JBC Papers in Press. Published on May 19, 2020 as Manuscript REV120.

013572
The latest version is at https://www.jbc.org/cgi/doi/10.1074/jbc.REV120.013572

Mechanisms of evolved herbicide resistance

Todd A. Gaines,1 Stephen O. Duke,2* Sarah Morran,1 Carlos A. G. Rigon,1 Patrick J. Tranel,3 Anita
Küpper,4 and Franck E. Dayan1

From the 1Agricultural Biology Department, Colorado State University, Fort Collins, CO 80523, USA;
2
National Center for Natural Products Research, School of Pharmacy, University of Mississippi, 38677
USA; 3Department of Crop Sciences, University of Illinois, Urbana, IL 61801, USA; 4Bayer AG,
CropScience Division, Frankfurt am Main, Germany

Running title: Mechanisms of evolved herbicide resistance

*To whom correspondence should be addressed: Stephen O. Duke: National Center for Natural Products
Research, School of Pharmacy, University of Mississippi, 38677 USA; sduke@olemiss.edu; Tel. +1
(662) 832-1594.

Keywords: Cytochrome P450, glutathione-S-transferase, herbicide metabolism, reduced translocation,

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target-site resistance, non-target-site resistance, cross-resistance, multiple resistance, plant evolution,
selection pressure

ABSTRACT the individual level to produce higher resistance

The widely successful use of synthetic herbicides
over the past 70 years has imposed strong and
widespread selection pressure, leading to the
evolution of herbicide resistance in hundreds of
weed species. Both target-site resistance (TSR) and
non-target-site resistance (NTSR) mechanisms
have evolved to most herbicide classes. TSR often
involves mutations in genes encoding the protein
targets of herbicides, affecting the binding of the
herbicide either at or near catalytic domains or in
regions affecting access to them. Most of these
mutations are non-synonymous single-nucleotide
polymorphisms, but polymorphisms in more than
one codon or entire codon deletions have also
evolved. Some herbicides bind multiple proteins,
making TSR mechanisms more difficult to evolve.
Increased amounts of protein target, by increased
gene expression or by gene duplication, is an
important, albeit less common, TSR mechanism.
NTSR mechanisms include reduced absorption or
translocation and increased sequestration or
metabolic degradation. The mechanisms that can
contribute to NTSR are complex and often involve
genes that are members of large gene families. For
example, enzymes involved in herbicide
metabolism–based resistances include cytochromes
P450, glutathione-S-transferases, glucosyl and
other transferases, aryl acylamidase, and others.
Both TSR and NTSR mechanisms can combine at
levels. The vast array of herbicide-resistance
mechanisms for generalist (NTSR) and specialist
(TSR and some NTSR) adaptations that have
evolved over a few decades illustrate the
evolutionary resilience of weed populations to
extreme selection pressures. These evolutionary
processes drive herbicide and herbicide-resistant
crop development and resistance management
strategies.
Plants that are not wanted at a particular time
and/or place (weeds) have been managed mostly
with synthetic herbicides for more than 70 years.
Before that time, weeds were largely controlled
with laborious manual weeding and often
environmentally damaging tillage. Adoption of
synthetic herbicides reduced the cost and
increased the efficacy of weeding, thereby
contributing to the yield increases and efficiency
of agriculture seen since the middle of the last
century. However, as with antibiotics, the utility of
synthetic weed killers is being threatened by wide-
spread evolution of resistance to most chemical
classes of herbicides that act on most of the more
than 25 molecular targets of current commercial
herbicides (1).
The main goal of this review is to provide an
update on the rapidly evolving topic of
mechanisms of evolved herbicide resistance in

