Table S2. Overview of differentially expressed genes between control and transgenic switchgrass plants.
CP-UT, control plants without 2,4-DNT treatment; CP-T, control plants with 20 mg·L-1 2,4-DNT treatment for 2 days; TP-UT, transgenic plants (NfsI_OE-02 and -14) without 2,4-DNT treatment; TP-T, transgenic plants (NfsI_OE-02 and -14) with 20 mg·L-1 2,4-DNT treatment for 2 days.
Table S3. Expression profiles of two intersections (IV and VI) involved in NfsI-mediated detoxification of 2,4-DNT in switchgrass.Fig. S1. Alignmentof nucleotide sequences of the wild type NfsI(WT_NfsI) and codon-optimized NfsI (CO_NfsI). The WT_NfsI sequence (GenBank accession No. M6308.1) was retrieved from the Enterobacter cloacae genome sequence. The nucleotide sequence of WT_NfsI was optimized based on the codon preference of switchgrass and the CO_NfsI was synthesized.
Fig. S2. UV-Vis and mass spectra of intermediates of 2,4-DNT transformed by NfsI in vitro. (A-D) The UV-Vis spectra of peak 1-4. (E-G) The mass spectra of peak 1, 2, and 4.
Fig. S3. Autoformation of peak 3 from peak 1. (A) HPLC analysis of peak 3 autoformation with time. (B) The concentration of peak 1 and 3 with time. Peak 1 (HAMNT) was produced from the NfsI-mediated reduction reaction and purified by HPLC.
Fig. S4. Effect of 2,4-DNT on root length and ROS content of wild type switchgrass plants. (A) The wild type switchgrass plantlets were grown on MS0 medium supplied with various concentrations of 2,4-DNT for 14 days. (B) Root length of the wild type switchgrass plantlets were measured after 2,4-DNT treatment. (C) ROS content of the wild type switchgrass plantlets were measured after 2,4-DNT treatment.
Fig. S5. Impact of 2,4-DNT on root growth and development.Roots morphology of control (A) and transgenic (B) switchgrass plants after 6 days incubation with 20 mg·L-1 2,4-DNT.
Fig. S6.Transcriptome analysis of control switchgrass plants responses to 2,4-DNT treatment. Analysis of volcano plots (A) and gene ontology term (B) of differentially expressed genes (DEGs) between control switchgrass plants with and without 2,4-DNT treatment. The control switchgrass plantlets were exposed to 20 mg·L-1 2,4-DNT. After 2 days of treatment, total RNAs were extracted and subjected to transcriptome analysis through RNA-sequencing. Each sample included three vegetatively propagated copies. Values are mean ± SE (n = 3).