Introduction
Dinitrotoluene (DNT) is a
nitroaromatic compound extensively used in manufacturing munitions,
polyurethane foams, automobile
airbags, and other important chemical products (Rickert et al., 1984).
Due to a history of long-term manufacturing, civilian and military use,
and waste steam leaks, DNT has entered the soil and water, which
threatens the health of residents and the environment around DNT
manufacturing and processing facilities. DNT exists as six isomers, with
2,4- and 2,6-DNT being the major forms that are moderately to highly
toxic to humans, animals, and plants (Gong et al., 2003). The mixture of
2,4- and 2,6-DNT has been classified as a Class B2 carcinogen to humans
and animals by the United States Environmental Protection Agency
(EPA). Among them, 2,4-DNT is
predominantly employed as an intermediate for production of polyurethane
(Lent et al., 2012). It is also a byproduct from the synthesis of
2,4,6-trinitrotoluene (TNT), which is one of the most widely used
explosives used for civilian and military purposes around the world. In
addition, 2,4-dinitrotoluene-3-sulfonate (DNTS) produced from TNT
refining is a major toxic component in TNT red water and its
contaminated soils (Tsai, 1991). Both 2,4-DNT and its sulfonate have
long-term environmental persistence and high-energetic toxicity and
mutagenicity to most organisms (Gilbert, 1977). Thus, they have been
considered as the priority pollutants that must be removed from soil and
groundwater contaminated by manufacturing and processing of polyurethane
and TNT.
Biological remediation including phytoremediation and microbial
remediation is a safe, economic, and highly efficient strategy for
degradation of
nitroaromatic
compounds. Previous studies have suggested that the detoxification
pathway of DNT and other nitroaromatic explosives in plants might go
through three phases: functionalization (hydrolysis, oxidation, and
reduction), transformation (conjugation by glycosyl transferases or
glutathione transferases), and compartmentalization (transportation to
cell wall or vacuole) (Rai et al., 2020; Rao et al., 2009). However, the
detailed molecular mechanism of
detoxification of these
nitroaromatic compounds remains
elusive in plants. Previous studies have suggested that the high
toxicity of nitroaromatic compounds to plants results from the
predominant accumulation of excessive reactive oxygen species (ROS) in
plant tissues, which damages normal physiological activities in plant
cells (Brentner et al., 2010; Johnston et al., 2015). Deficiency ofArabidopsis MONODEHYDROASCORBATE REDUCTASE 6 (MDHAR6) can reduce
the production of ROS in the mitochondria and confer Arabidopsishigh tolerance to TNT (Johnston et al., 2015). In addition,
overexpression of UDP-glycosyltransferases (UGT743B4 andUGT73C1 ) and glutathione transferases (GSTU24 and
GSTU25 ) in Arabidopsis leads to accumulation of the conjugation
products of TNT and reduces its cytotoxicity (Gandia-Herrero et al.,
2008; Gunning et al., 2014). Although some efforts have been made to
alleviate the pollution of TNT by phytoremediation over recent decades,
only one bioengineering work has been made for DNT detoxification by
expressing a cyanobacterial flavodoxin in tobacco plants (Tognetti et
al., 2007).
In contrast, DNT detoxification microbes and their degradation mechanism
have been investigated extensively. Many microbes includingEnterobacter cloacae ,Saccharomyces sp , Cadida sp , and white-rot fungus
participate in DNT degradation, which provide enriched sources for
identification and characterization of genes involved in detoxification
of these nitroaromatic compounds (Koder and Miller, 1998; Ziganshin et
al., 2007; Kist et al., 2020). Previous studies have suggested that a
dioxygenase is responsible for the first step of the DNT degradation
pathway. However, this dioxygenase is encoded by four genes
(dntAa , Ab, Ac, Ad ) in Burkholderia cepacian R34,
which makes it difficult to be applied for plant bioengineering (Johnson
et al., 2002). Microbial type I nitroreductase (NR) is an
oxygen-insensitive flavoprotein that catalyzes the NAD(P)H-dependent
reduction of nitro groups to hydroxylamino and/or amino groups on
nitroaromatic compounds. These type I NRs consist of two main groups,
NfsA (group A) and NfsB (group B)
(Roldán et al., 2008). Moreover, members of
NfsB can reduce both 2,4- and
2,6-DNT (Williams et al., 2019), while members of NfsA only convert
2,4-DNT (Rich et al., 2018). NfsI is a member of NfsB NR identified fromEnterobacter cloacae (Zajc, 1999). The recombinant NfsI protein
can convert both TNT and DNT in vitro (Bryant et al., 1991).
Overexpression of NfsIsuccessfully improves the degradation efficiency of TNT in transgenic
poplar, tobacco, and wheatgrass (Brentner et al., 2010; Zhang et al.,
2017b; Zhang et al., 2019). These findings shed light on
phytoremediation through transgenic plants engineered with bacterial
nitroaromatic compound degradation
genes. However, it is still unknown if the plants engineered withNfsI could detoxify DNT and its sulfonates.
Switchgrass (Panicum virgatum L.) is a perennial C4 tall grass
that has been used as lignocellulosic feedstock for forage and biofuel
production. Switchgrass can increase soil organic carbon and facilitate
a larger microbe population in margin lands. Moreover, switchgrass is
well adapted to various soil types with excellent drought, cold, saline,
and heavy metal tolerance (Song et al., 2018). Therefore, switchgrass
has great potential for
phytoremediation because of its extensive root system, vigorous growth,
high ability to stress tolerance, and low-input requirements (Rai et
al., 2020). A recent study has shown that ectopic expression of
bacterial flavodoxin-cytochrome P450 XplA coupled with flavodoxin
reducatase XplB in switchgrass can dramatically improve the
removal efficiency of RDX in transgenic plants (Zhang et al., 2017a).
Unfortunately, overexpression of the bacterial NfsI in
switchgrass cannot degrade TNT due to low transcription of NfsI(Zhang et al., 2017a).
Here, we synthesized a codon optimized bacterial NfsI for
overexpression in switchgrass and studied its function in DNT and DNTS
detoxification for the first time. The transgenic switchgrass withNfsI overexpression significantly alleviated the 2,4-DNT-induced
root growth inhibition and reduced ROS content.
The removal efficiency for DNT
achieved 94.1% in NfsI overexpressing transgenic switchgrass
plants, which was 1.7-fold higher than that of control plants. In
contrast, overexpression of NfsI in switchgrass barely improved
the removal capacity for DNTS, supporting the in vitro enzyme
kinetics analysis that suggests that the recombinant NfsI has
approximate 100-fold higher catalytic efficiency for 2,4-DNT than DNTS.
Furthermore, overexpression of NfsI in switchgrass partially
alleviated the impact of 2,4-DNT on expression profiling of genes
involved in plant detoxification. Our work suggests that engineeringNfsI in plants may have great application potential for DNT
phytoremediation in the future.