1
weeds, as there has been no recent comprehensive
review on this topic. We hope that this review will
inspire plant molecular biologists and biochemists
to determine more clearly how these resistance
mechanisms evolve and the biochemical and
physiological changes in weeds imparted by
resistance mutations. Such information will be
useful in resistance management and in design of
herbicide molecules for which evolution of
resistance is more problematic for weeds. We
provide discussions of the implications of
herbicide resistance mechanisms for the
development of new herbicides and herbicide-
resistant crops.
We summarize the wide array of resistance
mechanisms that weeds have evolved to survive
the intense selection pressure imparted by
commercial herbicides. Herbicide resistance
mechanisms can be broadly divided into two
categories, referred to as target-site resistance
(TSR) mechanisms and non-target-site resistance
(NTSR) mechanisms. Herbicide efficacy is
generally dependent on how much of the herbicide
enters a plant cell and how long its active form
remains available to interact with its site of action
(also called target-site). A full understanding of
the mechanism of resistance to a herbicide requires
understanding that herbicide’s mechanism of
action (MOA). MOA of herbicides is not
discussed in detail in this review, but we have
provided a summary of the molecular targets and
MOAs of the herbicides mentioned in this review
(Table 1). There are 26 molecular target sites of
the more than 260 commercial herbicide active
ingredients that are recognized by the Herbicide
Resistance Action Committee, an industry
organization that monitors herbicide resistance
(2). Of these target sites, resistance has evolved
globally (in 92 crops in 70 countries) to 167
herbicides representing about 23 of these targets,
with 512 weed species evolving resistance to one
of more herbicides (3). This review will not
provide an encyclopedic elaboration for each of
these cases but will give examples of different
mechanisms of resistance that often cross
herbicide classes.
NTSR mechanisms include all mechanisms
that reduce the concentration of active herbicide
remaining available to interact with the target site
protein, as well as mechanisms that allow the plant
to cope with inhibition of the target site. NTSR
mechanisms include reduced herbicide uptake and
translocation, increased herbicide sequestration,
and enhanced degradation or metabolism of the
herbicide to less-toxic compounds. On the other
hand, TSR mechanisms alter the amino acid
sequence and/or expression level of the target
enzyme, reducing the herbicide’s ability to inhibit
the enzyme or requiring a greater herbicide
concentration to achieve adequate inhibition.
Under intense selection pressure from highly
effective herbicides, all possible mechanisms
conferring a greater chance of survival and
reproduction to the individual may be selected.
More than one mechanism may be operating to
confer resistance, including combinations of TSR
and NTSR mechanisms. Several resistance
mechanisms can co-exist within a species, within
a population, and even within a single individual.
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Different resistance mechanisms combine through
cross-pollination between individuals. Species
with high levels of cross-pollination are more
likely to accumulate diverse resistance
mechanisms (to a single herbicide, or to multiple
herbicides), and this process can occur more
rapidly than in self-pollinated species.
Target site mechanisms
A single nucleotide mutation in the gene
encoding a protein bound by a herbicide can result
in a single amino acid change, disrupting the
ability of the herbicide to bind to the protein
without disabling the enzyme function. Generally,
there are few amino acids in or near the herbicide
binding-site of most target-site proteins where an
amino acid substitution will result in TSR. Most
target-site mutations occur in or near the
herbicide-binding site, but some mutations occur
elsewhere in the protein structure. Target-site
mutations are identified by the amino acid and its
position in the protein, numbered from the
protein’s start codon. In some cases, the mutation
can confer very high-level resistance, and in other
cases, the mutation confers lower level (but
significant) resistance. Some target-site mutations
reduce normal enzymatic function, and other
mutations retain nearly full enzymatic function. In
addition to single nucleotide substitutions, whole-
codon deletions can also reduce herbicide binding
to the target-site enzyme. It is important to note
that the same molecular mechanism (e.g., a single
2
nucleotide polymorphism (SNP) leading to an
amino acid change) forms the basis of resistance
for many different herbicide sites of action.
TSR mechanisms are specialist mechanisms,
specific to a single site of action. Whether a
specific target-site mutation that confers resistance
to a given herbicide also confers resistance to
different chemical families within the same site-
of-action group varies, depending on how the
specific herbicides interact with the target protein.
TSR can also be due to increased expression of the
target-site gene, producing more enzyme than can
be substantially inhibited by typical herbicide
application rates. Increased gene expression can be
due to regulatory changes increasing transcription
and/or increased genomic copy number of the
target-site gene, also resulting in increased
transcription.
Single nucleotide polymorphism
Non-synonymous SNPs imparting resistance
to a herbicide target site is the most common
mechanism of TSR. These mutations may be
within or in the proximity of the catalytic domain
of an enzyme and affect a herbicide’s ability to
compete for the binding of a substrate, or these
mutations may affect other domains of enzymes
and proteins.
Mutations affecting herbicide binding
The first discovered target-site mutation was
for the photosystem II (PSII)-inhibiting
herbicides, which compete with plastoquinone for
binding on the D1 protein encoded by the psbA
gene and thereby inhibit PSII electron transport
(4). Amino acid substitutions in the psbA gene
typically confer high-level resistance to a single
chemical family, but not to herbicides from other
families or groups. For example, the single amino
acid change Ser264Gly confers high level
resistance to the triazine herbicides in numerous
species around the world (e.g., (5)) but moderate
to no resistance to the other families of PSII
inhibitors, including the triazinones, which are in
the same group as the triazines. The substitution of
Gly for Ser at position 264 prevents triazine
binding, but also compromises plastoquinone
binding and impairs photosynthesis, resulting in a
strong fitness penalty (4). A Ser264Thr
substitution confers resistance to triazines and
ureas, but not to the nitrile or triazinone families.
Additional psbA resistance-imparting mutations
include Val219Ile, Asn266Thr, Phe255Ile, and
Ala251Val.
The dinitroaniline herbicides (such as
trifluralin and oryzalin) bind to plant tubulin
protein and disrupt meristem development by
depolymerizing microtubules (1). The first
reported mutation affecting binding of
dinitroaniline herbicides to plant microtubules was
found in an -tubulin gene transcript from
goosegrass (Eleusine indica) that encoded a
Thr239Ile substitution (6). This substitution
conferred resistance to trifluralin and oryzalin
when transformed in maize, demonstrating that
this mutation was the molecular basis of
dinitroaniline herbicide resistance (6).
Substitutions of Val202Thr (7) and Arg243 to Met
or Lys (8) have been reported for dinitroaniline
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resistance in annual ryegrass (Lolium rigidum). A
substitution of Met268Thr has also been identified
in E. indica, and both Thr239Ile and Leu136Phe
have been identified in green foxtail (Setaria
viridis) (9), demonstrating that substitutions at
multiple amino acid positions in a target site gene
can confer resistance, depending on the molecular
structure of the target site protein and where the
herbicide binds to the protein.
Acetyl CoA carboxylase (ACCase; EC
6.4.1.2) is a key enzyme for fatty acid biosynthesis
pathways. The plastidic form of ACCase in
grasses is inhibited by the
aryloxyphenoxypropionate (APP),
cyclohexanedione (CHD), and phenylpyrazoline
(PPZ - pinoxaden is the only member) herbicide
chemical families (1). Eight mutations have been
reported, all contained within the carboxyl
transferase domain of the ACCase enzyme.
Known mutations occur at seven positions:
Ile1781Leu or Val, Trp1999Cys or Leu,
Trp2027Cys, Ile2041Asn or Val, Asp2078Gly,
Cys2088Arg, and Gly2096Ala or Ser (10).
Substitutions at positions Ile1781 and Asp2078
have been reported most often, and these
substitutions confer resistance to APP, CHD, and
PPZ herbicides. Ile1781 is within the binding site
for the three ACCase herbicide chemical families,
explaining the resistance pattern to all three
classes. Asp2078 is not within the binding site but
occurs next to Ile1781, so the substitution of Gly
for Asp at 2078 can cause a large effect on
resistance level due to the substantial change in the
3
structure of the binding site. Mutations at positions
Ile2041 and Gly2096 confer resistance only to
APP herbicides, while mutations at Trp2027 can
confer resistance to APP and PPZ herbicides (11).
Somatic mutations
Target site mutations have also been identified
in the invasive aquatic weed Hydrilla verticillata
conferring resistance to fluridone, an inhibitor of
phytoene desaturase (PDS; EC 1.3.99.31), an
ezyme essential for carotenoid synthesis (12). H.
verticillata is a dioecious plant (male and female
flowers are on separate plants) and only the female
form was introduced to the United States.
Consequently, resistance has evolved through the
selection of a mutation in meristematic tissue that
was able to regenerate into whole plants and
spread to other lakes (13). The specific mutations
in PDS are Arg304 to Ser, Cys, or His. These
mutations have arisen through somatic variation,
with the consequence that H. verticillata
populations within a confined body of water
normally contain a single resistance mutation. The
three mutations confer cross-resistance to the
PDS-inhibiting herbicide norflurazon, but also
confer negative cross-resistance (i.e., increased
sensitivity) to three different PDS-inhibitors
(beflubutamid, picolinafen, and diflufenican) (14).
This finding further emphasizes that the effects of
amino acid substitutions are dependent on the
target site protein structure and vary across
different herbicide site of action groups.
An interesting aspect of somatic mutations
imparting resistance to herbicides is that the lack
of sexual reproduction may make the resistance
trait more stable in a population than in species
dependent on sexual reproduction due to a lack of
genetic recombination. Consequently, the three
distinct populations with variable resistance to
fluridone have remained in the same abundance in
some of these lakes despite an 8-year period with
no fluridone selection pressure (15). While the
aquatic environment in which this case evolved
may be partly responsible for this to occur, some
terrestrial weeds also can reproduce vegetatively,
suggesting the possibility of somatic mutations
creating herbicide resistance in these species.
Mutations affecting access to target site
Acetolactate synthase (ALS; EC 2.2.1.6) (also
called acetohydroxyacid synthase or AHAS) is a
key enzyme in the synthesis of branched chain
amino acids. Several chemical families have ALS
as their site of action, including the sulfonylureas
(SUs) and imidazolinones (IMIs). Twenty-one
combinations of weed species by ALS inhibitor
resistance-endowing amino acid substitutions
have been reported to date, with 127 total unique
(by species) occurrences of the different
substitutions (3). Resistance-imparting
substitutions at Pro197 have been reported most
frequently, followed by mutations at Trp574. As
with ACCase target site mutations, some of the
ALS mutations confer very high-level resistance,
and the resistance spectrum across chemical
families varies by mutation. General patterns are
that the Trp574 mutation confers resistance to SUs
and IMIs, the Ser653 mutation confers resistance
to IMIs but not SUs, and the Pro197 mutation
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confers resistance to SUs but not IMIs (depending
on the specific amino acid substitution; some
Pro197 mutations do confer resistance to IMIs).
The known ALS resistance mutations do not occur
at the substrate binding site, but instead occur at
amino acid positions where the ALS herbicides
can bind to and block an access channel within the
enzyme through which the ALS substrates must
move (16). This is the biochemical reason why
ALS mutations generally have little effect on the
normal catalytic activity, in contrast to mutations
in the psbA gene that significantly affect normal
biochemical function. The SUs and IMIs bind to
partially overlapping sites in the ALS enzyme, but
have different modes of binding (Figure 1). This is
important from an evolutionary perspective,
because rotating different ALS chemical families
in the field will likely select for the same mutation,
while rotating among different PSII-inhibiting
chemical families may select for different
mutations. In the case of ALS-inhibiting
herbicides, rotations to other ALS-inhibiting
chemical families provide the same selection
pressure from an evolutionary perspective and are
not functional in slowing the evolution of
resistance. In support of this concept, resistance to
ALS-inhibiting herbicides has occurred rapidly
following introduction of these herbicides.
Rotations among different PSII-inhibiting
chemical families may in some cases slow the
evolution of TSR, because the amino acid
substitution conferring resistance to one family
does not confer resistance to another family.
4
 The rarer a mutation is within a population
prior to herbicide selection, the longer it will take
for the mutation to be selected and reach a high
frequency within the population. A study of a
herbicide-susceptible L. rigidum population found
that SU target site resistance allele frequency
within previously untreated populations was as
high as 1.2 × 10-4, and IMI target site resistance
allele frequency was as high as 5.8 × 10-5 (17).
Similarly, a mutagenesis experiment in
Arabidopsis found ALS resistance (SU and IMI)
at a frequency of 3.2 × 10-5 in progeny of M1 lines
(first generation after chemical mutagenesis) and
with no detectable glyphosate resistance in
250,000 M1 progeny screened (18). These
experimental results are corroborated by the
relatively fast initial evolution of resistance to
ALS-inhibitors following their introduction, and
the relatively slower initial evolution of resistance
to glyphosate following its introduction (3).
The relatively higher number of mutations
imparting resistance to various classes of ALS-
inhibiting herbicides in comparison to other
herbicide groups is due to the fact that these
molecules do not compete with the substrate for
the catalytic domain of the enzyme. Instead, they
block the opening of the channel leading to the
catalytic domain (Figure 1). Consequently,
mutations imparting resistance often have no
impact on the kinetic properties of ALS.
Glyphosate inhibits 5-enolpyruvylshikimate-
3-phosphate synthase (EPSPS; EC 2.5.1.19), a key
enzyme in the shikimate pathway, and mutations
in the EPSPS gene have been reported in weeds.
