**3. Discussion**

In plants, characterization of yellow foliar mutants will present an interesting opportunity to understand the complex photosynthesis process. The *yl* mutant reported here showed reduced Chl *a*, *b* and total Chl contents (Figure 1B). Our study identified a point mutation in the *YL* gene responsible for the mutant phenotype (Figure 3B). In addition, we discovered decreased PSII activity and a decreased net photosynthesis rate of *yl* leaves (Figure 1D,E). These results reveal that *YL* plays an important role in the photosynthetic process. This conclusion is supported by two pieces of evidence: (1) Strong expression of *YL* was detected in the trifoliate leaves; (2) The *yl* mutant lacked stacked thylakoids in the chloroplast, where photosynthesis takes place. The YL protein was homologous to maize ORRM1. In addition, the *Mu* transposon insertional mutant in *ZmORRM1* showed defects in the major photosynthetic enzyme complexes [20]. However, it is not known whether YL could influence similar photosynthetic proteins.

Based on bioinformatic analysis and protein subcellular localization experiments, we conclude that *YL* encodes a 390-amino-acid chloroplast-localized protein (Figures 4A and 5B,C). Identification of the C-terminal RNA recognition motif of YL and the high level of homology with AtORRM1 and ZmORRM1 strongly suggest that YL functions in RNA editing. ORRM1 controlled the extent of editing in 62% of the chloroplast sites in *Arabidopsis* and 81% of sites in maize [20]. In our study, YL was required for the editing of 42% of chloroplast sites in 12 chloroplast transcripts (Figure 6A, Tables S1 and S2). Moreover, we compared RNA editing alterations in soybean, *Arabidopsis* and maize *orrm1* mutants. Notably, the three completely lost editing sites, *ndhB*-737, *ndhD*-674 and *rpoB*-551, in the *yl* mutant also exhibited a pronounced reduction in editing in *Arabidopsis* or maize *orrm1* mutants (Figure 6A,B, Tables S1 and S2). In contrast, the editing of *ndhF*-290 exhibited no change in the *Arabidopsis orrm1* mutant, whereas we observed a slight increase in the editing of *ndhF*-290 in the *yl* mutant (Figure 6A, Table S1). Consequently, it seems that ORRM1 showed species-specific functions in plants. These results suggest that YL may be involved in chloroplast RNA editing.

Twelve chloroplast transcripts, which encode components of the Clp protease proteolytic subunit, NDH complex, cytochrome *b6f* complex, PSII complex, RNA polymerase or ribosomal proteins, could not be edited in the *yl* mutant (Figure 6C). The RNA editing defect in transcripts encoding the components of the Clp protease proteolytic subunit, NDH complex, and RNA polymerase or ribosomal proteins may not be the main reason for the *yl* mutant phenotype. Although the *Arabidopsis orrm1* mutant exhibited severe editing defects in transcripts encoding these proteins, it did not show any phenotypic deficiencies [20]. In addition, partial knockout of *clpP* in tobacco resulted in an asymmetric, slender leaf shape but normal leaf color [34]. Moreover, no obvious phenotype was observed in tobacco mutants with disrupted NDH complexes [35].

The reduction in *petB*-611 and *psbL*-2 editing may have large contributions to the *yl* phenotype. The *petB* gene encodes cytochrome *b6*, which is one of the major subunits of the cytochrome *b6f* complex mediating electron transfer between PSII and I [36–38]. In the *yl* mutant, defective editing at *petB*-611 resulted in a change from the wild type residue at position 204 (Leu) to Ser (Figure 6B). The Leu204

residue belongs to the D helix span, which is involved in heme binding [37,39]. Leu204 to Ser204 alteration likely disrupts the assembly of cytochrome *b6f* complexes, as suggested by the behavior of *petB* mutants in *Chlamydomonas reinhardtii* [37]. The *yl* mutant is phenotypically similar to a tobacco *petB* mutant in which the portion of the *petB* coding region was replaced with an *aadA* cassette [38]. Both *yl* and *petB* mutants display yellow (pale green) leaf color and reduced chlorophyll contents and grana stacks. The *psbL* gene encodes a conserved low molecular weight protein of PS II [40]. In wild type soybean, the initiator codon (AUG) of *psbL* is formed by a C to U editing of the ACG codon. In the present *yl* mutant, defective editing at *psbL*-2 may partially influence the formation of the initiator codon (AUG). The lack of *PsbL* in tobacco has been implicated as impairing the assembly of PSII [41,42]. The reduced Fv/Fm in the *yl* mutant is similar to the behavior of *Arabidopsis* mutants deficient in RNA editing sites in plastid transcripts encoding PSII proteins, including *orrm6* mutants [21].

The cytochrome *b6f* and PSII complexes belong to the main components of the photosynthetic electron transfer chain [2]. The *yl* mutant, exhibiting RNA editing deficiency at *petB*-611 and *psbL*-2, may be unable to assemble functional cytochrome *b6f* and PSII complexes. Since the localization of cytochrome *b6f* and PSII complexes is predominantly in the grana regions of the thylakoid membrane system, this inference may account for lacked grana in *yl* chloroplast. In soybean, some mutants with defects in photosynthetic electron carrier proteins display a yellow phenotype [4,8]. For instance, the mutation in the PSII extrinsic protein GmpsbP leads to a lethal-yellow phenotype, extremely low Fv/Fm, and failure of proplastid differentiation into normal chloroplasts with grana [8]. The *yl* mutant showed reduced Fv/Fm, indicating that the *yl* mutant cannot utilize the absorbed light in photochemistry as effectively as the wild type. When more light energy is absorbed than is converted in plants, the photosynthetic organism is subjected to photooxidative stress, known as photoinhibition, leading to pigment bleaching, inactivation of electron transport and damage of the reaction center [43,44]. A recent study has shown that a leaf yellowing mutant phenotype in soybean may be largely due to the abnormal light absorption in the photosynthesis process [45]. Thus, photoinhibition may also be the cause of decreased Chl contents and net photosynthesis rate in the *yl* mutant.

#### **4. Materials and Methods**
