MORF9 Functions in Plastid RNA Editing with Tissue Specificity

Abstract

RNA editing in plant mitochondria and plastids converts specific nucleotides from cytidine (C) to uridine (U). These editing events differ among plant species and are relevant to developmental stages or are impacted by environmental conditions. Proteins of the MORF family are essential components of plant editosomes. One of the members, MORF9, is considered the core protein of the editing complex and is involved in the editing of most sites in chloroplasts. In this study, the phenotypes of a T-DNA insertion line with loss of MORF9 and of the genetic complementation line of Arabidopsis were analyzed, and the editing efficiencies of plastid RNAs in roots, rosette leaves, and flowers from the morf9 mutant and the wild-type (WT) control were compared by bulk-cDNA sequencing. The results showed that most of the known MORF9-associated plastid RNA editing events in rosette leaves and flowers were similarly reduced by morf9 mutation, with the exception that the editing rate of the sites ndhB-872 and psbF-65 declined in the leaves and that of ndhB-586 decreased only in the flowers. In the roots, however, the loss of MORF9 had a much lower effect on overall plastid RNA editing, with nine sites showing no significant editing efficiency change, including accD-794, ndhD-383, psbZ-50, ndhF-290, ndhD-878, matK-706, clpP1-559, rpoA-200, and ndhD-674, which were reduced in the other tissues. Furthermore, we found that during plant aging, MORF9 mRNA level, but not the protein level, was downregulated in senescent leaves. On the basis of these observations, we suggest that MORF9-mediated RNA editing is tissue-dependent and the resultant organelle proteomes are pertinent to the specific tissue functions.

Keywords: Arabidopsis thaliana; MORF9; RNA editing; organelles; tissue-specific.

Figure 1

A T-DNA insertion line of Arabidopsis at the MORF9 locus and generation of transgenic plants. (a) Schematic of the T-DNA insertion morf9 mutant and the transgenic constructs for overexpression of a GFP-tagged MORF9 or GFP alone in wild-type (WT) plants and complementation (Pmorf9:MORF9-myc) of the T-DNA insertion morf9 mutant. Not drawn to scale; (b) MORF9 transcript levels in the morf9 mutant and MORF9 complementation plants by qRT-PCR. The relative transcript levels of MORF9 with respect to ACTIN were normalized to those in WT plants. Error bars indicate the standard error (SE) of three biological replicates; (c) Western blots showing protein expression in the indicated plants using antibodies against GFP or MYC. The same membranes were stained with Ponceau for a loading control.

Figure 2

Phenotypes of morf9 mutant and transgenic plants. (a) Plants at 12 days after germination in MS medium and at 35 days after transfer to soil; (b) Root morphogenesis of the morf9 mutant and transgenic plants. The lower panel shows the average root length of 10 to 20 plants with standard errors; (c,d) Imaging and quantification of the leaf initiation fluorescence yield (F0) and efficiency of photosystem II (Fv/Fm) in plants with indicated genetic background at day 12 after germination, respectively. Data represent the mean of at least five plants with SE.

Figure 3

Flower phenotype and seed germination in the morf9 mutant and MORF9 transgenic plants. (a,b) Representative images of flowers in 56-day-old plants. Scale bars = 10 mm; (c–e) Flower dimensions of the indicated plant lines; n = 18–24; (f) Seed germination rate on the 5th day. Three independent experiments with n = 1500; (g) Cotyledon greening rates in the 5-day-old plants. Three independent experiments with 100 seedlings each; (h) Open cotyledons in 5-day-old plants. Three independent experiments with 100 seedlings each. Error bars indicate the standard errors of the replications. Asterisks indicate significant differences from the WT according to two-tail Student’s t test (* denotes p < 0.05, ** for p < 0.01 and *** for p < 0.001).

Figure 4

MORF9 transcript and protein levels in various tissues and organs. (a) The transcript levels of MORF9 in various tissues and organs retrieved from public datasets [33]; (b) Protein levels of MORF9 in various organs in Pmorf9:MORF9-myc plants, determined by using an antibody against MYC. The same membrane was subsequently probed with an anti-ACTIN antibody for loading control. Ponceau staining prior to immunodetection shows total proteins on the membrane. Arrows indicate the expected bands.

Figure 5

Percent editing of 24 plastid RNA sites in various organs of WT and morf9 seedlings. (a) Nucleotide sequence profiles of the RNA editing sites in leaf, flower, and root most significantly affected by morf9 mutation; (b) A heatmap of RNA editing efficiencies for multiple editing sites in various organs of WT and morf9 plants. Leaf and root samples were collected in seedlings after 21 days of growth in sugar-free medium. For flower sampling, 56-day-old plants were used.

Figure 6

Summary of relative plastid RNA editing efficiency in the indicated organs in morf9 plants as compared to WT plants. The left panel shows the average ratio of morf9 mutant’s to WT line’s editing efficiency from three replicates. The right panel plots the fold change relative to the WT, showing standard errors of the replicates. Full dataset are provided in Supplemental Table S2.

Figure 7

Alteration of MORF9-associated RNA editing efficiency during leaf senescence in WT Arabidopsis. (a) MORF9 mRNA levels of subsequent rosette leaves of mature plants; (b) MORF9 mRNA levels of leaf 7; (c). MORF9 mRNA levels of rosette leaves of young and aging plants; (d). Western blot of MORF9-MYC fusion protein in rosettes of plants expressing the tagged MORF9 driven by the native promoter. The Ponceau staining of the same membrane and Western blotting of ACTIN by anti-ACTIN antibody were used to control gel loading; (e) Plastid RNA editing efficiency in leaves of 12-day-old and 21-day-old plants. Full dataset are provided in Supplemental Table S2.