GUN1 interacts with MORF2 to regulate plastid RNA editing during retrograde signaling

Abstract

During development or under stress, chloroplasts generate signals that regulate the expression of a large number of nuclear genes, a process called retrograde signaling. GENOMES UNCOUPLED 1 (GUN1) is an important regulator of this pathway. In this study, we have discovered an unexpected role for GUN1 in plastid RNA editing, as gun1 mutations affect RNA-editing efficiency at multiple sites in plastids during retrograde signaling. GUN1 plays a direct role in RNA editing by physically interacting with MULTIPLE ORGANELLAR RNA EDITING FACTOR 2 (MORF2). MORF2 overexpression causes widespread RNA-editing changes and a strong genomes uncoupled (gun) molecular phenotype similar to gun1 MORF2 further interacts with RNA-editing site-specificity factors: ORGANELLE TRANSCRIPT PROCESSING 81 (OTP81), ORGANELLE TRANSCRIPT PROCESSING 84 (OTP84), and YELLOW SEEDLINGS 1 (YS1). We further show that otp81, otp84, and ys1 single mutants each exhibit a very weak gun phenotype, but combining the three mutations enhances the phenotype. Our study uncovers a role for GUN1 in the regulation of RNA-editing efficiency in damaged chloroplasts and suggests that MORF2 is involved in retrograde signaling.

Keywords: GUN1; MORF2/RIP2; PPR; RNA editing; retrograde signaling.

Fig. 1.

GUN1 is required for the regulation of RNA-editing efficiency at multiple sites in plastids under NF treatment, and it interacts with MORF2. (A) RNA-editing levels of different sites in plastid altered by gun1 mutation under NF treatment. Col6-3 is the wild type. The x axis indicates different editing sites. The y axis represents the C to T (equal C to U in RNA) editing level. Data are mean ± SEM (three biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001, two-tailed Student’s t test. (B) Sequencing chromatograms show RNA-editing profiles of representative sites indicated by red arrows. The peak for C is in blue and the peak for T is in red. (C) GUN1 interacts with MORF2 as shown by BiFC assay. nYFP: N-terminal YFP; cYFP: C-terminal YFP. nYFP(Chloro) and cYFP(Chloro) represent chloroplast-targeted nYFP and cYFP, respectively. Chlorophyll red autofluorescence indicates the localization of chloroplasts. Bright-field images correspond to the protoplast cells. Merged images show the colocalization of YFP and chloroplasts. GUN1-cYFP/nYFP(Chloro) and MORF2-nYFP/cYFP(Chloro) cotransformations are negative controls. (Scale bar, 10 μm.)

Fig. 2.

Overexpression of MORF2 confers a gun phenotype and regulates RNA editing under NF treatment. (A) The qPCR analysis of retrograde signaling marker gene expression indicates that MORF2OX lines have gun phenotypes. Total RNAs were isolated from 5 μM NF-treated seedlings grown under 24 h light (100 µmol⋅m⁻²⋅s⁻¹) at 22 °C for 5 d. The x axis indicates different samples. The y axis shows the relative expression level, and expressions in Col6-3 are set to 1. The PPI2 gene is the negative control. Data are mean ± SEM (three biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, two-tailed Student’s t test. (B) Phenotype of MORF2OX seedlings grown on normal medium under long-day (LD) conditions (16 h light/8 h dark, 22 °C) for 14 d. (Scale bar, 0.5 cm.) (C) The leaf phenotype of 5-wk MORF2OX plants grown in soil under LD. The leaves are the rosette leaf, the first and the second cauline leaf from left to right, respectively. (Scale bar, 1 cm.) (D) The RNA-editing profile overlap between NF-treated MORF2OX(s) and gun1-9 seedlings. The x axis indicates the editing sites. The y axis shows the editing levels. Data are mean ± SEM (three biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, two-tailed Student’s t test.

Fig. 3.

Transcriptome data indicate that MORF2OX(s) regulates nuclear gene expression similarly to gun1-9 during retrograde signaling. (A) The Venn diagram shows the differentially expressed gene overlap among Col6-3 (Col6-3 NF vs. Col6-3 LS), gun1-9 (gun1-9 NF vs. Col6-3 NF), and MORF2OX(s) [MORF2OX(s) NF vs. Col6-3 NF] under NF treatment. (B) The Venn diagram shows overlapped DEGs that are down-regulated in Col6-3 (Col6-3 Down) but have a higher expression level in gun1-9 (gun1-9 Up) and MORF2OX(s) [MORF2OX(s) Up] compared with wild type under NF treatment. (C) The Venn diagram shows overlapped DEGs that were up-regulated in Col6-3 (Col6-3 Up), but had a lower expression level in gun1-9 (gun1-9 Down) and MORF2OX(s) [MORF2OX(s) Down] than in the wild type under NF treatment. P values in A–C show the statistical significance of the overlap between two groups of genes in Venn diagrams. (D–F) The hierarchical clustering of expression levels of GUN1-dependent (D), MORF2/GUN1-overlapping (E) retrograde signaling genes, and differentially expressed nuclear-encoded photosynthesis genes (F) in different samples. LS, untreated; NF, NF-treated. Heatmaps show the Z-score value of log2-transformed [(average reads per kilobase of transcript per million mapped reads) + 0.001] of each gene.

Fig. 4.

MORF2 interacts with the site-specificity factors OTP81, OTP84, and YS1 as shown by BiFC assay. cYFP, C-terminal YFP; nYFP(Chloro), chloroplast-targeted nYFP; nYFP, N-terminal YFP. MORF2-nYFP interacts with OTP81-, OTP84-, and YS1-cYFP showing the YFP complementation in chloroplasts. Chlorophyll red autofluorescence indicates the localization of chloroplasts. Bright-field images correspond to protoplast cells. Merged images show the colocalization of YFP and chloroplasts. OTP81-, OTP84-, or YS1-cYFP cotransformed with nYFP(Chloro) are negative controls. (Scale bar, 10 μm).

Fig. 5.

otp81, otp84, and ys1 single and triple mutants have weak gun phenotypes. The qPCR analysis of retrograde signaling marker gene expression in otp81, otp84, and ys1 single and triple mutants. Col-0 is the wild type. Total RNAs were isolated from 5 μM NF-treated whole seedlings grown under 24 h light (100 µmol⋅m⁻²⋅s⁻¹) at 22 °C for 5 d. The x axis indicates the samples. The y axis shows the relative expression level of marker genes, and their expressions in Col-0 are set to one. The PPI2 gene is the negative control. Data are mean ± SEM (three biological replicates). *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significant, two-tailed Student’s t test.