Most described EPSPS target site mutations in
weeds are located at the Pro106 residue, using the
Arabidopsis numbering system, from the start of
the mature enzyme (reviewed by 19). Known
resistance mutations in weeds include Pro106 to
Ser, Thr, Ala, or Leu (19). Glyphosate inhibits
EPSPS by competing with the normal substrate
phosphoenolpyruvate (PEP) for binding to the
enzyme (Figure 2). Glyphosate binding is almost
irreversible, so once bound, that unit of EPSPS is
blocked. The Pro106 residue is not directly
involved in a molecular interaction with
glyphosate or PEP, but it provides part of the
molecular structure at the active site (20), and
changing the Pro106 to a different residue changes
the spacing in the active site. This increases the
inhibitory constant (Ki, the concentration of
inhibitor required to decrease reaction rate to half
of the uninhibited value) for glyphosate. This
increase in Ki for EPSPS with the mutation is the
reason for resistance, as more glyphosate is
required to inhibit an equivalent amount of
enzyme; however, the structural change also
increases the Michaelis constant for PEP (K m, the
substrate concentration required for effective
catalysis to occur).
An EPSPS mutant with a higher Km requires
higher PEP concentration to achieve the same
reaction velocity as the wild-type EPSPS with
normal Km. This means that the selectivity factor
for PEP binding over glyphosate binding is
affected, due to changed affinity for PEP, and a
higher PEP concentration is necessary under
normal conditions to maintain the same reaction
rate. This effectively reduces the catalytic activity
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of EPSPS, a possible reason why this target site
mutation may be rarer within populations
compared to ALS target site mutations (which do
not generally affect the Km or catalytic activity of
ALS). A mutation at Thr102Ser conferred
glyphosate resistance in tridax daisy (Tridax
procumbens) (21). This mutation imparted lower
affinity to glyphosate, but also higher affinity for
PEP.
Multiple nucleotide polymorphisms
A concomitant mutation, generated by point
mutation of the maize EPSPS and commercialized
as GA21 glyphosate-resistant maize (22), of both
Thr102Ile and Pro106Ser (TIPS) resulted in
structural changes that retained high affinity for
PEP and made the enzyme insensitive to
glyphosate inhibition (23) (Figure 2). The first
known naturally-occurring case of this TIPS
double mutation evolved in E. indica (24), in
which the Pro106Ser had previously evolved. This
double mutation confers a much higher level of
glyphosate resistance, and maintains the affinity
for PEP at a similar level as the wild-type enzyme
(25), although the TIPS mutation was found to
have a high fitness cost in E. indica (26). The
double TIPS mutation has also been reported in
hairy beggarticks (Bidens pilosa) from Mexico
(27). A double Thr102Ile and Pro106Thr (TIPT)
mutation was found in greater beggarticks (Bidens
subalternans) from Brazil, a tetraploid species in
which the TIPT mutation was found in only one of
the two genomes (28). Recently, a triple amino
5
acid substitution in EPSPS was found in smooth
pigweed (Amaranthus hybridus) in Argentina,
with a Thr102Ile, Ala103Val, and Pro106Ser
(TAP-IVS) allele conferring high resistance to
glyphosate (29,30). This stepwise evolution of
mutations is an excellent example of how
herbicide resistance is rapid evolution, as many
gene families over evolutionary time have evolved
by this same process involving the incremental
accumulation of mutations that alter and improve
enzymatic activity and efficiency. When a
herbicide with a single site of action is used
repeatedly, evolutionary processes leading to
higher resistance levels and more efficient
resistance mechanisms are expected to occur.
Receptor/Co-receptor interactions
Synthetic auxin herbicides mimic the
endogenous auxin hormone indole-3-acetic acid
(IAA) and deregulate growth and development
processes. Synthetic auxins bind to a receptor
protein (auxin F-box, or AFB) and to a co-receptor
protein (Aux/IAA) to deregulate gene expression
controlling plant growth, as well as binding to
other auxin-binding proteins. In the case of
synthetic auxin herbicides, mutations that reduce
herbicide binding to auxin-binding proteins, AFB
proteins, or Aux/IAA proteins (the sites of action
for synthetic auxins) could have roles in conferring
resistance in weeds. For example, assays of auxin-
binding protein preparations isolated from auxinic
herbicide-susceptible and resistant wild mustard
(Sinapsis arvensis) found similar binding for the
normal substrate indole-3-acetic acid (IAA), but
differences in binding were found for several
auxinic herbicides, and these differences
correlated with the whole-plant resistance
phenotypes (31).
Multiple auxin-binding proteins interact with
native auxins and synthetic auxin herbicides, and
evidence indicates that some mutations confer
resistance specifically to certain chemical families
of synthetic auxins. For example, mutations in the
Arabidopsis TIR1 homolog, an AFB protein,
confer resistance to the picolinate class of
synthetic auxins such as picloram but not to 2,4-D
(32).
Aux/IAA transcriptional repressors are co-
receptors for synthetic auxin herbicides and part of
the target site complex. A double-nucleotide TSR
substitution was discovered in the IAA16 gene of
kochia (Bassia scoparia), changing a GGT (Gly)
at amino acid position 127 to an AAT (Asn) (33).
This double mutation is located in the conserved
degron region II of the Aux/IAA protein and
confers resistance to dicamba.
From an evolutionary perspective, rotating
different synthetic auxin herbicide chemical
families may be an effective resistance
management practice, as TSR mechanisms may be
highly specific to certain chemical families and
may not necessarily confer resistance across
different families.
Codon deletion affecting topology of target site
A codon deletion is the removal of three
nucleotides from the coding sequence of a gene.
The coding frame is unaffected by a codon
deletion, and a single amino acid is removed from
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the protein encoded by the allele carrying the
codon deletion. Codon deletions conferring
herbicide resistance are rare relative to single
nucleotide substitutions, with the only known
example to date applying to herbicides that inhibit
protoporphyrinogen oxidase (PPO; EC 1.3.3.4), a
key enzyme in chlorophyll and heme biosynthesis.
Common waterhemp (Amaranthus
tuberculatus) resistant to PPO inhibitors
(including lactofen, fomesafen, and others) had a
three-nucleotide deletion in its PPX2 gene, which
encodes a PPO enzyme (34). It is thought that the
three-nucleotide deletion was fostered by its
occurrence within a region containing bi-repeats
of three nucleotides. These short simple repeat
(SSR) regions are typically associated with
insertion and deletion events. Most plant species
do not have a SSR region in the homologous
location of PPX2, suggesting they are not
predisposed for the same mutation (35). Palmer
amaranth (Amaranthus palmeri), however, is one
of the few weeds with a homologous SSR region
(36) and, in fact, resistance to PPO inhibitors due
to the same codon deletion was subsequently
documented in this species (37). The codon
deletion, which specifically results in removal of a
glycine at position 210 was functionally validated
to confer resistance using a transgenic E. coli
system (34) and also has been subjected to
biochemical and structural analysis (38). Deletion
of glycine 210 is proposed to partially unravel an
helix adjacent to the PPO active site, raising the
Ki for the herbicide and enlarging the active site
6
cavity (Figure 3). This structural change confers
broad cross resistance to PPO-inhibiting
herbicides at both the enzyme and whole-plant
level, although resistance magnitudes vary among
herbicides by up to 10-fold (39). Efforts are
underway to develop new PPO-inhibiting
herbicides that overcome the Gly210 PPO deletion
(40). To date, a codon deletion has not been
reported to confer herbicide resistance in any weed
species other than A. tuberculatus and A. palmeri.
Increased expression of target site genes
Up-regulation
Baerson et al. (41) found elevated EPSPS
expression (2.5 to 3 times higher) in glyphosate-
resistant L. rigidum. The EPSPS protein extracted
from resistant and susceptible individuals was
equally sensitive to glyphosate inhibition, but
basal enzyme activity was higher in the resistant
population. The authors concluded that higher
EPSPS mRNA expression was resulting in higher
EPSPS production, but they were uncertain
whether the magnitude of over-expression
accounted for the observed resistance level. No
evidence was found to indicate EPSPS gene
duplication in the glyphosate resistant population
using DNA blot hybridization, despite the
observed 2.5 to 3 times higher EPSPS expression
(41). In glyphosate-resistant populations of
horseweed (Conyza canadensis) and hairy
fleabane (C. bonariensis), basal EPSPS mRNA
expression was 2-fold higher than in glyphosate-
susceptible populations when measured using
northern blots.
Other examples of target site gene up-
regulation have been reported. In three ALS-
resistant barleygrass (Hordeum leporinum)
populations from Western Australia, ALS enzyme
activity was 3-fold higher than in an ALS-
susceptible population (42). In addition, the three
populations also had a mutation at Pro-197
resulting in a serine substitution. While this
mutation confers ALS resistance on its own, the
increased ALS expression may also contribute to
the observed resistance level. It is not known if the
up-regulation of ALS protein activity is due to
altered gene expression regulation, gene
duplication, and/or reduced enzyme turnover rates.
A johnsongrass (Sorghum halepense) population
resistant to the ACCase-inhibiting herbicides
sethoxydim and quizalofop had 2- to 3-fold higher
ACCase enzyme activity relative to an ACCase-
susceptible population (43). The I50 (herbicide
concentration required to inhibit 50% of enzyme
activity in vitro) was similar between resistant and
susceptible populations, but the higher activity
was maintained across a range of herbicide
concentrations in vitro. However, the study did not
determine whether the increased ACCase
enzymatic activity was due to ACCase gene
duplication or up-regulation of ACCase
transcription.
Target site gene duplication
Gene duplication, the heritable replication of a
coding segment of DNA resulting in one or more
additional gene copies within the genome of an
organism (44), is a common process in the
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evolutionary history of plants and is vital for
generating genomic diversity (45). Increased gene
expression at the mRNA level is the immediate
result of gene duplication, and as mutations
accumulate in duplicated gene copies over time,
duplicated gene copies can begin to have
variations in function or acquire new functions
(44). The term gene amplification is also
frequently used synonymously with gene
duplication. Gene amplification has been defined
in some literature as the non-heritable replication
of a segment of DNA, such as in cases of cancer
tumors where amplified gene copies are not
inherited in the progeny of the individual, and gene
duplication indicates the heritable replication of a
segment of DNA (44).
The initial examples of EPSPS gene
duplication related to glyphosate resistance were
obtained in cell culture studies. The involvement
of gene duplication in glyphosate-resistant A.
palmeri (46) was the first case identified in a weed
population (Figure 4). The EPSPS gene in a
glyphosate-resistant A. palmeri population was
duplicated from 4- to over 100-fold relative to a
susceptible population. Expression of EPSPS
mRNA and EPSPS protein corresponded with the
increased genomic EPSPS copy number. The
increased EPSPS expression means that more
EPSPS is present at the target-site than the typical
applied concentration of glyphosate can inhibit
(19). Enough uninhibited EPSPS remains
following typical glyphosate applications such that
the plant can survive. In the studied A. palmeri
7
population, duplicate copies of the EPSPS gene
appeared to be present on all chromosomes (2n =
34).
In a fascinating example of molecular genetic
variation leading to adaptation, the duplicated
EPSPS gene was contained within a >300 kb
replicon containing multiple additional open
reading frames for other genes and various types
of repetitive DNA elements (47). The replicon was
found to have a circular structure existing outside
the chromosome, termed an extra-chromosomal
circular DNA (eccDNA) (48). The eccDNA can
attach to the chromosomes to be transmitted both
at mitosis and at meiosis, explaining the observed
variation in heritability of EPSPS gene copy
number in A. palmeri (49,50). The eccDNA
sequence across glyphosate-resistant A. palmeri
populations from across the U.S. was nearly
identical, suggesting the possibility of a single
origin of the eccDNA for glyphosate resistance
followed by seed- and pollen-mediated gene flow
(47,51). EPSPS gene duplication has been
reported in many glyphosate-resistant weed
species. Glyphosate resistance via EPSPS gene
duplication can be viewed as an example of
convergent evolution across these diverse plant
species (52). Populations of B. scoparia and A.
tuberculatus have fewer duplicated EPSPS
copies than A. palmeri, on the range of 4 to 10-fold
(Table 2).
Despite having a lower quantity of duplicated
EPSPS copies, A. tuberculatus populations have
a similarly high level of resistance as the A.
palmeri populations (Table 2). Both EPSPS
mRNA expression and EPSPS protein levels have
been reported to have a linear correlation with
EPSPS genomic copy number in B. scoparia and
A. tuberculatus populations (57,58), as well as in
Italian ryegrass (Lolium multiflorum populations)
(59). In the case of L. multiflorum, EPSPS protein
expression level correlated very well with
population level resistance; as EPSPS protein
expression increased, so did the dose required to
achieve 50% reduction in plant growth (59).
Comparisons of resistance level across species are
problematic, as the various reports were conducted
under different experimental conditions and
estimated LD50 and GR50 (concentrations causing
50% mortality and growth reduction, respectively)
parameters are not directly comparable. However,
available evidence suggests that higher EPSPS
copy number confers higher glyphosate resistance
in A. palmeri (e.g., (50), L. multiflorum (59), and
B. scoparia (60). Continued observation and
monitoring of EPSPS copy number in populations
over time will be needed to determine whether
EPSPS copy number may increase following
continued glyphosate selection pressure.
Recently, a population of glyphosate-resistant
spiny amaranth (Amaranthus spinosus) was
reported in which EPSPS gene duplication and
sequence data revealed that the EPSPS gene in
glyphosate-resistant A. spinosus individuals is
identical to glyphosate-resistant A. palmeri EPSPS
(61) and not glyphosate-susceptible A. spinosus.
This result indicates that the EPSPS gene has
transferred through inter-specific cross-
pollination. The two species are most closely
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related to each other among Amaranthus species
(62), and the two species can hybridize and
produce fertile hybrids (63). This is important
from an evolutionary perspective, because a
genetic trait that may be extremely rare within
