Results
Biodegradation metabolites of 2,4-DNT and DNTS transformed by NfsI in vitro
We initially conducted codon optimization forEnterobater cloacae NfsI to improve its expression in switchgrass (Fig. S1 ). Thereafter, we produced the recombinant NfsI in E.coli and examined the biodegradation metabolites of 2,4-DNT by LC-PDA/ESI-MS/MS. Our results showed that the NfsI transformed 2,4-DNT in 2/4-hydroxyl amino-4/2-mononitrotoluene (HAMNT, Peak 1) through a two-electron reduction (Fig. 1A, Fig. S2A, E ). Furthermore, HAMNT was reduced to 2/4-amino-4/2-mononitrotoluene (AMNT, Peak 2) or formed a novel compound by autonomous polymerization (Peak 3) (Fig. 1A, B, Fig. S2B, C, F, Fig. S3 ). In addition, we analyzed the biodegradation metabolites of DNTS and found that the NfsI only transformed DNTS into 4-hydroxyl amino-4/2-mononitrotoluene-3-SO3-(HAMNTS, Peak 4) (Fig. 1C, D, Fig. S2D, G ).
NfsI had higher catalytic efficiency toward2,4-DNT than DNTS in vitro
To evaluate the catalytic efficiency of NfsI toward 2,4-DNT and DNTS, we next examined NfsI enzyme kinetic characterization in vitro . Compared with DNTS, the NfsI had lower Kmtoward 2,4-DNT (256.9 ± 68.9 μM vs. 2814.1 ± 586.9 μM), while it had higher Vmax toward 2,4-DNT (183.0 ± 18.3 μM·s-1·mg-1 vs. 19.4 ± 1.6 μM·s-1·mg-1) (Table 1 ). Moreover, the NfsI had higher Kcat/Km toward 2,4-DNT (6.83 ± 0.29) than DNTS (0.07 ± 0.22), suggesting that the NfsI is highly efficient at detoxifying 2,4-DNT rather than DNTS in vitro (Table 1 ). Thus, we selected 2,4-DNT as the NfsI substrate for further studies.
Effects of pH value and NADPH concentration on NfsI enzymatic activity
Both pH value and NADPH concentration had significant effects on NfsI enzymatic activities in vitro . Therefore, we next decided to examine the optimum pH and NADPH concentration for NfsI toward 2,4-DNT. Our results showed that NfsI enzymatic activities initially increased and then decreased from pH 4.0 to 9.0. The optimum pH was around 6.0 at which the NfsI reached its highest activity (Fig. 2A ). We further studied the effect of NADPH concentration on NfsI enzymatic activity at pH 6.0. The NfsI activity was dramatically elevated as the NADPH concentration increased from 50 μM to 200 μM until a limiting rate was reached (Fig. 2B ).
Generation and identification ofNfsI overexpressing transgenic switchgrass plants
To improve uptake efficiency of 2,4-DNT and DNTS in plants, we overexpressed the codon optimizedNfsI in switchgrass plants. All transgenic lines were produced from a single genotypic embryogenic switchgrass callus line through Agrobacterium -mediated transformation, which excluded the potential influence of the genetic background of switchgrass on 2,4-DNT and DNTS tolerance as well as biodegradation. Eighteen independent positive transgenic switchgrass lines were identified by genomic PCR with NfsI specific primers. The control plants were generated with the pANIC6B empty vector which was used as the backbone for constructing NfsI -overexpressing vector. There was no obvious morphological or developmental difference between transgenic and control switchgrass plants in our greenhouse condition (Fig. 3A ).
Overexpression of NfsI in switchgrass enhanced plant tolerance against 2,4-DNT
We next decided to study if engineeringNfsI in switchgrass has potential for remediation of 2,4-DNT. Two transgenic lines NfsI_OE-02 and -14 with highestNfsI transcript abundance were selected for further investigation (Fig. 3B ). Firstly, we regenerated numerous plantlets from wild type, control, and transgenic switchgrass plants by immature inflorescence-derived callus cultures. These uniform plants were employed for examining the tolerance to 2,4-DNT. Our results showed that 2,4-DNT had significant impacts on the growth and development of wild type switchgrass plants. They exhibited obvious etiolation and wilting symptoms after 14 days when incubated with more than 5 mg·L-1 2,4-DNT (Fig. S4A ). In addition, as 2,4-DNT concentration increased from 2 to 40 mg·L-1, their root length decreased gradually, while the ROS content increased (Fig. S4B, C ). Furthermore, we measured the root length and ROS content ofNfsIoverexpressing transgenic switchgrass plants. Compared with control plants, the root length of transgenic switchgrass plants increased by 32.2-46.1%, and their ROS contents decreased by 21.8-35.6% at a high 2,4-DNT level (20 mg·L-1) (Fig. 4 ). Therefore, our results indicate that the NfsI overexpressing transgenic switchgrass plants have a higher capacity to tolerate 2,4-DNT than control plants.