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).