Stress-induced evolution of herbicide 
resistance and related pleiotropic effects
Authors: William E. Dyer
This is the peer reviewed version of the following article: see full citation below, which has been 
published in final form at https://dx.doi.org/10.1002/ps.5043. This article may be used for non-
commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Dyer, William Edward. "Stress-induced evolution of herbicide resistance and related pleiotropic 
effects." Pest Management Science 74, no. 8 (August 2018): 1759-1768. DOI:10.1002/ps.5043.
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Stress-induced evolution of herbicide
resistance and related pleiotropic effects
William Edward Dyer*
Abstract
Herbicide-resistant weeds, especially those with resistance to multiple herbicides, represent a growing worldwide threat 
to agriculture and food security. Natural selection for resistant genotypes may act on standing genetic variation, or on 
a genetic and physiological background that is fundamentally altered because of stress responses to sublethal herbicide 
exposure. Stress-induced changes include DNA mutations, epigenetic alterations, transcriptional remodeling, and protein 
modifications, all of which can lead to herbicide resistance and a wide range of pleiotropic effects. Resistance selected in this 
manner is termed systemic acquired herbicide resistance, and the associated pleiotropic effects are manifested as a suite of 
constitutive transcriptional and post-translational changes related to biotic and abiotic stress adaptation, representing the 
evolutionary signature of selection. This phenotype is being investigated in two multiple herbicide-resistant populations of the 
hexaploid, self-pollinating weedy monocot Avena fatua that display such changes as well as constitutive reductions in certain 
heat shock proteins and their transcripts, which are well known as global regulators of diverse stress adaptation pathways. 
Herbicide-resistant populations of most weedy plant species exhibit pleiotropic effects, and their association with resistance 
genes presents a fertile area of investigation. This review proposes that more detailed studies of resistant A. fatua and other 
species through the lens of plant evolution under stress will inform improved resistant weed prevention and management 
strategies.
Keywords: herbicide resistance; non-target site resistance; plant stress response; systemic acquired herbicide resistance; weeds
other pleiotropic effects. Plants have evolved under a wide range
of biotic and abiotic stresses and in response have evolved adap-
tive strategies to deal with both. To combat biotic invasions by
pathogenic bacteria and fungi, they have evolved highly effec-
tive innate and induced immune responses.5 The components and
pathways of pattern-triggered immunity6 and systemic acquired
resistance (SAR)7 are well characterized, involving recognition, sig-
nal transduction, and transcriptional reprogramming in response
to biotic challenge. Systemic acquired acclimation describes a con-
stitutive upregulation of proteins that confer enhanced resistance
to subsequent abiotic stresses.8 There are a number of commonal-
ities between the two responses,9,10 including cell-surface recep-
tors that recognize stress-related molecules, components of signal
transduction cascades, and the central role of reactive oxygen
species (ROS).11 Priming of pattern-triggered immunity and SAR
can require multiple, consecutive exposures to modest, perhaps
hormetic (see below) stresses,6 as has been noted for the evo-
lution of NTSR.4,12 An intriguing and potentially related initiator
of NTSR was recently proposed: microbial plant endophytes that
either directly metabolize xenobiotics or prime host plant stress
responses that include NTSR.13
When exposed to sublethal herbicide doses by improper appli-
cations or environmental constraints, susceptible plants respond
in much the same as they do to other abiotic stresses (reviewed
in Alberto et al.14). Stress resulting from abiotic sources including
several herbicides15,16 induces rapid ROS generation, which in
turn impacts multiple pathways and cellular components.4,17
SAR and systemic acquired acclimation are characterized by
the constitutive, heritable upregulation of genes/enzymes for
redox maintenance, xenobiotic metabolism, and other protective
pathways,18,19 likely as a result of rapid yet persistent changes
in abiotic stress-specific20 transcription factors and non-coding
RNAs.21 Similarly, stress-induced changes in protein PTMs like
phosphorylation and cysteine oxidation modify enzymatic and
regulatory activities, allowing plants to rapidly adjust carbon
flux.22,23 Plant transcriptome, proteome, and PTM changes caused
by herbicide stress are quite similar to those resulting from abiotic
and biotic stresses,24–31 supporting the existence of the SAHR
phenotype proposed here. Although not the focus of this review,
herbicide safeners also induce similar gene expression changes
likely through oxylipin-mediated signaling,32 and the unclear
relationship between safeners and NTSR evolution is currently
under investigation.33,34 Given that systemic acquired acclimation
and SAR are better understood than NTSR, this review proposes
that the plant stress literature may be fertile ground for gen-
erating hypotheses about NTSR evolution and its pleiotropic
effects.
The widely accepted theory for the origin of NTSR is that her-
bicides impose Darwinian selection on standing genetic variation
in weed populations, and sublethal doses select for pre-existing
individuals able to evolve resistance.35,36 Surviving individuals are
thought to subsequently accumulate additional alleles for NTSR
and/or MHR in succeeding generations, mostly through outcross-
ing. However, because the majority of the world’s worst annual
weeds are self-pollinating,37,38 an additional theory is proposed
here that does not rely on outcrossing and incorporates a deeper
understanding of the plant stress response. As discussed further
below, the theory proposes that stress imposed by sublethal herbi-
cide exposure creates a profoundly altered physiological state that
provides novel sources of variation upon which selection for MHR
and NTSR can act, without the need for additional alleles obtained
through outcrossing.
