Overexpression of NfsI in switchgrass had global effects on expression profiling of genes responsible for 2,4-DNT
To assess the global effects of engineering NfsI in switchgrass on expression profiling of genes responsible for 2,4-DNT, we first examined the transcriptome of root samples of control switchgrass plants that were exposed for 2 days in a liquid culture medium supplied with 20 mg·L-1 2,4-DNT. The RNA-seq analysis revealed that 2,4-DNT treatment induced 4,740 out of 85,522 (5.5%) genes differentially expressed in the control plants, with 3,002 genes upregulated and 1,738 genes downregulated, respectively (Fig. S6A, Table S2 ). The differentially expressed genes (DEGs) were subjected to Gene Ontology (GO) analysis, revealing that most enrichment pathways were related to the plasma membrane, oxidation-reduction process, toxin catabolic process, and secondary metabolites biosynthesis (Fig. S6B ).
We further compared the transcriptome profiling of treated and untreated control (CP-T and CP-UT) and transgenic (TP-T and TP-UT) switchgrass plants. The matrix analysis of the four sets of DEGs revealed that 732 genes clustered in intersection IV, which were upregulated in CP-T/CP-UT intersecting set but downregulated in TP-T/CP-T intersecting set (Fig. 6A ). Moreover, there were 353 genes in intersection VI, which were downregulated in CP-T/CP-UT intersecting set but upregulated in TP-T/CP-T intersecting set (Fig. 6A ). Our results suggest that overexpression of NfsI in switchgrass can alleviate expression changes of 22.9% of the genes responsible for 2,4-DNT stress (Fig. 6A ). These genes were categorized into biological processes, and mainly enriched stress response, metabolic biosynthesis, gene expression regulation and protein modification, and growth-development response (Fig. 6B ). It is noteworthy that most of the genes among 353 candidates were involved in plant morphological formation and regulation processes (Fig. 6C and Table S3 ), implying that NfsI may promote recovery from the damage resulted from 2,4-DNT stress. In contrast, most of the 732 candidate genes participated in the secondary metabolism process of the plant. This finding suggests that some metabolic genes involved in 2,4-DNT detoxification were activated in switchgrass (Fig. 6D and Table S3 ).