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).