Enzymatic activity assay of NfsI at different pH values and NADPH concentrations
The effects of pH and NADPH on NfsI activity were determined by the methods described by Kim (Kim and Song, 2005). NfsI activities were examined after being incubated with reaction buffers at pH 4.0, 5.0, 6.0, 7.0, 8.0, 9.0. The reaction mixture contained 50 μg purified recombinant NfsI protein, 200 μM 2,4-DNT/DNTS, 200 μM NADPH, and 50 mM of sodium phosphate buffer in the final volume of 500 μL with a pH range of 4.0-9.0. NfsI activities were also detected after being incubated with reaction buffers containing different NADPH concentrations (50, 100, 200, 400, 800, 1600 μM). The reaction mixture contained 50 μg purified recombinant NfsI protein, 200 μM 2,4-DNT/DNTS, 50 mM sodium phosphate buffer (pH 6.0), and different NADPH concentrations in the final volume of 500 μL. After 30 min of incubation at 30 oC, the enzymatic reaction was terminated by 50 μL methanol, and the mixture was centrifuged at 17,000 g for 10 min. The supernatant was collected and filtered through 0.22 μm filter membrane for HPLC analysis.
Generation ofNfsI overexpressing transgenic switchgrass plants
The codon optimized NfsIsequence was cloned into pENTR vector infused with cMyc tag and then subcloned into pANIC6B vector via attL-attR recombinant reaction (Mann et al., 2012). The NfsI overexpression vector pANIC6B_NfsI _cMyc was finally transformed intoAgrobacterium tumefaciens strain EHA105 using the freeze-thaw method (Wise et al., 2006). The single genotype callus line induced from switchgrass cultivar Alamo was employed forAgrobacterium -mediated transformation, following the procedure as described by Wu (Wu et al., 2016). Hygromycin was used as the selectable reagent to generate NfsIoverexpressing transgenic switchgrass lines that were transplanted into soils and grown in the greenhouse. The control plants were generated with the pANIC6B empty vector.
Expression levels ofNfsI in transgenic switchgrass plants
The independent positive transgenic lines were identified by genomic PCR with specific hph and NfsI primers. The expected sizes of PCR products were 375 and 654 bp for hph and NfsI (Table S1). Stems at the E2 stage were collected from each plant and ground in liquid nitrogen. Approximate 200 mg stem samples were extracted for total RNA by TriZol extraction kits (TransGen Biotech, China) and subjected to reverse transcription with SuperMix (TransGen Biotech, China) after treatment with TURBO™ DNase I (Ambion, Austin, TX). The expression levels of NfsI were analyzed by quantitative real-time PCR (qRT-PCR). The primers used for qRT-PCR are listed in Table S1. The cycle thresholds were determined using a ABI PRISM 7900 HT sequence detection system (Applied Biosystems, Foster City, CA), and the data were normalized using the level of switchgrassPvUbq2 transcripts (GenBank accession No. HM209468).
Root length and ROS content
The two representative transgenic switchgrass lines, control, and wild type plants were micropropagated by node culture described by Alexandrova (Alexandrova et al., 1996). The well rooted uniform regenerated plantlets were subcultured in new 1/2 MS solid medium after their roots were cut off. Thereafter, the wild type plantlets were treated with different concentrations of 2,4-DNT (0, 2, 5, 10, 20, 40 mg·L-1) and DNTS (0, 2, 4, 8, 16 g·L-1), respectively. The control and transgenic switchgrass plantlets were further treated with 20 mg·L-1 2,4-DNT. The roots of switchgrass plantlets treated with 2,4-DNT and DNTS were photographed, and their length was measured after two weeks of growth. An enzyme-linked immunosorbent assay (ELISA) was employed for detecting ROS contents of switchgrass plantlets. Approximately 50 mg of the above fresh root samples were ground in the sodium phosphate buffer (0.01 M, pH 7.4). After centrifugation at 4,500 g, 4 °C for 20 min, the supernatants were collected for measurement of ROS contents following the protocol of ELISA kit (Shanghai Enzyme-linked Biotech, China). The DAB stain following the procedure was carried out as described by Johnston (Johnston et al., 2015).