*2.5. Intron Splicing of Mitochondrial Transcripts*

Next, the splicing efficiencies of the 20 mitochondrial introns were analyzed among the samples by qRT-PCR (Figure 3). The splicing efficiencies of several introns were higher in both N and SR than UR, such as *nad1* introns 2/3/4, *nad4* intron 3, *nad5* introns 2/3, *cox2* intron 1, and *ccmFc* intron 1. While the nuclear intron maturases responsible for the splicing of some of these introns were identified in *A. thaliana* [20,23,26], the scenario in soybean is complicated by the presence of six nuclear intron maturases in the soybean genome (Supplementary Table S4). It should be noted that the splicing efficiency of the intron 1 of *nad4* was greatly enhanced in N than in UR and SR in the following order (N > UR > SR).

**Figure 3.** Quantitative RT-PCR analysis of splicing efficiency of mitochondrial transcripts. (**a**) Splicing efficiencies of mitochondrial transcripts in nodule and stripped root samples were compared with that of the root sample. The ratio of spliced mRNA to unspliced mRNA is designated as the splicing efficiency. The splicing efficiencies of nodules or stripped roots were divided by the splicing efficiency of roots. (**b**) The relative abundance of spliced mitochondrial mRNAs of nodule and stripped root samples were compared with that of roots. The histogram shows the log2 ratios of spliced transcripts. Values are means SD of three biological replicates.

#### *2.6. In-Gel Activity Assay and Western Blotting*

Since qRT-PCR showed that there was more splicing of *nad4* intron 1 in N, we examined the abundance of NAD4 protein in our samples by Western blotting and in-gel activity assays (Figure 4). In-gel activity assay showed that the activity of protein complex II was similar among the three samples, and the complex I activity assay showed that while the activity of the monomeric complex I was similar among the three samples (Figure 4a,b), the activities of the supercomplex composed of complex I and dimeric complex III were lower in N (Figure 4a). In terms of protein abundance, while the level of NAD9 protein was similar among the three samples, a higher protein level of NAD4 was observed

in the N sample in comparison to the other two samples. The N sample also had a higher protein abundance of COXIII, which could be due to a 2× increase in its mRNA abundance (Table 2).

**Figure 4.** In-gel activity of the mitochondrial respiratory complexes separated by BN-PAGE. Complex I activity (**a**), Complex II activity (**b**), and Western blotting (**c**). UR, uninoculated roots; SR, stripped roots; N, nodules. I, complex I; I + III2, supercomplex composed of complex I and dimeric complex III; II, complex II. For Western blotting, 10 μg mitochondrial proteins were separated by SDS-PAGE and blotted with antibodies. Three biological replicates were run and the results were reproducible. The representative images are shown in (**a**–**c**).
