Ecotype allelic variation in C-to-U editing extent of a mitochondrial transcript identifies RNA-editing quantitative trait loci in Arabidopsis

Abstract

In higher plants, RNA editing is a posttranscriptional process that converts C to U in organelle mRNAs. Although RNA editing in mitochondria occurs much more frequently than in chloroplasts, editing of exogenously supplied RNA substrates in vitro and in organello has shown that editing in the two organelles shares some common features. In particular, the 20 nucleotides upstream of the editing site play an important role in specifying the C to be edited. Biochemical approaches have allowed the identification of features of cis-sequences necessary for RNA editing to occur, but have failed to identify any of the components of the mitochondrial editing machinery. In order to implement a genetic approach for identification of editing factors, we have identified a polymorphism in the editing efficiency of a mitochondrial site between two ecotypes of Arabidopsis (Arabidopsis thaliana), Columbia (Col) and Landsberg erecta (Ler). In rosette leaves, an editing site within the ccb206 mitochondrial gene is more highly edited in Col than in Ler. Depending on the development stage and tissue analyzed, the difference in editing extent varies between the two ecotypes; for example, in floral buds, editing extent does not differ. Single-point regression analysis of the editing efficiency in a sample of recombinant inbred lines derived from a cross between Col and Ler allowed the identification of two quantitative trait loci controlling this trait. A member of the pentatricopeptide repeat protein family that carries a putative mitochondrial transit sequence has been identified near a major quantitative trait locus on chromosome 4.

Figure 1.

Editing extent of ccb206 C24 in different tissues of Arabidopsis ecotype Col and Ler. Editing efficiency was assayed by PPE. A, PPE was performed on an RT-PCR product from the radiolabeled oligo PPE-ccb206-C24; extension of the primer was poisoned by ddCTP incorporation. PPE products were resolved on a 12% sequencing gel, which was then exposed on a phosphorimager screen. B, The percentage of edited transcript was determined by quantifying the radioactivity associated with edited and unedited ccb206 C24 using ImageQuant software (Molecular Dynamics). The editing efficiency is reported on the y axis and is the average of three independent PPE reactions. The error bars represent the sds of the three measurements. 0, PPE without template indicating the size of the radiolabeled primer; U, PPE with a cloned (genomic) unedited PCR product; E, PPE with a cloned edited RT-PCR product.

Figure 2.

Editing extent of ccb206 C24 in rosette leaves of F₁s generated from a cross between Arabidopsis ecotype Col and Ler. A, PPE results obtained for the parents Col and Ler and for four F₁ progeny. B, Editing efficiency of Col and Ler and of F₁s generated from a cross between the two ecotypes in both combinations. The value for Col and Ler is the same as the one reported in Figure 1B and comes from three different measurements. For the hybrids, the value is the average of assays of four different F₁ progeny for Col × Ler and of three different F₁ progeny for Ler × Col. The error bars represent the sds of the measurements for each genotype. 0, PPE without template indicating the size of the radiolabeled primer; U, PPE with a cloned (genomic) unedited PCR product; E, PPE with a cloned edited RT-PCR product; Col × Ler, F₁ generated from a cross with Col as a female; Ler × Col, F₁ generated from a cross with Ler as a female.

Figure 3.

Editing extent of ccb206 C24 in 30 RILs generated from a cross between Col and Ler. The average of the editing percentage obtained from three different PPE experiments was determined as indicated in Figure 1. The error bars represent the sds of the three measurements.

Figure 4.

Editing sites sharing some similarity in their putative cis-recognition sequences with ccb206 C24. These sites were identified by using Pratt software (see “Materials and Methods” for details). ccb206 C24 is shown in the top line for reference. Identical nucleotides shared by other sites are shown in gray-shaded background. Cs targeted for editing are in bold. The number in parenthesis refers to the nucleotide position of the edited C in the coding sequence of each gene A from the initiation codon having the position 1. *, orfx and ccb203 coding sequences are incomplete and do not start with an ATG codon.

Figure 5.

The ccb206 transcript pattern does not cosegregate with ccb206 C24 editing efficiency. Numbers below the figure refer to the editing extent of ccb206 C24.