Fig.  1.  The tsl1 mutant is sensitive to elevated ambient temperatures. A–H: Heat-sensitive phenotype of the tsl1 mutant plants. The wild-type (WT) and tsl1 mutant seedlings were germinated under normal growth temperature (22 °C) for 4 days and then kept at 22 °C (A, C and E) or transferred to an elevated temperature condition for 7 days (B, D and F), or recovered at 22 °C after grown at 29 °C for 7 days (G). Alternatively, 11-day-old seedlings grown at 22 °C were transferred to 29 °C for the indicated period and then photographed (H). Bar = 5 mm.

Fig.  2.  TSL1 regulates chloroplast development and function at elevated ambient temperatures. A–D: Impairment of chloroplast development and photosynthesis in the tsl1 mutant plants under high-temperature conditions. Wild-type (WT), tsl1 mutant, and genetic complemented transgenic plants (Line 3/4) were germinated at 22 °C for 4 days and then either kept at 22 °C or transferred to the growth chamber under elevated temperature conditions (26 °C) for 7 days. Ultrastructure of chloroplasts was examined under transmission electron microscopy (A and B). The chloroplast fluorescence of the whole plants was imaged using CF Imager after plants were dark-adapted for 30 min (C and D). The maximum photochemical efficiency of photosystem II (Fv/Fm) was measured with the highest value in red. Bar = 5 mm.

Fig.  3.  TSL1 is allelic to DG1. A–D: Genetic complementation analysis of the tsl1 mutant plants. Genomic sequences of two candidate genes, AT5G67570 (A and B, Line 1/2) and AT5G59900 (C and D, Line 11/12/13/14), including the upstream promoter sequences, were amplified from the wild-type (WT) plants and introduced into the tsl1 mutant plants, respectively. E–F: Sensitivity of dg1 mutants to an elevated ambient temperature. The WT, tsl1 and dg1 mutants, and different lines of transgenic plants were germinated at 22 °C for 4 days and then either kept at 22 °C (A, C and E) or transferred to growth chamber under elevated temperature conditions (29 °C) (B, D and F). After 7 days of additional growth, plants were photographed. G–H: Rescue of dg1 mutant phenotype by the mutated TSL1 in Arabidopsis. Genomic sequence of AT5G67570 was amplified from the tsl1 mutant plants and introduced into the dg1 mutant plants (Line 21/22). Plants were germinated at 22 °C for 4 days and then either kept at 22 °C (G) or transferred to 29 °C (H). After 10 days of additional growth, plants were photographed. Bar = 5 mm.

Fig.  4.  DG1 is important for chloroplast mRNA editing at elevated ambient temperatures. A–C: Impairment of mRNA editing in the tsl1 mutant plants under elevated temperature conditions. Wild-type (WT), tsl1 mutant, dg1 mutant, or DG1 complemented transgenic plants were germinated at 22 °C for 4 days and then either kept at 22 °C or transferred to the growth chamber under elevated temperature conditions (29 °C or 26 °C) for 7 days. Total RNA was extracted, and RT-PCR products were either directly sequenced (A or C) or TA cloned and then sequenced (B). The sequencing chromatograms are shown with nucleotide sequences listed. The editing sites are indicated with arrows. In TA cloning, 25 clones of each PCR products were sequenced.

Fig.  5.  DG1 is involved in chloroplast mRNA editing in response to heat stress. Wild-type (WT) andtsl1 mutant plants were grown at 22 °C for 8 days and then subjected to heat stress treatment (35 °C) for different time periods as indicated. Total RNA was extracted, and RT-PCR products were directly sequenced. The sequencing chromatograms are shown with nucleotide sequences listed. The editing sites are indicated with arrows.

Fig.  6.  DG1 interacts with MORF2 both in vitro and in vivo. A–C: Protein-protein interaction between DG1 and MORF2 in yeast two-hybrid (Y2H) assays (A), in vitro pull-down assays (B), and in vivo split-luciferase assays (C). In the Y2H assays, DG1 (AA 48–799) was fused with the DNA-binding domain (bait), and MORF2, MORF8, MORF9, or OZ1 was fused with the activation domain (prey) of GAL4. Activation of HIS3 expression was used as the interaction reporter. In the pull-down assays, GST-tagged DG1-C was used to pull down His-MORF2. In the split-luciferase assays, MORF2 was fused with the N-terminal (nLUC), while DG1 was fused with the C-terminal (cLUC) portion of firefly luciferase (LUC). Different combinations of constructs were infiltrated in tobacco leaves, and luciferase luminescence was observed. The mutated form (P289L) of DG1 (DG1M) was also used as a bait in the Y2H assay (D).

Fig.  7.  The C-terminus of DG1 is required for the interaction between DG1 and MORF2 in yeast two-hybrid assays. A: Domain structure of DG1. In the domain deletion experiment, various deletion fragments of DG1 were used as the baits. B and C: The long form (AA 48–799), N-terminal form (AA 1–250), PPR domain form (AA 251–594), and C-terminal form (AA 595–799) of DG1 were fused with the DNA-binding domain (bait), and MORF2 was fused with the activation domain (prey) of GAL4. Activation ofHIS3 expression was used as the interaction reporter. ∗, 3-AT (2.5 mM) was included in the medium to suppress HIS3 leakage.

Fig.  8.  DG1 directly binds to RNA targets in vitro. A and B: RNA EMSA assays. His-tagged truncated form of DG1 (His-DG1-PPR) was purified and incubated with biotin-labeled accD, ndhD, or petL RNA, and RNA EMSA assays were performed. Nonlabeled cold probes (competitors) were used to show the binding specificity. Arrows and arrow heads point to free probes and shifted bands, respectively. EMSA, electrophoretic mobility shift assay.

Fig.  9.  A hypothetical working model for the role of DG1 in chloroplast RNA editing in Arabidopsis. Besides the functions in RNA processing and splicing, the P-type PPR protein DG1 has a novel function in chloroplast RNA editing in wild-type (WT) Arabidopsis plants through direct binding to RNA targets such as ndhD and interacting with RNA editing factors such as MORF2. The mutated form of DG1 in tsl1 mutants remains functional at 22 °C but losses its function at 29 °C, most probably due to the weakened interaction between DG1 and MORF2 at an elevated ambient temperature.