populations (such as the eccDNA containing
EPSPS) has a selective advantage in multiple
species that are under similar selection
environments (repeated exposure to glyphosate).
Presumably genetic transfer between A. palmeri
and A. spinosus occurs at a low level in wild
populations, but the selective advantage of the
glyphosate resistance trait that initially evolved in
A. palmeri enabled transfer of this trait, plus
additional, unknown linked genetic traits, to A.
spinosus. This genetic transfer may have
evolutionary implications beyond loss of
glyphosate effectiveness in A. spinosus.
While EPSPS gene duplication in A. palmeri
has occurred via eccDNA, in B. scoparia a tandem
gene duplication has occurred (60). Several repeat
units were identified in a bacterial artificial
chromosome (BAC) assembly of the duplicated
locus, including a 56.1 kb repeat containing seven
predicted genes, and a 32.7 kb repeat containing
four predicted genes (64). Some of these co-
duplicated genes showed similar increased
expression as EPSPS. The border of the duplicated
region contained a mobile genetic element that
may have provided the initial DNA break to
initiate the tandem duplication process. The
potential evolutionary consequences of
duplicating and over-expressing other genes in
8
addition to EPSPS needs further exploration, as
does the relative stability of duplicated EPSPS
inheritance for both tandem duplication and
eccDNA mechanisms (65).
In addition to EPSPS gene duplication, the
target-site gene ACCase was found to have 5-7
fold higher gene copy number in a large crabgrass
(Digitaria sanguinalis) population resistant to five
ACCase inhibitor herbicides, resulting in 3- to 9-
fold higher ACCase transcript abundance (66).
Non-target site mechanisms
Reduced absorption
To be effective, herbicides must be absorbed
into cells of plants through the roots, in the case of
soil-applied herbicides, or from the leaves in the
case of foliar-applied herbicides (Figure 5).
Menendez et al. (67) provide an excellent
description of the factors involved in foliar
absorption and root absorption of herbicides.
Differences in root absorption of herbicides
between species have been attributed to root
morphology differences (68). There are no cases
of evolved resistance to soil-applied herbicides
due to reduced root absorption. Early work on
differential foliar absorption of herbicides between
species was attributed mainly to differences in
cuticle thickness and/or composition (68), but the
number and/or structures of leaf trichomes and
hairs have also been implicated (69). Hirsute
leaves are covered with hairy trichomes that can
retain spray droplets better than smooth, hairless
or glandless cuticles, thereby facilitating
absorption. Other leaves have lysigenous glands
involved in the production and storage of oily
secondary metabolites that can compartmentalize
lipophilic herbicides, preventing them from
reaching their site of action (70). Differences in
foliar absorption of herbicides between plants
have been attributed to leaf anatomical features
rather than any biochemical differences.
Decreased absorption is not a common NTSR
mechanism, but has been reported with resistance
of common sunflower (Helianthus annuus) to
imazethapyr and chlorimuron (71), prickly lettuce
(Lactuca serriola) to 2,4-D, annual bluegrass (Poa
annua) to atrazine (72), and L. multiflorum and S.
halepense to glyphosate (73,74). No differences
were found in cuticular wax amount per unit area
of leaf surface between two biotypes of L.
multiflorum with a three-fold difference in
glyphosate susceptibility and reduced absorption
in the less sensitive biotype (75). When reduced
absorption is implicated, it is most often only one
contributing factor to the overall resistance
mechanism. For example, resistance to glyphosate
in A. tuberculatus biotypes was due to both
reduced absorption and a herbicide-resistance
allele of the glyphosate enzyme target EPSPS (76).
Reduced translocation and vacuolar
sequestration
Many foliar-applied systemic herbicides rely
on translocation through the phloem for optimal
activity. These herbicides must cross the cuticle
barrier and enter the cells of mature source leaves
(symplast). This transport can involve active (i.e.,
protein-mediated) and/or passive diffusion
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processes. Once inside the symplast, systemic
herbicides translocate from source leaves to
younger sink leaves via the phloem, often along
with the movement of photosynthetic sugars.
Herbicide resistance due to reduced translocation
occurs when the herbicide is retained in source
leaves and prevented from translocating to the
growing points (Figure 5). Mechanisms that trap
the herbicide in source leaves (e.g., through
sequestration within vacuoles or leaf trichomes) or
prevent its normal movement to the growing
points across membrane barriers (through altered
activity of active membrane transporters) will
reduce the total amount of herbicide translocated,
thus conferring resistance. Reduced absorption
across the cuticle and reduced translocation out of
source leaves sometimes work in concert.
Reduced translocation of glyphosate is the
most prominent example of this NTSR mechanism
(65). In these plants, the amount of glyphosate
delivered to the meristems is lower than what is
necessary to be phytotoxic. Reduced glyphosate
translocation was first demonstrated in
glyphosate-resistant L. rigidum from Australia,
where glyphosate moved to the edges of treated
leaves, and less glyphosate translocated to the
meristems, relative to glyphosate-susceptible L.
rigidum (77). Glyphosate-resistant C. canadensis
had reduced translocation as well (78). This is due
to differences in cellular distribution of glyphosate
and subsequent phloem loading and translocation.
In these biotypes, glyphosate enters the
symplasm of source leaves normally but cannot
9
translocate to the meristems because it is rapidly
sequestered within the vacuole (79) (Figure 4).
The vacuole sequestration process is temperature-
dependent, with less sequestration occurring in C.
canadensis under colder temperatures (80). The
dependence on temperature suggests the
involvement of active membrane transporters.
Considerable research effort with both microarray
and transcriptomic sequencing (81,82) has
searched for a specific ABC transporter gene
suspected to be responsible for the vacuolar
sequestration, but no causative specific gene has
yet been confirmed.
The effect of lower temperatures on the
reduced translocation mechanism has also been
reported in S. halepense and L. multiflorum (83).
The temperature effect supports the hypothesis
that vacuole sequestration is an active process and
restricts glyphosate translocation by preventing
normal glyphosate movement into the phloem.
Reduced translocation may result from other
NTSR mechanisms in A. palmeri, A. tuberculatus,
and S. halepense (19). In these reports, reduced
cellular glyphosate absorption across the plasma
membrane may be associated with altered rate of
an active glyphosate transport process (Figure 4).
Reduced cellular uptake is not predicted to alter
translocation at the whole plant level, and
generally no changes in glyphosate translocation
have been observed in A. palmeri. Finally, reduced
chloroplast absorption of glyphosate has been
suggested (19) (Figure 4). This phenotype is
difficult to measure, as isolating intact chloroplasts
is technically difficult and may have unknown
effects on the physiological ability of the isolated
chloroplasts to exhibit any reduced absorption. To
date, reduced chloroplast glyphosate absorption as
a glyphosate resistance mechanism has not been
experimentally demonstrated.
Reduced translocation of paraquat from
treated source leaves to sink leaves has also been
reported for paraquat-resistant L. rigidum (84). In
this population, there was no difference in the
interaction of paraquat with the target site
Photosystem I complex (indicating a lack of TSR),
and no differences were found in the antioxidant
systems of superoxide dismutase or ascorbate
peroxidase (increased antioxidant activity may
allow a plant to tolerate the free radicals generated
by diversion of electrons from Photosystem I).
Because the population had reduced translocation
and the finding of Ge et al. (79) demonstrating
vacuole sequestration of glyphosate, intact
protoplasts of paraquat-resistant and -susceptible
L. rigidum were isolated. The paraquat
concentration was measured, as a method to
determine if the paraquat concentration within
single cells was higher in paraquat-resistant
protoplasts than paraquat-susceptible protoplasts
(85). Paraquat-resistant L. rigidum protoplasts
contained more paraquat than susceptible
protoplasts. Thus, the paraquat is likely
sequestered in the vacuole, in a similar process as
reported for vacuolar sequestration of glyphosate.
Reduced paraquat translocation attributed to
vacuole sequestration has also been reported in L.
multiflorum from California (86) and in two
Conyza spp. from California (87).
Characterization of a paraquat-resistant
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Arabidopsis mutant revealed a mutation in a gene
called Paraquat Resistant 1 (PAR1), a putative
amino acid transporter (88). The mutant, par1, had
similar cellular paraquat absorption as the normal
PAR1 genotype, but had reduced paraquat
concentration in the chloroplast. Overexpressing
the PAR1 homologue in rice resulted in paraquat
hypersensitivity, and silencing the rice PAR1
resulted in paraquat resistance, revealing that
reduction in paraquat transport into the chloroplast
can confer resistance. Natural polymorphic
variations in a gene (LHR1) for a plasma
membrane-localized polyamine transporter in
Arabidopsis caused variations in uptake of
paraquat that correlated with sensitivity to
paraquat (89). Endogenous polyamines have been
shown to play a role in paraquat-resistant E. indica
(90). Evolved paraquat resistance in weeds has not
yet been attributed to selection for orthologs of
PAR1 or LHR1.
Reduced translocation of 2,4-D was observed
in a 2,4-D-resistant population of L. serriola,
relative to a susceptible population (91), as well as
in 2,4-D resistant wild radish (Raphanus
raphanistrum) (92). Reduced dicamba
translocation was found in a dicamba resistant B.
scoparia population (93). Naturally-occurring
auxins such as IAA are polar and readily
translocate in the phloem, so it is reasonable that a
synthetic auxin such as 2,4-D or dicamba also
requires adequate translocation from the
application site to the target site in growing
meristems. Changes in auxin transport from cell to
10
cell via active transporters could play a role in
reduced 2,4-D translocation. Reduced
translocation of 2,4-D or dicamba would
presumably reduce the total concentration
achieved at the target site to low enough levels to
enable survival.
An unusual way for a plant to achieve reduced
translocation of a herbicide to the meristems has
been described as the phoenix phenomenon, which
is the result of reduced translocation caused by
more rapid action of the herbicide (Figure 6). One
of the assets of glyphosate as a herbicide is that it
acts slowly, allowing it to translocate to and kill
meristematic tissues. With herbicides that act
rapidly, translocation from treated plant organs is
limited because of the rapid action of the
herbicide. Giant ragweed (Ambrosia trifida)
evolved a rapid response to glyphosate that
prevents translocation of the herbicide to
meristems (94,95). Light- and/or sucrose-
dependent rapid withering and desiccation of
treated foliage occurs, followed by regrowth of the
plant from meristems that were not contacted by
the foliar spray – hence the “phoenix
phenomenon”. None of the other known
mechanisms of glyphosate resistance were found
(95). Reactive oxygen species (ROS) accumulated
within 30 min of treatment, only in the older leaves
of the resistant biotype of the weed. Treatment of
the leaf tissue with exogenous phenylalanine and
tyrosine (two aromatic amino acids that are
products of the shikimate pathway) prevents the
rapid effect, indicating that the effect may be
associated with EPSPS inhibition or to
physiological blocking of the rapid response by the
amino acids. The mechanism for accelerated
action of glyphosate in this biotype is unknown,
but it might be explained by rapid cessation of
carbon fixation due a rapid deregulation of the
shikimate pathway removing enough erythrose-4-
phosphate and phosphoenolpyruvate from the C3
carbon fixation pathway to stop it. In another
species with a rapid response to glyphosate, sugar
beet (Beta vulgaris), cessation of carbon fixation
is rapid, and the symptoms are similar to those of
the resistant A. trifida. Alternatively, perception of
glyphosate in mature, green plant cells of this
resistant biotype may trigger a rapid cell death
defense mechanism.
Rapid necrosis followed by regrowth was also
identified as a resistance mechanism to 2,4-D in a
Sumatran fleabane (Conyza sumatrensis) biotype
from southern Brazil (96). The symptoms after
2,4-D application are similar to those observed in
glyphosate-resistant A. trifida (i.e., necrosis in
older leaves and absent in younger leaves,
followed by regrowth from meristems). The 2,4-
D-resistant biotype was also resistant to
glyphosate, but not by the phoenix phenomenon.
Rapid necrosis symptoms did not occur with six
other auxinic herbicides (e.g., picloram). ROS
accumulation was much higher within 30 min after
2,4-D treatment than in a susceptible accession
and remained much higher for more than 7 h. The
resistant biotype did not show the normal epinasty
symptom (leaves bending downward) caused by
2,4-D, probably due to rapid cell death caused by
ROS inhibiting herbicide translocation.
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Metabolic alterations
Plants contain large numbers of genes
encoding enzymes that perform biochemical
reactions for the synthesis of secondary
metabolites and for detoxifying xenobiotic
compounds (e.g., herbicides) (97).
Serendipitously, some members of these gene
families can also detoxify herbicides. The
selective action of many herbicides (i.e., they
control weeds without damaging crops) often
depends on relatively rapid metabolism of the
active ingredients into harmless breakdown
products in crops compared to weeds. This
biochemical feature (differential rates of
detoxification) has been repeatedly exploited for
selective chemical weed control. Herbicide
detoxification is generally divided into three
phases (Figure 5). Phase I involves addition of a
functional group to the herbicide by oxidation,
reduction, or hydrolysis, often mediated by
cytochrome P450 monooxygenases (P450; EC
1.6.2.4). Phase II involves more complex changes
to a herbicide, such as conjugation, either to
glutathione mediated by glutathione-S-
transferases (GST; EC 2.5.1.18), or to glucose
mediated by glucosyltransferases (GT; EC 2.4).
Note that phase II enzymes such as GSTs and GTs
can directly detoxify some herbicides without
depending on phase I activation. The final step in
plants (phase III) involves compartmentalization
of the herbicide metabolites in the vacuole or
incorporation into cell walls (Figure 7). In general,
the same genes and biochemical mechanisms for
11
herbicide detoxification often exist in weeds that
are related to crops, with the critical difference
being that expression of these genes is lower in the
weeds. Thus, there is evolutionary potential to
select for increased expression and/or mutations of