Figure 1. Theoretical herbicide dose–response graph, showing the
hormetic stimulation region (A) and the no observable adverse effect
level (B).
Sublethal herbicide doses create a continuum of responses that
have implications for resistance evolution, as illustrated by a typical
dose–response curve for herbicides (or almost any inhibitor of any
organism; Fig. 1). The stimulatory effect seen at very low doses of
the response curve (region A) is termed hormesis, a poorly under-
stood but likely universal response.39 A recent plant study seek-
ing a mechanistic explanation showed that hormetic doses of a
disease resistance-inducing chemical enhanced Arabidopsis root
growth and suppressed the expression of photosynthesis- and
respiration-related genes, while higher sublethal doses induced
a very different suite of defense-related transcripts.40 This find-
ing, along with the fact that reduced herbicide applications can
select for resistance in Avena fatua41 and other species42 supports
the idea that hormetic and sublethal doses are part of a con-
tinuum that induces overlapping adaptive responses like NTSR.
Interestingly, point B in Fig. 1 is often termed the no observable
adverse effect level, although the absence of observable damage
should not suggest that this dose is physiologically neutral. In fact,
this dose of glyphosate induced numerous proteome changes in
the fungus Aspergillus nidulans with no other observable effects.43
Hormetic herbicide doses may play a part in resistance evolution in
weeds, because highly resistant and less resistant subpopulations
from a heterogeneous acetyl CoA carboxylase inhibitor-resistant
Alopecurus myosuroides metapopulation responded differentially
to hormetic herbicide doses (Belz R, personal communication). Fur-
ther, the recent widespread weed exposure to very low (perhaps
hormetic) volatilized dicamba doses in the Midwestern United
States may have implications for resistance evolution.
Plants exposed to (probably) hormetic doses and (certainly)
higher sublethal doses of herbicides experience stress, and
stress is well known to induce profound physiological changes.
Stress-induced mutagenesis,44,45 originally thought to operate
only in prokaryotes, is widely documented in plants in response
to both biotic and abiotic stressors. Mutations are caused by
rapid ROS generation, which causes random breaks in DNA46
and induces other systemic stress responses.15,16 The idea that
herbicides and other pesticides can increase mutation frequen-
cies is not new,47 although it is rarely discussed. In addition
to ROS-mediated DNA damage, sublethal herbicide exposure
can also lead to genetic and epigenetic changes such as gene
amplification48 and altered DNA methylation patterns.49 And as
discussed above, changes in ROS-mediated signal transduction,
transcriptional remodeling, and PTM patterns create a significantly
altered physiological environment during herbicide stress, with
attendant widespread pleiotropic effects.
If herbicide stress is like other abiotic stresses, weeds exposed
to sublethal doses unleash a vast array of heritable epigenetic,
biochemical, and physiological defense/repair mechanisms, and
together these changes provide novel sources of variation upon
which selection for NTSR can act. As shown for systemic acquired
acclimation and SAR, this variation allows selection on genome,
transcriptome, and proteome changes that confer a suite of
stress-response adaptations, resulting in NTSR and the SAHR
phenotype. As previously proposed,45 the genetic association
between stress and adaptation50 challenges widespread assump-
tions about the evolution of herbicide resistance. More specifically,
estimates for the initial frequency of resistance alleles and con-
stancy of mutation rates used in traditional population genetic
models51,52 may be very different in a stressed plant from those of
standing genetic variation, and revised values incorporated into
resistance models may lead to novel and useful predictions about
NTSR evolution.
3 PLEIOTROPIC FITNESS COSTS
Of the potential pleiotropic effects associated with resistance, fit-
ness costs as predicted by Fisher in 193053 have been the most
studied and have shaped our expectations about herbicide resis-
tance for decades. Under annual applications of herbicides with
the same mode of action, weed populations are expected to fix
resistance alleles regardless of their cost, because continued resis-
tance is necessary for survival. However, more realistic rotations
among modes of action, non-herbicide integrated weed manage-
ment strategies, and other weed suppression tactics cause fluc-
tuating selection pressure on resistance alleles with pleiotropic
fitness costs. Under varying selection pressures, overall popula-
tion genetic variation and cycling of allele frequencies should
be maintained.54 Thus, the theoretical relationship between NTSR
alleles and pleiotropic fitness cost alleles can become disassoci-
ated over time, or their effects modified through compensatory
adaptation.55,56 Understanding this relationship is essential to pre-
dict the evolutionary fate of NTSR alleles.
