WT) and sot5 plants. Bars = 5 mm at left and 100 µm at right. B, Scanning electron microscopy analysis of adaxial and abaxial surfaces of the first true leaves from 25-d-old wild-type and sot5 plants. Enlargements of the larger and smaller squares in the top row are shown in the middle and bottom rows, respectively. Bars = 1 mm in the top row, 200 µm in the middle row, and 50 µm in the bottom row. C, Comparison of palisade cell size (left) and number (right) from the first true leaves of 25-d-old wild-type and sot5 plants. The data represent means ± sd of 10 leaves. Asterisks indicate a significant difference between the wild type and sot5 (Student’s t test, ** P < 0.05). Bars = 50 µm. D, sot5 enhances the leaf adaxial-abaxial polarity of as2-1. White arrows indicate filamental leaves. Bars = 1 cm.

SOT5 encodes EMB2279, a PPR protein. A, Map-based cloning of SOT5. SOT5 was mapped between two markers, F26G16-a and T5I8-d, on chromosome 1. The molecular markers used for fine-mapping are shown. Numbers indicate the recombinant sot5 mutants in 900 F2 individuals from crosses between sot5 (Col-0 ecotype) and Ler. B, Model for the SOT5 (AT1G30610) gene structure. The flanking sequences of the seventh intron are shown below the gene model. The G-to-A point mutation is labeled with an asterisk and red font in sot5 at the first position of the seventh intron. The position of the T-DNA insertion in emb2279-2 (SALK_088420) is shown above the gene model. The arrows show the primers used for RT-PCR. WT, Wild type. C, RT-PCR analysis of SOT5 transcripts in Col-0 and sot5 by using the primer pair shown in B. There are three bands, designated a, b, and c, in sot5. Genomic DNA (gDNA) was used as a temperate control, while the expression of ACTIN was used as a positive control. D, Overexpression of SOT5 cDNA complemented the phenotype of sot5. Bar = 1 cm. E, The null mutation emb2279-2 is embryo lethal. The top image shows the albino seeds (arrows) in a young emb2279-2 heterozygote silique. The bottom image shows the aborted seeds (arrows) in an older emb2279-2 heterozygote silique. Bars = 1 mm. F, Allelic test of sot5 and emb2279-2. The F1 plant of sot5/emb2279-2 appears albino and is seedling lethal. Bar = 1 cm. G, Knockdown lines of SOT5 by the artificial microRNA technique exhibit a leaf virescent phenotype. Bar = 1 cm.

Subcellular localization of SOT5 and tissue expression patterns of SOT5. A, Microscopy analysis of the YFP fusion protein SOT5_1-120-YFP transiently expressed in Arabidopsis protoplasts. The green fluorescence of SOT5_1-120-YFP was overlapped with chloroplast autofluorescence in merged images. DIC, Differential interference contrast microscopy. Bars = 10 µm. B, RT-PCR analysis of SOT5 expression in various tissues and seedlings. Total RNA was extracted from rosette and cauline leaves, stems, inflorescences, siliques, and roots of 40-d-old plants. Total RNA was extracted from 5-d-old seedlings grown in light or darkness or etiolated seedlings exposed to light for 4 h. Genomic DNA was used as a template control, and ACTIN expression was used as a loading control of RNA.

SOT5 is required for the splicing of the plastid rpl2 and trnK introns. A, Schematic SOT5 protein containing 11 PPR motifs. B, Predicted 11 ribonucleotides targeted by the 11 PPR motifs shown in A. In each repeat, the two key amino acid residues and their nucleotide targets are shown. X indicates unpredictable; two nucleotides with a slash mean optional; the red nucleotides are precisely predicted. C, RT-qPCR analysis of relative expression levels of the plastid genes containing the predicted target sequences or group IIA introns in sot5. Three biological replicates were analyzed. D, Splicing efficiency analysis of 14 plastid introns in sot5 by RT-qPCR. Three biological replicates were analyzed. E, RT-PCR analysis of intron retention in eight plastid genes. Genomic DNA (gDNA) was used as a template control. S, Spliced; U, unspliced. F, RT-PCR analysis of rpl2 intron splicing in wild-type (WT), amiR-SOT5/sot5, 35S:SOT5/sot5, and sot5 plants. G, Analysis of SOT5 transcript levels and splicing efficiency of rpl2 in wild-type and amiR-SOT5 plants by RT-qPCR. H, Northern-blot analysis of plastid trn genes that contain introns in different genotypes. Lanes 1 to 6 indicate the wild type, sot5, thf1, thf1 sot5, 35S:SOT5/sot5, and amiR-SOT5, respectively. The ethidium bromide-stained gel under each blot is shown as a loading control. Two biological replicates were analyzed, and one representative result is shown.