these key genes in weeds, enabling enhanced
herbicide metabolism to confer resistance.
Prominent examples of herbicides that are
selective due to differential detoxification between
crops and weeds include the selective ALS and
ACCase inhibitors. Some of these, including
chlorsulfuron, diclofop-methyl, and fenoxaprop-
P-ethyl, are used to control grass weeds in wheat.
For these herbicides, rapid metabolism and crop
safety in wheat involves detoxification pathways
including P450, GT, and GST. For example,
metabolism of the ACCase inhibitor diclofop-
methyl to non-toxic metabolites in wheat occurs
via P450-mediated aryl hydroxylation, followed
by glucose conjugation, likely mediated by GT
(e.g.,(98)). These wheat-like metabolic pathways
also exist in grass weeds that have considerable
intra-specific variation for herbicide
susceptibility. In susceptible weed populations the
detoxification rates are generally too slow to
prevent herbicide phytotoxicity. However, some
weed species such as blackgrass (Alopecurus
myosuroides) in Europe, and L. rigidum in
Australia have evolved wheat-like rapid
detoxification pathways (e.g., 99,100). These
weed populations have long histories of repeated
exposure to wheat-selective herbicides and have
evolved enhanced metabolism-based resistance.
Enhanced herbicide metabolism is especially
problematic from a weed management standpoint
because the detoxification systems that confer
resistance to one herbicide can sometimes have
activity on other herbicides with the same or
unrelated sites of action. The term cross-resistance
is defined as when a single mechanism (such as
enhanced metabolism) confers resistance to more
than one herbicide with the same or of a different
site of action group. The term multiple resistance
is defined as when multiple, distinct mechanisms
have combined within an individual (or
population), and the individual (or population) is
resistant to herbicides from more than one site of
action group. When considering enhanced
metabolism, it may often be difficult to distinguish
phenotypically whether a single metabolic
mechanism is conferring cross-resistance to other
herbicide groups, or if multiple distinct metabolic
mechanisms are present. The key concept is that
some enhanced metabolism mechanisms,
including P450 and GST, can confer broad-
spectrum resistance, with known examples of
cross-resistance due to a single mechanism and
multiple resistance due to accumulation of
multiple, distinct mechanisms. From an
evolutionary biology perspective, enhanced
metabolism can be considered a broad-spectrum,
generalist adaptive response. Critically, enhanced
metabolism mechanisms can also combine with
other mechanisms including TSR and reduced
translocation to confer higher levels of resistance.
Cytochrome P450-mediated herbicide metabolism
Cytochrome P450 monooxygenases are
membrane-bound proteins localized in the
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endoplasmic reticulum and are one of the largest
gene families in all organisms (101). These
enzymes have crucial roles in the synthesis of
hormones, lipids, and metabolism of endogenous
and exogenous substances (Figure 8). The number
and diversity of P450s in plants is higher than in
other organisms. While in humans there are
approximately 54 P450 genes (0.1% of genome),
in plants the number is higher, with 246 in
Arabidopsis (1% of genome) and 328 in rice (0.5-
1% of genome). Weeds have been found to have
high diversity in P450 genes, with 917, 323, and
277 in barnyardgrass (Echinochloa crus-galli), C.
canadensis, and L. rigidum, respectively. The
greater diversity of the P450 genes in plants has
likely evolved for chemical defense, mainly for
degradation of many xenobiotics and by chance
these P450 genes also detoxify herbicides (102).
These genes have gained importance in agriculture
as the basis for herbicide selectivity in crops due
to natural metabolic processes. For example,
different P450 monooxygenases metabolize ALS
inhibitors in rice (103), cinmethylin in wheat
(104), 4-hydroxyphenylpyruvate dioxygenase
(HPPD; EC 1.13.11.27) inhibitors in maize (105),
and other mode-of-action herbicides in different
crops (106), at a faster rate than weeds. The
implication is that genetic variation exists in plants
for the expression level of P450 genes and crops
have higher expression of P450 genes that
metabolize herbicides. It should be noted that the
herbicide discovery process often identifies
candidate herbicides that are metabolized faster in
12
some weeds than in crops; these herbicides would
not be effective and are not commercialized.
However, the wide use of herbicides for weed
control has selected biotypes with the same ability
to inactivate herbicide molecules, threatening
weed management worldwide. The most
important reactions catalyzed by these enzymes
are either aryl- or alkyl hydroxylation, the first step
in the metabolism of xenobiotics (107). In general,
P450s insert molecular oxygen on a herbicide
molecule to be more reactive or more soluble
using an electron from NADPH P450 reductase.
As a result, herbicide molecules are metabolized
to products with reduced or modified
phytotoxicity in weeds with metabolism-based
resistance mechanisms.
Extensive work with P450 inhibitors provides
strong evidence for the role of various P450
isozymes in herbicide metabolism. Application of
a P450 inhibitor prior to herbicide exposure will
block P450 activity. When the resistance
mechanism is due to enhanced metabolism by
P450s, inhibition of the P450 activity enables the
herbicide to reach the target site in a high enough
concentration to cause normal phytotoxicity. P450
inhibitors have been used in various resistant
weeds to test for P450 herbicide metabolism. In L.
rigidum populations resistant to multiple
herbicides through metabolism-based
mechanisms, the P450 inhibitor malathion restores
chlorsulfuron activity (108). The herbicide
amitrole, structurally similar to the P450 inhibitor
1-aminobenzotriazole, restores diclofop-methyl
activity. However, the P450 inhibitors have
specificity for P450 isoforms. Pre-treatment with
piperonyl butoxide (PBO), but not malathion,
increased control of resistant P. annua with
fenoxaprop (109), indicating that PBO inhibited
specific P450 enzymes critical for fenoxaprop
metabolism. Although P450 genes found in the
past decade were mostly related to metabolism of
ACCase, ALS, and PSII inhibitors, more recently
P450 inhibitors have inhibited the P450
metabolism of several classes of herbicides (i.e.,
HPPD, PPO, synthetic auxins, and carotenoid
synthesis inhibitors) (110-113). Critically, P450s
have potential to metabolize herbicides from
different mode of action groups, creating
unpredictable patterns of cross-resistance.
Successful isolation of microsomes with
herbicide metabolism activity from weeds was
achieved in late watergrass (Echinochloa
phyllopogon) by first inducing P450 activity with
a sub-lethal herbicide dose, demonstrating P450
activity in metabolizing bispyribac-sodium,
fenoxaprop-p-ethyl, and thiobencarb (114). E.
phyllopogon populations are also resistant to
clomazone and penoxsulam via enhanced
oxidative metabolism from P450 activity
(115,116). Expression of several P450 genes was
measured using quantitative PCR in a metabolism-
based resistant E. phyllopogon population relative
to a susceptible population, and substantial
induction of the P450 genes occurred following
sub-lethal herbicide treatment (117). Analysis of
twelve candidate P450 genes from resistant E.
phyllopogon revealed that CYP81A12 and
CYP81A21 had the highest transcription levels in
the resistant biotype (118). These P450 genes were
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transformed into Arabidopsis, and transgenic
plants exhibited resistance to bensulfuron-methyl
and penoxsulam (118). Recently, P450 genes were
cloned from E. phyllopogon and transformed into
both Arabidopsis and E. coli (119). The ability to
assay the membrane-bound P450 proteins in
bacteria is a major advance in available tools to
functionally validate candidate resistance genes
from weeds. The E. phyllopogon CYP81As
metabolized 18 herbicides from 13 different
chemical classes. The recombinant expression of
CYP81As in E. coli metabolized different
herbicides by demethylation or hydroxylation
reactions in unrelated herbicide groups belonging
to ALS, ACCase, PDS, PSII, PPO, HPPD, and 1-
deoxy-D-xylulose-5-phosphate synthase inhibitors
(119), demonstrating the incredible potential of
P450 enzymes to metabolize herbicides and confer
cross-resistance.
The generally broad substrate recognition of
plant P450 enzymes poses a threat for weed
management due to potentially unpredictable
cross-resistance patterns. While CYP81A12 and
CYP81A21 can metabolize ALS inhibitors through
demethylation, the same genes are also involved in
cross resistance to ACCase inhibitors such as
diclofop-methyl, tralkoxydim, and pinoxaden
through hydroxylation (120). The same
CYP81A12 and CYP81A21 genes, as well as
CYP81A15 and CYP81A24, confer resistance to
clomazone (121). Other enzymes from this P450
family, such as CYP81A6, were found to
metabolize bentazon, sulfonylureas, and also
13
quinclorac in rice. The P450 enzymes of family
CYP81A appear to be “super P450s” able to
metabolize different chemical classes within
different herbicide mechanisms of action in the
same weed species. However, other P450 enzymes
of families CYP72, CYP71, CYP70, and CYP96
have also been found to metabolize different
herbicides.
Fewer examples of enhanced P450
metabolism have been identified in eudicot species
(typically broadleaf species). The reasons may be
biological, such as lower P450 activity or fewer
P450 genes in eudicots, or the reason may be due
to less research into enhanced herbicide
metabolism for eudicots. Recently, reports of
metabolism-based herbicide resistance in eudicots
are increasing. More rapid initial metabolism of
chlorimuron was found in an ALS-resistant A.
hybridus population (122). A survey of ALS-
resistant A. palmeri in Georgia found that more
than half of the populations had no ALS target site
mutations and exhibited enhanced ALS herbicide
metabolism (123). Resistance to HPPD inhibitors
has been reported in A. tuberculatus (124,125), A.
palmeri (110,126) and recently in R. raphanistrum
(127). The resistant biotypes metabolized HPPD
inhibitors at a faster rate than the susceptible
populations through hydroxylation reactions,
indicating a P450 role in the resistance mechanism
in the broadleaf species (110,127,128). Recently,
A. tuberculatus biotypes from Nebraska with
resistance to tembotrione and 2,4-D, as well as A.
palmeri from Arkansas with resistance to
fomesafen, were controlled when a P450 inhibitor
was applied (111,113,129). As research efforts
into metabolism-based resistance expand, more
examples of broadleaf species with enhanced
herbicide metabolism will likely be reported, and
the candidate genes in eudicot species can be
identified.
More work is needed to improve
understanding of P450 evolution and regulation
for herbicide resistance. The role of gene copy
number variation for cytochrome P450 should be
studied, especially in polyploid weeds, as a
pathway to generate novel allelic variation and
increased expression. Chromosome duplication in
polyploid species may generate more potential
variation for the evolution of metabolic resistance
through P450s. Little is known about the structure-
activity relationship between the many P450s
enzymes and which substrates (herbicides) they
may degrade. Biochemical modeling predicting
the tertiary structure of known P450s from weeds
would enable docking simulations with different
herbicides and anticipate classes of herbicides
more likely to succumb to metabolic degradation.
Transcriptional regulation of P450 genes is
another area where more research is needed.
Although a trans-element was proposed to control
the expression of both CYP81A12 and CYP81A21
genes in the resistant E. phyllopogon (118), we
lack studies to functionally demonstrate
transcription factors, activator elements, repressor
elements, or epigenetic modifications that may be
regulating P450 gene expression in metabolic
resistant weeds.
Glutathione-S-transferases (GST)
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The enzyme super-family of GSTs are
involved in herbicide detoxification by
conjugating glutathione to the herbicide molecule,
rendering the herbicide non-toxic. This
conjugation reaction can occur directly to the
active herbicide or following the activity of other
enzymes such as P450s. GST activity was first
identified on triazine herbicides, including
atrazine in corn (130). Additional herbicides
known to be glutathione-conjugated by GSTs
include chloroacetamides (such as alachlor and
metolachlor), sulfonylureas (such as chlorimuron
ethyl), diphenylethers (such as fluorodifen), and
aryloxyphenoxypropionates (such as fenoxaprop-
ethyl) (131). E. phyllopogon populations from
California are resistant to fenoxaprop-ethyl via
enhanced GST-mediated glutathione conjugation
(132), as are A. myosuroides populations from the
UK (133). Not all herbicides within a given
chemical group are equally susceptible to GST-
mediated glutathione conjugation. For example,
while GST acts directly on the ACCase inhibitor
fenoxaprop ethyl (133), structurally similar
ACCase inhibitor herbicides, such as diclofop-
methyl, have no chemical features that can be
glutathione-conjugated and are instead able to be
ring-hydroxylated by P450s (98). The chemical
structure of the herbicide determines whether GST
is able to conjugate the herbicide with glutathione.
Recently a GST (AmGSTF1), from multiple
herbicide resistant A. myosuroides with enhanced
GST activity, was cloned and expressed in
transgenic Arabidopsis (134). The transgenic lines
14
had increased resistance to chlorotoluron, alachlor,
and atrazine, and also had increased antioxidant
flavonoid and anthocyanin content. In additional
experiments, a GST inhibitor (4-chloro-7-nitro-
benzoxadiazole) synergized with the herbicides
fenoxaprop and clodinafop in multiple-resistant A.
myosuroides populations and restored herbicide
activity (134), providing additional evidence for
the role of increased GST expression as a
resistance mechanism. A. myosuroides
populations in Europe were found to be resistant
to flufenacet via enhanced GST-mediated
metabolism (135). Populations of Lolium spp.
resistant to flufenacet were found to conjugate
glutathione to flufenacet for metabolic
detoxification, and overall GST activity was
increased in protein extracts (136).
Glycosyl transferases (GT) and other transferases
The enzyme family of GT have roles in Phase
II of herbicide metabolism, via conjugation of
glucose to herbicide metabolites after initial Phase
I modification (typically hydroxylation or
demethylation) (137). For example, monocot
species (most often grasses) are tolerant to
synthetic auxins in part because glycosylation of
hydroxylated rings of auxinic herbicides tends to
be irreversible, highlighting the importance of GT
in protecting a plant from herbicide activity (69).
To date, no reports are known of herbicides for
which the first step of metabolism involves GT;
however, increased GT expression may be
necessary as a secondary biochemical step for
metabolic resistance in weeds (138,139).
On the other hand, sensitive eudicots tend to
utilize other transferases, such as amino acid
transferases, to catalyze a reversible conjugation
of amino acid residues to auxinic herbicides, but a
