**2. Results**

## *2.1. RNA Sequencing and Di*ff*erentially Expressed Mitochondrial Genes*

To study the differences in mitochondrial transcripts between soybean nodules and uninoculated roots, we collected nodule (N) samples 28 days after rhizobium inoculation. Stripped roots (SR), main roots after the removal of nodules, and uninoculated roots (UR) of the same age were also harvested. We extracted total RNA from N, SR, and UR for RNA sequencing (RNA-seq) analysis (Table 1). The BioProject accession numbers of the RNA-seq data are PRJNA627909 (UR) and PRJNA626514 (N and SR), respectively. Here, we focused on mitochondrial gene expression and identified 93 gene transcripts (Supplementary Table S1). Besides transcripts encoding for hypothetical proteins, we identified 14 transcripts differentially expressed between N and UR, SR and UR, or SR and N (fold change > 1.5, *p*-value < 0.05 using Student's *t*-test). These include transcripts of cytochrome c biogenesis C/FN (*ccmC*, *ccmFN*), cytochrome c oxidase subunit III (*cox3*), NADH dehydrogenase subunit 1/2/4L/5 (*nad1*, *nad2*, *nad4L*, *nad5*), and some ribosomal proteins (Table 2). Compared to UR, eight transcripts, including six *nad* transcripts and two ribosomal protein transcripts, were significantly upregulated in SR, while the abundance of six transcripts including *cox3*, *nad1*, *nad5*, and three ribosomal protein transcripts was significantly higher in N. On the contrary, *ccmFN* was significantly down-regulated in N compared with UR. The abundance of four transcripts, including *cytochrome c biogenesis C*/*FN* (*ccmC*, *ccmFN*) and *ribosomal proteins L5* and *S14*, was lower in N than in SR, whereas the abundance of *cox3* and *rps12* transcripts was higher in N when compared with SR and UR.


**Table 1.** Summary of RNA-seq sequencing data.



 samples were sequenced average presented t-tests were out decreased (fold change > 1.5 or < 0.067, *p*-value < 0.05) are shown in bold.
