*2.5. Dramatic Defects of Chloroplast RNA Editing in yl*

To further understand the function of *YL*, we performed DNA resequencing and RNA sequencing (RNA-seq) for the wild type to screen out soybean chloroplast editing sites, obtaining 44 predicted sites from 22 chloroplast transcripts (Table 2, Tables S1 and S2). In addition, the *rpl23*-89 site was identified through comparative analyses with *Arabidopsis* chloroplast editing sites (Table 2). Most of these sites were verified by direct sequencing of PCR products of transcripts or the corresponding genomic DNA carrying them (Table S2). Among the 45 editing sites, 44 sites were C-to-U conversions, 43 sites were in the coding regions of genes, and most of them caused alteration of the encoded amino acids (Table 2).


**Table 2.** RNA editing sites in soybean chloroplast transcripts.


**Table 2.** *Cont.*

<sup>a</sup> Position is given with respect to the initiation codon of each chloroplast transcript. <sup>b</sup> Codon site is the order in the amino acid codon. <sup>c</sup> RNA editing sites are in introns of the chloroplast gene. The position here is given with respect to the initiation codon of each gene.

The RNA-seq method was combined with direct sequencing of PCR products of transcripts carrying chloroplast RNA editing sites to compare the RNA editing between wild type and *yl* leaves. The results showed that the editing was completely abolished for *ndhB*-737, *ndhD*-674 and *rpoB*-551 in the *yl* mutant (Figure 6A,B, Tables S1 and S2). These deficiencies caused changes in the encoded amino acid residues from Leu, Leu and Leu in the wild type to Pro, Ser and Ser in the *yl* mutant, respectively. In addition, the editing of 14 sites was decreased by 10% to 90% in the *yl* mutant (Figure 6A, Tables S1 and S2). However, the *ndhF*-290 and *rpoB*-566 sites exhibited higher editing levels in the *yl* mutant compared with the wild type (Figure 6A, Tables S1 and S2).

**Figure 6.** RNA editing at multiple chloroplast sites is impaired in the *yl* mutant. (**A**) Eighteen sites exhibit a significant alteration in editing of more than 10% in the *yl* mutant through RNA-seq analysis.

(**B**) The three abolished editing sites and *petB*-611 in the *yl* mutant. Rectangular frames indicate defective editing sites. The corresponding amino acids are underlined. (**C**) Percentage of altered editing sites/transcripts in the *yl* mutant. Each bar shows a transcript color-coded in accordance with the complex to which it belongs.

We then investigated the distribution of the affected editing sites in the *yl* mutant. The 19 affected editing sites were distributed in 12 chloroplast transcripts encoding components of the Clp protease proteolytic subunit, NDH complex, cytochrome *b6f* complex, PSII complex, RNA polymerase or ribosomal proteins. As shown in Figure 6C, the percentage of altered editing sites per transcript varied from 36.4% to 100%, suggesting that the effect of the *YL* mutation on editing was site specific but not transcript specific.