herbicidally active metabolite may be recovered
upon hydrolysis (140).
Similarly, several other classes of transferases
may be involved in Phase II metabolism.
However, enzymes such as malonyl-transferase
typically impart herbicide selectivity, but have not
been demonstrated to be directly involved in
evolved herbicide resistance (141).
Aryl acylamidase
The mechanism of natural tolerance of rice
(Oryza spp.) to the PSII inhibitor herbicide
propanil is high levels of aryl acylamidase (AA;
EC 3.5.1.13), an enzyme that hydrolyzes propanil
to 3,4-dinitroaniline, a non-phytotoxic compound.
Studies of a wild-type rice and a rice mutant with
no AA activity indicated that the normal role of
this enzyme in plants is in nitrogen metabolism
relating to asparagine. Weedy rice (feral forms of
cultivated rice, often called red rice) is also
tolerant to propanil by the same mechanism.
Among Oryza species, tolerance to propanil
correlates with AA activity (142). Resistance to
propanil by elevated AA activity has evolved in
junglerice (Echinochloa colona) (143) and E.
crus-galli (144). The AA inhibitors anilofos,
piperophos, and carbaryl synergize with propanil
activity in propanil-resistant E. crus-galli (144).
Not all evolved resistance to propanil is by
enhanced AA activity, as resistance to propanil in
rice sedge (Cyperus difformis) is a single amino
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acid change of the D1 protein of PSII (145).
Aldo-keto reductase
Some plant species, especially legumes,
metabolize glyphosate to aminomethyl
phosphonic acid (AMPA) and glyoxylate, whereas
others (grasses in particular) apparently have little
capacity for this degradation pathway or any other
transformation of the herbicide (146). Neither the
enzyme nor the gene for the glyphosate
oxidoreductase (GOX) that is considered
responsible for glyphosate degradation in plants
have been identified. AMPA is very weakly
phytotoxic, so sufficiently rapid degradation of
glyphosate to AMPA should provide resistance.
Because glyphosate is a very slow-acting
herbicide, evolution of such a resistance
mechanism would seem likely. However,
numerous studies have found no differences in
glyphosate degradation in glyphosate-resistant
weeds. Pan et al. (147), however, recently found
that the mechanism of glyphosate resistance of an
E. colona biotype involves elevated levels of aldo-
keto reductase (AKR; EC 1.1) due to upregulation
of two AKR genes, resulting in more rapid
metabolism of glyphosate to AMPA. Earlier,
overexpression of an AKR gene from rice provided
glyphosate resistance to tobacco, and silencing this
gene in rice caused hypersensitivity to glyphosate
(148). Whether AKRs are the GOX enzymes that
cause accumulation of AMPA in other weeds and
crops remains to be determined (146).
15
-Cyanoalanine synthase
An example of the broad-spectrum resistance
that can be conferred by enhanced metabolic
activity is quinclorac resistance in E. phyllopogon.
Quinclorac is a unique auxin-type herbicide
because it has activity on grasses. Normally, most
synthetic auxin herbicides have excellent activity
on eudicot species but no or very little activity on
grasses. Quinclorac stimulates ethylene synthesis
in sensitive grass species, which results in
accumulation of cyanide and subsequent toxicity
(149). The enzyme β-cyanoalanine synthase (β-
CAS; EC 4.4.1.9) detoxifies the cyanide generated
upon ethylene synthesis, but normally β-CAS
activity is insufficient to protect against the toxic
accumulation of cyanide. Surprisingly, E.
phyllopogon populations in California exhibited
quinclorac resistance before quinclorac had been
used commercially (150), due to their selection
history with multiple other herbicides and
resulting enrichment for enhanced metabolism
mechanisms. These quinclorac-resistant
populations were found to produce less ethylene
after quinclorac treatment than susceptible
populations. The P450 inhibitor malathion
synergized with quinclorac on the resistant
populations, suggesting that enhanced P450
activity detoxified quinclorac and inhibition of this
P450 metabolism enabled quinclorac to reach
phytotoxic concentrations. The mechanism is even
more complicated, however, as β-CAS activity
was 2- to 3-fold higher in resistant populations.
The malathion treatment did not change ethylene
production in the resistant nor the susceptible
populations, but malathion treatment did inhibit β-
CAS activity in the resistant population.
Therefore, malathion is also an inhibitor of β-CAS
activity, in addition to P450 activity. β-CAS
functions to process accumulated cyanide and
metabolize it to non-toxic forms. The enhanced
activity of this protective mechanism in the
resistant population was inhibited by malathion,
demonstrating that enhanced β-CAS activity can
confer metabolic resistance to quinclorac. An
additional, unknown mechanism makes the
resistant populations less sensitive to the
stimulation of ethylene production by quinclorac.
NTSR Summary
The diverse mechanisms that can contribute to
NTSR are complex and involve several different
gene types, many of which exist in plants as gene
families. This means that identifying the specific
genes involved in a particular case of resistance
can be difficult. Gene family members can be
difficult to distinguish using traditional
sequencing techniques and can be subject to
specific expression regulation at various
developmental stages or following herbicide
treatments. Sequence variation among gene family
members occurs normally and plays an important
evolutionary role in functional diversification, so
mutations altering substrate specificity for
herbicide detoxification may also occur. The most
important gene families for NTSR characterized to
date are P450s and GSTs. Unraveling the complex
mechanisms involved in NTSR and understanding
their relationship to overall plant stress response
pathways is a clear priority for future research
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(151). To that end, ongoing research is utilizing
next generation sequencing technology and
transcriptomics to help unravel the molecular and
genetic basis of NTSR, particularly for
metabolism-based resistance (138,152). An
improved understanding of the evolution and
regulation of NTSR could guide an additional
classification system for herbicides, old and new,
according to their metabolism or sequestration by
specific NTSR mechanisms.
Importance of genomics and transcriptomics in
understanding NTSR
Understanding the molecular and genetic basis
of resistance mechanisms can help explain
patterns of cross-resistance across different
herbicide modes of action due to a single
mechanism. Target site mutations are specific for
herbicides within the same mode of action. NTSR
mechanisms, however, can confer resistance
across dissimilar modes of action. For example,
similarities in the resistance mechanism could
provide an explanation for the concomitant
evolution of paraquat resistance following
recurrent selection with low glyphosate rates in L.
rigidum (153). A more thorough understanding of
cross-resistance patterns across different modes of
action would be highly beneficial for developing
improved herbicide rotation recommendations,
avoiding repeated selection with herbicides that
are vulnerable to shared resistance mechanisms
despite having different modes of action.
16
 “Omics” approaches (genomics,
transcriptomics, proteomics, and metabolomics)
hold great potential for making rapid advances in
our understanding of NTSR mechanisms (154). Of
these omics approaches, transcriptomics have
provided the most insights thus far. It is generally
thought that NTSR is often mediated by increased
expression of one or more genes (e.g., a gene
encoding a P450 or an ABC transporter) and,
consequently, a transcriptomics approach is
ideally suited to identify such genes (155). One of
the downfalls of a transcriptomic approach,
however, is that it usually yields several “false
positives,” requiring significant downstream
research to validate candidate genes.
Consequently, scientists typically opt to select the
most obvious candidates (such as genes annotated
as coding potential metabolism enzymes) for
follow-up study, reducing the likelihood that novel
NTSR genes will be discovered. Additionally,
identification of a gene that is overexpressed and
confers herbicide resistance provides insight into
the physiology of the resistance mechanism, but
not directly into the evolution of the mechanism.
Specifically, transcriptomics is not well suited to
determine the molecular change that caused
overexpression of the gene.
Draft genomes have recently been obtained for
some of our most important herbicide-resistant
weeds, including A. tuberculatus (156), C.
canadensis (157), B. scoparia (64), R.
raphanistrum (158), E. crus-galli (159), and E.
indica (160). Although some of these genomes are
still highly fragmented, those of A. tuberculatus,
C. canadensis, and E. crus-galli have N50s greater
than 1.5 Mb (N50 is the length of a
computationally-assembled contiguous sequence
for which 50% of the sequences in an assembly are
longer and 50% are shorter; longer N50 indicates
a better assembly). Such high-quality genomes
enable identification of candidate resistance genes
through approaches such as genetic mapping of
quantitative trait loci (QTL) in biparental mapping
populations and genome-wide association studies
(GWAS) in existing populations (161). Such
genomic approaches could identify herbicide-
resistance genes even if they are not
overexpressed, as well as potentially identify
trans-acting factors that cause overexpression of
genes identified through transcriptomics
approaches.
Genetics of herbicide resistance
The inheritance and genetic basis of different
resistance mechanisms has evolutionary
implications, such as how rapidly selection can
occur and how frequent mechanisms may be in
unselected populations. As described above, many
herbicide-resistance traits, particularly TSR,
require a change of a single enzyme and,
consequently, are inherited as single-gene traits.
Furthermore, herbicide-resistance alleles often act
in an additive to dominant fashion. Because of this
single-gene, additive-to-dominant nature of many
herbicide resistances, their evolution can occur
rapidly, and they can spread effectively by both
seed and pollen (162).
The additive-to-dominant nature of many
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herbicide-resistance traits means that the
resistance allele often will be selected in
heterozygous plants, particularly in cases where
the magnitude of resistance is high. For example,
single ALS amino acid changes can confer
resistance ratios of 100-fold or more, relative to
sensitive biotypes (e.g., (163)). In such cases,
heterozygotes and homozygous-resistant plants
will have similar responses to typical field doses
of the herbicide, even if the resistance allele is not
completely dominant. Resistance in this case can
be considered “functionally” dominant. In
contrast, single amino acid changes to EPSPS
confer very modest levels of resistance (typically
less than 10-fold (19)). In E. indica, an EPSPS
target-site mutation was additive (164).
Consequently, in this case, plants heterozygous for
an EPSPS mutation potentially could be controlled
by full recommended rates of glyphosate and using
reduced rates would foster resistance evolution.
Known examples of herbicide resistance
inherited as a recessive trait are restricted to TRS
to herbicides that bind tubulin (107) and to a few
cases of resistance to auxinic herbicides (165). As
expected, because of the recessive nature of these
traits, they have been observed almost exclusively
in self-pollinated weed species. However,
outcrossed weeds that have evolved recessive
resistance include yellow starthistle (Centaurea
solstitialis) resistant to some auxinic herbicides
(166) and L. rigidum resistant to dinitroaniline
herbicides (167). Population-genetics theory
predicts that in an outcrossed population the
17
probability of a resistant plant occurring for a
recessive mutation in a reasonably sized field (e.g.,
30 ha) is essentially nil, even with a relatively high
mutation rate (1 x 10-6 gametes per locus per
generation) and high weed density (500 m-2) (162).
So how were C. solstitialis and L. rigidum able to
evolve recessive resistance? In the case of C.
solstitialis, it was reported that the species is not
completely outcrossed, and even the very low
selfing rate (<0.1%) would be sufficient for
generating a few plants with the homozygous-
recessive mutation (166). Lolium spp., although
predominantly self-incompatible (female flowers
on the plant reject pollen shed from the same plant,
requiring pollination from a different individual),
also can exhibit some degree of self-pollination
(168). In addition, dinitroaniline herbicide
resistance in L. rigidum was not completely
recessive; heterozygous plants exhibited some
resistance to low herbicide doses (167).
The discussion of herbicide-resistance
inheritance so far has included only nuclear genes.
An important exception to nuclear inheritance of
herbicide resistance is maternal inheritance of
resistance to PSII inhibitors, reported over 40
years ago (169). The basis for this maternal
inheritance is now known to be a herbicide-
insensitive D1 protein, which is encoded by a
chloroplastic gene and, therefore, transmitted only
maternally in most species (5). For species that are
predominantly self-pollinated, the lack of pollen-
mediated dispersal of this resistance trait is
probably of limited consequence. In outcrossing
species, however, dissemination of nuclear-
inherited traits would be favored over maternally
inherited traits. This may explain why nuclear-
encoded NTSR to atrazine is more common in A.
tuberculatus (a dioecious—and, therefore,
outcrossed—species) than is maternally inherited
atrazine TSR (170). However, TSR to PSII
inhibitors also is known to incur a significant
fitness penalty (4), and this also would disfavor the
evolution of this resistance.
Inheritance of glyphosate resistance conferred
by EPSPS duplication in some cases is observed to
be consistent with a single-gene model. This
apparent single-gene inheritance occurs because
the multiple EPSPS copies are found together in
the genome as tandem repeats (60). In other cases,
notably in A. palmeri, the EPSPS copies are
present as eccDNA and, consequently, inheritance
does not follow a single-gene model. In fact, the
EPSPS copy number appears to be unstable in
genetic transmission, as segregating F2 A. palmeri
progeny were highly variable for EPSPS copy
number, from less than the parental copy number
to greater than the sum of both parents (50).
NTSR traits, such as enhanced herbicide
metabolism and reduced translocation, sometimes
show inheritance consistent with a single-gene
model. Examples include atrazine resistance in
velvetleaf (Abutilon theophrasti) and A.
tuberculatus, paraquat resistance in L. rigidum,
glyphosate resistance in C. canadensis, and 2,4-D
resistance in oriental mustard (Sisymbrium
orientale) (171-174). More often, however NTSR
appears to be mediated by multiple genes.
The multigenic nature of NTSR often has been
described as “creeping” resistance, in which the
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magnitude of resistance slowly increases over
repeated herbicide selection, presumably due to
the stacking of small-effect resistance alleles in the
population (175). Empirical support for creeping
resistance is provided by low-dose selection of
experimental populations. Such studies, conducted
