Figure Legends
Fig. 1.M etabolites of 2,4-DNT and DNTS transformed by NfsI in vitro. HPLC analysis of metabolites of 2,4-DNT (A) and DNTS (B) transformed by the purified recombinant NfsI in vitro . The proposed pathway for the reduction of 2,4-DNT (C) and DNTS (D) by NfsI. Peak 1, 2/4-hydroxyl amino-4/2-mononitrotoluene (HAMNT); Peak 2, 2/4-amino-4/2-mononitrotoluene (AMNT); Peak 3, a novel compound derived from HAMNT; Peak 4, 4-hydroxyl amino-4/2-mononitrotoluene-3-SO3-(HAMNTS).
Fig. 2. Effects of pH value and NADPH concentration on NfsI enzymatic activity. The enzymatic activity of NfsI was examined at different pH values (A) and NADPH concentrations (B).
Fig. 3. Morphological and molecular characterization of transgenic switchgrass plants overexpressing bacterialNfsI. (A) Representative switchgrass plants from control (Ctrl) and transgenic (NfsI_OE-02 and -14) switchgrass plants are shown. (B) Quantitative real-time PCR analysis of NfsI transcript abundances in control and transgenic switchgrass plants. The control plants were generated with the pANIC6B empty vector. Stems at the R1 stage were collected. The expression level of switchgrassPvUbiq2 was used as the reference for normalization. Values are mean ± SE (n = 3).
Fig. 4. Effect of 2,4-DNT on root length and ROS content of control and transgenic switchgrass plants. (A) Root length of the control and transgenic switchgrass plantlets were measured after 2,4-DNT treatment. (B) In situdetection of ROS in roots of control and transgenic switchgrass plantlets by DAB staining. (C) ROS content in roots of the control and transgenic switchgrass plantlets were measured after 2,4-DNT treatment. The control and transgenic (NfsI_OE-02 and -14) switchgrass plantlets were exposed to 0, 2, and 20 mg·L-12,4-DNT. After 14 days of treatment, the photographs of roots stained with NBT were taken. Root length as well as ROS content were also measured. Each sample included six vegetatively propagated copies. Values are mean ± SE (n = 3).
Fig. 5. Uptake of 2,4-DNT and DNTS by control and transgenic switchgrass plants. (A) 2,4-DNT concentration in 1/2 MS liquid culture medium with time. The control and transgenic switchgrass plants at E2 stage were exposed to 1/2 MS liquid medium supplied with 20 mg·L-1 2,4-DNT, respectively. The blank was 1/2 MS liquid medium supplied with 20 mg·L-1 2,4-DNT without plants. (B) DNTS concentration in liquid culture medium with time. The control and transgenic switchgrass plants at E2 stage were exposed to 1/2 MS liquid medium supplied with 1000 mg·L-1 DNTS, respectively. The blank was 1/2 MS liquid medium supplied with 1000 mg·L-1 DNTS without plants.
Fig. 6. Transcriptome analysis of transgenic switchgrass plants responses to 2,4-DNT treatment. (A) Venn diagram and intersecting sets analysis for upregulated and downregulated genes among the control and transgenic switchgrass plants with and without 2,4-DNT treatment. CP-UT, control plants without 2,4-DNT treatment; CP-T, control plants with 20 mg·L-12,4-DNT treatment; 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-12,4-DNT treatment for 2 days. The horizontal bars represent different intersections of four sets of samples (I-VIII). The vertical bars represent the gene number of the intersection. (B) Gene ontology term enrichment of differentially expressed genes (DEGs) of intersection IV and VI. (C) KEGG pathway enrichment of DEGs in intersection VI. (D) KEGG pathway enrichment of DEGs in intersection IV. The control and transgenic (NfsI_OE-02 and -14) plantlets were exposed to 0 and 20 mg·L-12,4-DNT, respectively. After 2 days 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).