Discussion
2,4-DNT is an important intermediate to produce polyurethane foams and
dyes. It is also found in the environment as a mixture with TNT since
2,4-DNT is a byproduct of TNT biosynthesis. Although DNT and TNT are
broadly used in civilian and military activities, both are highly toxic
xenobiotics that threaten human health and environmental safety.
Phytoremediation is a low-cost technique to clean up these nitroaromatic
compounds (Doty, 2008). Previous studies have suggested that engineering
bacterial NfsI in plants can detoxify TNT efficiently (Van
Dillewijn et al., 2008; Zhang et al., 2017; Hannink et al., 2001).
However, it remains unknown if this strategy could be employed for
detoxification of DNT and its derivatives. It had been shown thatEnterobater cloacae NfsI, a
member of the group B type I NRs, can convert both TNT and DNT in
vitro . A recent study also indicates that the cleanup rate of N.
tabacum plants for 2,4-DNT is significantly increased after inoculation
with 2,4-DNT degrading P. putida strains (Akkaya, 2020). These
works shed light on the effectiveness of genetically modifying plants
for DNT remediation. Switchgrass is a perennial tall grass with great
potential for phytoremediation of nitroaromatic compounds (Zhang et al.,
2019). Unfortunately, overexpression of Enterobater cloacae NfsIin switchgrass plants did not improve their resistance to TNT. Thus,
challenges remain for engineeringNfsI in switchgrass for the
detoxification of DNT and its derivatives.
It has been suggested that the expression levels of genes driven by 35S
promoter in switchgrass are lower than maize ubiquitin (ZmUbq) promoter
(Mann et al., 2011). Therefore, we re-overexpressedNfsI in switchgrass under
the control of ZmUbq promoter to increase NfsI transcript
abundance. Moreover, we employed a codon optimization technique to
improve transcription and translation efficiency of NfsI in
switchgrass since NfsI is a bacterial derived NR . Finally,
we generated NfsI overexpressing transgenic switchgrass plants
and improved their tolerance and detoxification to 2,4-DNT. The uptake
rate of 2,4-DNT increased from ~56% in the control
plants to ~94% in transgenic lines NfsI_OE-02 and -14
(i.e., a relative increase of 67%). Compared with 2,4-DNT, NfsI
exhibited little conversion capacity to DNTS neither in vitro norin vivo , implying that sulfonate has a crucial effect on nitro
reduction of 2,4-DNT. Therefore, the enzyme specificity to nitroaromatic
compounds should be fully considered before engineering NRs in
plants for detoxification of these highly toxic pollutants.
NfsI reduces TNT to 2-hydroxyl-4,6-dinitrotoluene (HADNT) through
two-electron reduction and then transforms HADNT to
2-amino-4,6-dinitrotoluene (ADNT). The reduction pathway of 2,4-DNT is
similar to that of TNT, and both HAMNT and AMNT were produced during
2,4-DNT reduction catalyzed by NfsI. Most strikingly, a novel compound
that likely formed by an autonomous polymerization was detected by
LC-PDA/ESI-MS/MS, suggesting HAMNT and its derivatives are the greatest
sources of 2,4-DNT reduction. Compared with 2,4-DNT, DNTS were only
transformed to HAMNTS by NfsI in vitro . Furthermore, the enzyme
kinetics analysis indicates that NfsI has much higher affinity and
catalytic efficiency for 2,4-DNT than DNTS. Given the fact that DNTS is
a sulfonate of 2,4-DNT, we speculated that the presence of the sulfonate
may block NfsI from binding to its substrate. Although the crystal
structures of many bacterial NRs have been determined (Chauviac et al.,
2012), the selectivity mechanism of NRs for their nitrated substrates
warrants further investigation.
Overexpressing NfsI in
switchgrass alleviated the impact of 2,4-DNT on root length and ROS
production significantly. This is similar with the previous observation
that overexpressing NfsI in Arabidopsis , tobacco, and
poplar can partially restore the inhibition of TNT on root elongation
and reduce ROS accumulation (Van Dillewijn et al., 2008; Hannink et al.,
2001). Although the morphological and physiological responses of
genetically modifying plants to
nitroaromatic pollutants have been
assessed widely, there are few studies involved in their transcriptome
responses. One study indicated that TNT treatment can induce more than
500 genes that are differently expressed (at least 5-fold change) in
wild type Arabidopsis roots. Serial analysis of gene expression
further reveals multiphase mechanisms of TNT detoxification inArabidopsis including
oxidative and reductive processes,
conjugation reactions, and sequestering within the vacuole and/or cell
wall (Reference, Ekman 2003). Genes involved in these detoxification
processes were also differentially expressed in our control switchgrass
treated with 2,4-DNT. It suggests that other plant species might employ
a similar mechanism for detoxification of nitroaromatic pollutants.
Furthermore, we found that overexpression of NfsI in switchgrass
was able to restore approximately
22.9% of DEGs induced by 2,4-DNT treatment by different extents. Among
them, genes involved in reactions of oxidation and reduction,
conjugation, and sequestering are worthy of functional characterization
in the future.
In conclusion, the bacterial NfsI was successfully overexpressed
in switchgrass, which is a multiple purpose crop used for forage and
biofuel production as well as phytoremediation.
Overexpression of NfsI in
switchgrass can remove 94.1% of 2,4-DNT from liquid culture medium
after 6 days which is 1.7-fold higher than that of control plants.
Moreover, our transcriptome analysis suggests that approximately 22.9%
of switchgrass DEGs induced by 2,4-DNT might be involved in
NfsI-mediated detoxification in switchgrass. Therefore, these genes are
potential candidates for deciphering molecular mechanisms underlying
switchgrass responses to nitroaromatic compounds. Further investigation
will lead to more novel targets being discovered and engineered to
improve switchgrass tolerance and detoxification to nitroaromatic
pollutants in the future.