with different species and different herbicides,
have consistently demonstrated the ability of
plants to evolve increased herbicide resistance (in
some cases by many fold) after only two or three
generations (176-178). That such a shift in
herbicide response was due to the accumulation of
multiple alleles was supported by a follow-up
study on one of the selected populations that
indicated at least three loci were involved. It is also
noteworthy that when similar selection protocols
were followed on an outcrossed weed (L. rigidum)
and selfed weeds (wild oat, Avena fatua), the
selfed weed’s shift was only about 5% of that of
the outcrossed weed (178). This is consistent with
the idea that the resistance evolution occurred by
stacking together diverse alleles initially
distributed among different plants.
The evolution of NTSR also could involve
epigenetic factors (179). For example, changes in
the methylation status of a gene could affect its
expression. Epigenetic involvement in evolved
herbicide resistance is an understudied topic,
although differential methylation of EPSPS
recently was reported between glyphosate-
resistant and sensitive biotypes of C. canadensis
(180). It is not yet clear, however, if this
differential methylation contributes to resistance.
18
 Predicting and mitigating herbicide resistance
requires understanding not only the genetics of the
resistance traits, but also the origins of the genetic
diversity that fuels resistance evolution (181). We
are now moving into an era in which population
genomics approaches should be able to shed
increased light on, for example, the importance of
new mutations vs. standing genetic variation in the
evolutionary history of herbicide resistance (182).
Influences of ploidy
Understanding resistance mechanisms in
polyploid species is complicated by the presence
of multiple genomes and associated regulatory
processes, ranging from whole-genome to single
allele, that are not a consideration in diploids. In
particular, allele dosage, or the relative
contribution of orthologous or homoeologous
genes (homoeologs are genes with homologous
sequence/function that are present in the different
genomes of a polyploid) in a polyploid individual,
can influence the effect of target site mutations in
weed species.
The influence of poidy has been documented
for herbicide resistance in multiple species. An
ACCase inhibitor-resistant population of
hexaploid A. fatua had three different mutations in
ACCase (at positions 1781, 2078, and 2088) that
segregated independently, indicating one mutation
present for each homoeologous loci (183). When
occurring alone, each allele conferred a lower
ACCase resistance level than the same mutation
confers in diploid species. The mutations showed
an additive effect when co-expressed in vitro,
suggesting in single mutant lines the contribution
of susceptible ACCase alleles were diluting the
effect of the resistance allele. Similar allele dose
effects were observed in IMI-resistant hexaploid
wheat (184). Resistance mutations on ALS genes
on two of the three sub-genomes (referring to one
of the multiple genomes within a polyploid)
resulted in higher resistance than when the
mutation was present on only one sub-genome.
More recently, an allotetraploid P. annua
population was identified with resistance to
glyphosate via a Pro106Leu mutation in EPSPS
and 7-fold increased EPSPS copy number (185).
Interestingly, both the target site mutation and
duplicated EPSPS genes were present in
homoeologs only from the supine bluegrass (Poa
supina) progenitor sub-genome. The P. supina
sub-genome is known to contain higher nucleotide
substitution rates than the early meadow-grass
(Poa infirma) progenitor sub-genome (186), and
this expression dominance toward a more dynamic
sub-genome may represent a resistance evolution
process present in this species. Despite these
examples just described, studies are limited on the
influence of ploidy on resistance mechanisms, and
further research is needed to understand the
complexities of herbicide resistance and evolution
in polyploid weeds.
Implications for herbicide-resistant crops
The wide range of mechanisms of evolved
resistance to herbicides by weeds provides a
wealth of potential genes for production of
herbicide-resistant crops. However, not since the
production of triazine-resistant canola by
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backcrossing canola with an evolved triazine-
resistant weed (187), has a gene from a weed been
used to produce a commercial herbicide-resistant
crop. The rapid evolution of resistance to ALS and
ACCase inhibitors by single nucleotide
polymorphisms provided a clue that crops resistant
to these herbicides could be generated by
mutagenesis and breeding. This approach has been
especially successful in producing imidazolinone-
resistant rice, wheat, oilseed rape, sorghum, maize,
sunflower, and sugarbeet. Imidazolinone-resistant
crops have been the only significant commercial
success with this technology, even though crops
resistant to ACCase inhibitors and sulfonylurea
ALS inhibitors have been generated by such
mutation breeding approaches (188).
Relatively few herbicide-resistance genes
have been utilized to produce commercial,
transgenic, herbicide-resistant crops, and most of
them have been of bacterial origin. These include
bacterial genes for a glyphosate-resistant form of
EPSPS and those that degrade glyphosate,
glufosinate, 2,4-D, bromoxynil, and dicamba. A
non-bacterial exception is a TIPS form of EPSPS
that was engineered from a plant EPSPS gene and
is used in some glyphosate-resistant maize
cultivars (22). It took fifteen more years after use
as a transgene for the TIPS form of EPSPS to
evolve in a weed (24). A bacterial gene with a
single nucleotide polymorphism encoding an
isoxaflutole-resistant HPPD is being used as a
transgene for isoxaflutole-resistant crops (189).
However, another company is developing crops
19
resistant to triketone HPPD inhibitor herbicides
through use of a mutant triketone-resistant HPPD
gene from oats (190).
For the most part, researchers developing
transgenic herbicide-resistant crops became
involved with crops resistant to a particular
herbicide before a weed had evolved resistance to
that herbicide or the genes for weed resistance had
been discovered. A major consideration in
development of herbicide-resistant crops is the
strong benefit of the transgene providing
resistance to only one herbicide or class of
herbicides (e.g., glyphosate or imidazolinones)
controlled by the same company. The very high
cost of developing and obtaining regulatory
approval of such a crop can preclude production of
a crop that is resistant to products belonging to a
competing company. If transgenic, herbicide-
resistant crops continue to be developed, the
burgeoning information on the molecular biology
of resistance to herbicides evolved by weeds might
influence the choice of future transgenes. For
example, targeted mutagenesis on a codon
involved in resistance to certain PDS inhibitors
identified a double mutation not likely to occur in
nature that imparts even greater resistance to these
herbicides (14). Interestingly, this mutation
provided negative cross-resistance to other classes
of PDS inhibitors. This may be particularly useful
in managing volunteer plants as well as weeds that
may evolve resistance by selection for this
mutation.
What we know about the evolutionary biology
of herbicide-resistant weeds will influence what
herbicides are chosen for future herbicide-resistant
crops and how resulting crops are managed. An
impetus to the development of glyphosate-
resistant crops in the early 1990s was the false
view that evolution of weeds resistant to
glyphosate would be almost impossible because
more than one codon in EPSPS would have to
mutate simultaneously to provide robust TSR
(191). The unparalleled success of glyphosate-
resistant crops and the resulting massive selection
pressure for glyphosate resistance helped to
overcome the difficulty that weeds had in evolving
resistance. Thus, even if a herbicide is selected
because mutations imparting resistance are less
likely to evolve, resistance management must be
part of the stewardship of future herbicide-
resistant crops because sustained, strong selection
pressure over a wide area can overcome any
perceived genetic barrier.
Implications for herbicide discovery
Decades of non-diversified chemical weed
control and the resistance challenges that arose
from it have taught us several hard lessons about
the importance of herbicide stewardship. Research
into the evolution of resistance provides valuable
insights for future herbicide discovery, as certain
resistance mechanisms are more prevalent within
certain modes of action than others. These findings
can be used to pro-actively influence the
development of less resistance-prone herbicides.
For TSR, the rate and likelihood at which a
point mutation conferring resistance occurs is
determined by both the target protein and the
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inhibitor. Many herbicidal molecules compete
with substrates that are crucial for plant survival
by emulating their volume, surface geometry, and
binding properties. Compounds such as
glyphosate and transition state analogues such as
glufosinate tightly fit into the active site of the
target enzyme (competitive binding) where they
form strong hydrogen bridges. Point mutations in
this highly conserved area would render the
enzyme dysfunctional. In contrast, herbicides
inhibiting ACCase, ALS, PPO, or PDS adopt
different binding modes than the substrate so that
a mutation can have differential effects on
inhibitor and substrate binding. The enzyme’s
reaction would still take place despite an amino
acid change in the inhibitor binding domain –
where genetic diversity within and among species
is more likely. However, changes can come at a
cost of a reduced turnover rate (kcat) for the
substrate, as seen with the Gly210 PPX2 deletion.
Therefore, the vast majority of resistance-
conferring point mutations are found in
hydrophobic binding sites away from the active
reaction site. ALS point mutations, for example,
are found in the non-conserved terminal ends of
the ALS sequences (192). Not all point mutations
confer resistance to all compounds from the same
chemical class. For example, while the 1781
mutation in A. myosuroides confers resistance to
all ACCase inhibitors, the 2027 mutation confers
resistance to aryloxyphenoxypropionates, but not
to cyclohexanediones (10). The closer the
mutation is to the active site, the more likely it is
20
for a point mutation to negatively affect the
enzyme’s catalytic rate but also to confer
resistance to all molecules of a site of action rather
than to just one chemical class.
Sometimes, several homologues for the same
target exist in a plant. Resistance via point
mutations would be less likely to evolve if these
isoforms were expressed at the same time, non-
redundant in function, and can be inhibited by the
same molecule. In the case of a point mutation in
one of the target enzyme isoforms, the inhibition
of the remaining enzymes would then still lead to
the death of the plant. Alternatively, one herbicide
may inhibit two different target proteins (e.g.,
auxinic herbicides). The probability of an
individual to evolve two coincidental, independent
and catalytically competent point mutations in
each target enzyme is equal to the product of the
frequencies of the single mutation. Hence,
molecules that fully inhibit several targets that
individually would lead to plant death, are
preferred. Likewise, target site mutations are less
likely to occur if they need to be homozygous to
confer resistance (recessive inheritance). In that
case an identical mutation would need to be
present in both the paternal and maternal alleles.
They are also less likely to spread if they impart a
fitness penalty for the plant, and if compensatory
mutations rarely occur, as in the case of TSR to
PSII inhibitors (193,194). Duplication of the gene
for the herbicide target protein, likely guided by
transposons or non-homologous recombination, is
a resistance mechanism that is particularly
difficult to predict in herbicide discovery
strategies.
The physicochemical properties of a molecule
define how it binds the protein target, as well as
strongly influence uptake, translocation, and
degradation pathway before it reaches the target
site. For example, a major pathway of herbicide
metabolism is through oxidation via P450s, which
can add hydroxyl groups to a molecule. This
reaction is more difficult for small and polar
molecules, which allow for strong hydrogen
bridges, inhibiting the active site of the target only.
In contrast, hydrophobic parts of the molecule, like
aryl or alkyl moieties, are more prone to oxidation,
as there is a positive correlation between
lipophilicity and KM for P450-catalyzed reactions
(195). Prevention of this metabolic degradation
can be achieved by introduction of halogens into
strategic positions of the molecules. A second
major pathway of metabolic degradation is the
nucleophilic attack of electrophilic centers in the
inhibitor molecule, such as through GSTs. Such
soft regions can be circumvented by the
introduction of heteroatoms such as oxygen at the
respective place of the molecule. However, these
changes can alter a molecule’s binding properties.
Once Phase I metabolism has taken place, phase II
metabolism, such as glycosylation, cannot be
prevented. The biggest challenge in pesticide
discovery lies in creating a biologically active
molecule that is toxicologically benign to non-
target organisms and is degraded in the
environment as well as by non-target organisms.
Normalized by use frequency, strong
differences in the rates of herbicide resistance
evolution between different modes of action have
Downloaded from http://www.jbc.org/ by guest on May 19, 2020
been observed (196). Glyphosate has been such an
excellent herbicide because it tightly occupies the
active site of the EPSPS-S3P complex only,
outcompeting the substrate PEP. Its low
dissociation rate allows for almost irreversible
inhibition of the enzyme. Heteroatoms and
phosphonates that cannot be oxidized leave it inert
for metabolism by most metabolic enzymes.
Hence, resistant plants have generally had to
“resort” to the fairly complicated and comparably
slow-to-evolve mechanism of EPSPS gene
duplication and even slower-to-evolve double
codon mutations in EPSPS to survive. In the end,
any type of persistent selection pressure will be
eventually met by adaptation, and the potential
number of resistance mechanisms are many. Even
if all currently known resistance mechanisms
could be addressed, new, unpredictable
mechanisms may evolve, such as the “phoenix
phenomenon” mechanism of glyphosate and 2,4-
D resistance. Therefore, it is futile to set a goal of
finding a “resistance-proof” herbicide.
Nevertheless, the considerations listed in Table 3
should be considered in herbicide discovery
strategies. Weed biology, herbicide application
rates and herbicide mixtures all influence the type
and rate at which resistance mechanisms evolve
(197). We can aim to delay the evolution of
resistance to newly developed molecules by
choosing sensible targets and optimized
molecules. We must recognize, however, that
doing so will not replace the necessity to practice
integrated resistance management.
21
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: All authors contributed substantially to the writing of this review.

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Table 1. Herbicides mentioned in the text with their molecular targets and mechanisms of resistance.
Only the mechanisms provided in the text are listed. Additional herbicides to which resistance has
evolved are found in ref. (3).

Inhibited process/molecular target Herbicide chemical Mechanism(s) of

class/herbicide1 resistance
Amino acid synthesis
ALS; branched chain amino acids Imidazolinones
imazethapyr TSR and NTSR
imazaquin TSR
Sulfonylureas
benzulfuron TRS and NTSR
chlorimuron TSR and NTSR
chlorsulfuron TSR and NTSR
Pyrimidinyl benzolates
bispyribac TSR and NTSR
Triazolopyrimidine

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penoxsulam NTSR
EPSPS; shikimate pathway function glyphosate TSR and NTSR
Glutamine synthase glufosinate NTSR
Auxin mimics
F-box proteins Phenoxycarboxylates
2,4-D TSR and NTSR
dicamba TSR and NTSR
quinclorac NTSR
Picolinates
picloram TSR
Carotenoid synthesis
HPPD isoxaflutole NTSR
Triketones
mesotrione NTSR
tembotrione NTSR
Lycopene cyclase amitrole NTSR
Phytoene desaturase Diphenyl heterocycles
fluridone TSR
Phenyl-ethers
beflubutamid no resistance
diflufenican no resistance
picolinafen no resistance
N-Phenyl heterocycles
norflurazon TSR
Deoxy-D-xyulose phosphate synthase clomazone NTSR
Cell division
-Tubulin Dinitroanilines
oryzalin TSR
trifluralin TRS
Chlorophyll synthesis
PPO Diphenyl ethers
lactofen TSR
fomesafen NTSR

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 Fatty acid synthesis
ACCase Aryloxyphenoxypropionates
diclofop-methyl TSR and NTSR
fenoxaprop-P-ethyl TSR and NTSR
clodinofop NTSR
quizalofop TSR
Cyclohexanediones
sethoxydim TSR
tralkoxydim TSR and NTSR
pinoxaden NTSR
Fatty acid thioesterase cinmethylin NTSR
Very long-chain fatty acid synthases thiobencarb NTSR
Chloroacetamides
alachlor NTSR
metolachlor NTSR
Photosynthesis
Photosystem II D1 protein Amides

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propanil TSR and NTSR
Nitriles
bromoxynil NTSR
Triazines
atrazine TSR and NTSR
Ureas
chlorotoluron NTSR
bentazon NTSR
Photosystem I energy diversion paraquat NTSR

1
the chemical class of herbicides underlined are not provided, as they are the only representatives
of their chemical class

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Table 2. EPSPS gene duplication and glyphosate resistance level (LD50, dose required to cause 50%
mortality) reported in glyphosate-resistant weed species. Note that some values for LD50 were measured
in different populations and reported in different studies than EPSPS copy number [adapted from (19)].
EPSPS Relative Genomic LD50 (R/S)
Species Copy Number Range
Bassia scoparia 3–9 2-8
Chloris truncata 32-48(53) 2.4-8.7
Hordeum glaucum 9-11(54) 2.8-6.6
(55)
Bromus diandrus 10-36 4.7
Amaranthus spinosus 26-37 5
Amaranthus tuberculatus 2-8 5-19
Lolium multiflorum 15 – 25 12-13
Amaranthus palmeri 2 – 160 15-40
Eleusine indica 28(56) -

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36
Table 3. Features of an ideal herbicide from a resistance management perspective.

- Inhibits several different targets

- Inhibits several homologous and non-redundant target genes
- Fits tightly and deep into enzyme’s active site
- Only fits into enzyme’s active site
- Does not bind to hydrophobic pockets around the active site in case the natural substrate does not
contain any lipophilic parts
- Builds strong hydrogen bridges
- Small polar molecule with low structural complexity
- Low lipophilicity
- Non-reversable binding, strong enzyme affinity (low KM or P50 values)
- High volume overlap with substrate for good competition

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 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 1. Crystal structure of Arabidopsis ALS. A) view of the homodimer with chain A (gold) and chain
B (slate colors) (Adapted from McCourt et al. (16)). The herbicide imazaquin is located at the entrance of
the channel leading to the catalytic domain of the enzyme. B) Closer view of the interface between the two
ALS monomers with imazaquin positioned at the entrance. The area in red highlights the position of Ser653
imparting resistance to imidazolines but not to sulfonylureas.

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Figure 2. Interaction of glyphosate with EPSP synthase. A) Interaction between shikimate-3-
phosphate (S3P) and glyphosate (GLY) (yellow dotted line) within the catalytic domain of EPSP
synthase. B) Location of the TIPS double mutation in glyphosate-resistant EPSP synthase relative
to the S3P-glyphosate complex. Leucine is shown in pink and serine is shown in slate. C) Mutation-
induced structural changes in EPSP synthase. In the ternary complex, the mutations cause a shift
of the Cα atom of Gly96 toward the phosphonate moiety of glyphosate, seen most drastically in
the TIPS enzyme (pink), thereby narrowing the inhibitor binding site (residue numbers are for E.
coli EPSPS, and is equivalent to Gly101, Thr102, and Pro106 in plants) (Adapted from Funke et
al., (23)).

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 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 3. View of the catalytic domain of PPO. The porphyrin substrate is centered on top of α-helix 8
(green) and stabilized by several interactions with residues lining the pocket. The yellow spheres represent
the position of Gly210, the deletion of which confers TSR. The two groups of pink spheres represent
Arg128 and Gly399, which can be substituted to impart TSR.

40
 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 4. Summary of the mechanisms of resistance to glyphosate. Observed (regular font) and
putative (italicized font) glyphosate resistance mechanisms. Glyphosate (red circles) crosses the plasma
membrane (blue) to enter the cytoplasm and is transported into the chloroplast (green) to the target-site
enzyme, EPSPS, in herbicide-sensitive plants. Expression of EPSPS variants with 1-3 amino acid
differences can confer resistance to the herbicide (19, 26, 29). Target gene duplication of EPSPS produces
more EPSPS protein that remains sensitive to glyphosate, requiring proportionally more glyphosate to cause
complete inhibition of the extra enzyme (52). Other routes to resistance include sequestration in the vacuole
and enhanced metabolism by aldo-keto reductases (79, 80, 147). Altered import/export from the chloroplast
and/or cytoplasm may also alter glyphosate effectiveness, but these mechanisms remain hypothetical and
have not been documented in weeds. The phoenix phenomenon shown in Figure 6 results in reduced
translocation and involves a currently unknown mechanism triggering cell death upon glyphosate
application.

41
 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 5. Summary of non-target-site resistance (NTSR). Plants can evolve resistance to a herbicide by
reducing its absorption, altering its translocation and/or sequestration, developing a rapid necrosis of the
foliage (phoenix phenomenon), or via degradation of the active ingredient through the phases I, II and III
of metabolism.

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 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 6. The phoenix phenomenon in plants treated with glyphosate. Both giant ragweed (Ambrosia
trifida) biotypes were sprayed with 0.7 kg ha-1 glyphosate. Glyphosate-susceptible A. trifida at 2 days (A)
and 21 days (B) after glyphosate treatment, behaving like most plants treated with glyphosate. Growth stops
but no injury is observed for the first few days. Glyphosate-resistant A. trifida at 2 days (C) and 21 days
(D) after glyphosate treatment. In plants exhibiting the phoenix phenomenon, older leaves dessicate very
rapidly, trapping most of the glyphosate in dead tissues, and the new shoots emerge undamaged from the
glyphosate treatment. Cover image from (95) with permission from John Wiley.

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 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 7. Summary of herbicide metabolism in a plant cell. Herbicides are normally taken through the
three phases of metabolism as they are detoxified by plant cells. Typically, phase I introduces small
functional groups on the structure of the active ingredient, phase II attaches a number of water-soluble
metabolites via the action of several types of transferases, and phase III moves the conjugated metabolites
to the vacuole (or the cell wall) for compartmentalization and further degradation. Active transport
sometimes requires ABC transporters (or other transporter types) to move the herbicide metabolites across
membranes.

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 Downloaded from http://www.jbc.org/ by guest on May 19, 2020
Figure 8. Examples of the reactions catalyzed by plant cytochrome P450 monooxygenases involved
in herbicide metabolism. Functional groups either on the substrates of P450 monooxygenases or on the
products of the reactions catalyze by these enzymes are shown in red.

45
 Mechanisms of evolved herbicide resistance
Todd A Gaines, Stephen O. Duke, Sarah Morran, Carlos A. G. Rigon, Patrick J Tranel,
Anita Küpper and Franck E Dayan
J. Biol. Chem. published online May 19, 2020

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