The first well-characterized resistant weed species (Senecio vul-
garis L. with target site resistance to photosystem II inhibitors57)
fit Fisher’s prediction of fitness costs exactly58 and shaped our
expectations for the next 20 years. However, more recent examples
from multiple disciplines show that fitness costs are not neces-
sarily a consequence of evolved resistance to insecticides59 or
fungicides,60 and the situation for herbicides remains unclear.61
In entomology, there are ongoing controversies concerning the
fitness of laboratory- versus field-selected insecticide-resistant
populations59 and in weed science, determining how to properly
make fitness comparisons has been discussed at length.61,62 Thus,
the correlation between resistance and fitness costs is not abso-
lute, but the co-occurrence of other pleiotropic effects with resis-
tance is consistent across many species, as shown below. For target
site resistance, pleiotropic effects are likely conferred by the resis-
tance gene, while NTSR pleiotropic effects are due to combinations
of known and unknown genes.
4 PLEIOTROPIC EFFECTS OF RESISTANCE
Table 1 shows some known pleiotropic effects associated with
herbicide resistance. For the purposes of this review, the effects are
restricted to those with a non-obvious association with resistance
mechanisms, are not necessarily associated with fitness costs, and
are constitutively present prior to herbicide exposure. Populations
with both target site and non-target site resistance are included, to
illustrate that unexpected pleiotropic effects are associated with
both. It should be noted here that it is unclear if the pleiotropic
effect is directly conferred by resistance gene(s), or if it is associated
with the action of other genes unrelated to resistance.
Whole-plant pleiotropic effects include a phenological asso-
ciation between seed dormancy/germination and resistance in
several species. MHR A. myosuroides, Lolium rigidum, and Kochia
scoparia populations had lower germination than their suscep-
tible counterparts in certain environments. By contrast, aceto-
lactate synthase inhibitor-resistant K. scoparia germinated more
rapidly than susceptible counterparts at low temperatures, indi-
cating that this association may be complex. Studies of 32 natural
Ipomoea purpurea populations showed that glyphosate-resistant
populations displayed lower anther–stigma distances and thus
had higher rates of self-pollination than susceptible populations.
Although such changes in seed germination and pollination rates
would no doubt create fitness effects in all environments, negative
cross-resistance of triazine-resistant populations to other herbi-
cides would confer a fitness benefit only under ongoing herbicide
selection.
At the biochemical level, triazine-resistant populations of several
species display altered photosynthetic inhibition characteristics,
chloroplast membrane lipid composition, and elevated heat tol-
erance, features that do not seem to be directly related to their
well-characterized target site mutations. Paraquat resistance is
associated with the monogenic upregulation of several ROS detox-
ification enzyme activities in Conyza bonariensis, indicating the
involvement of a pleiotropic transcription factor. At the molecu-
lar level, essentially all transcriptome analyses of resistant popu-
lations show some kind of constitutive gene expression changes
unrelated to biochemical resistance mechanisms. For example,
several species display upregulation of SAR-associated transcripts
for protein kinases and transcription factors involved in disease
resistance, salicylic acid response, and others. In particular, MHR
A. fatua contains a number of constitutive transcript, protein, and
PTM changes related to the stress response, as discussed below.
This phenotype may be more common than currently recognized,
because most transcriptome and proteome surveys to date have
focused on known mechanisms of resistance, to the exclusion of
other possibilities. Pleiotropic effects of genes conferring resis-
tance to fungicides83 and insecticides84 are well documented, and
so their presence in herbicide-resistant populations should not be
surprising.
Resistance conferred by amplification of the gene encoding
the herbicide target enzyme deserves extra attention, due to
the high potential for pleiotropic effects. Detailed investigations
of glyphosate-resistant Amaranthus palmeri populations reveal
that resistant plants possess multiple copies of a 297-kb cassette
containing one copy of the 5-enol-pyruvylshikimate 3-phosphate
synthase gene and 72 additional open reading frames.48 The
amplified cassette shows little homology to sequences from
glyphosate-sensitive A. palmeri plants, indicating that it is a
recent evolutionary event and was not present before glyphosate
use. Among the additional predicted cassette genes are those
similar to stress response genes, while others are involved in
transposon mobility and DNA replication. (Molin W, and Saski
C, personal communication). If shown to be functional, the
potential for pleiotropic effects of these additional genes is
Table 1. Constitutive pleiotropic effects associated with target site and non-target site herbicide resistance
Species Resistance Target (T) or non-target (N) Pleiotropic effects Reference
Whole plant effects
Lolium rigidum MHR Both Altered seed germination 63
Alopecurusmyosuroides ALS inhibitors T Altered seed germination patterns 64
Kochia scoparia Glyphosate T Reduced seed longevity and germination 65
Kochia scoparia ALS inhibitors ? Faster germination at low temperatures 66
Ipomea purpurea Glyphosate ? Reduced anther–stigma distance 67
E. crus-galli; C. canadensis Triazines T Negative herbicide cross-resistance 68
Biochemical effects
S. vulgaris; C. album Atrazine T Altered chloroplast lipids 69
Glycinemax (cell culture) Atrazine T Altered chloroplast lipids; heat tolerance 70
Brassica napus Atrazine T Increased sensitivity to photoinhibition 71
Brassica campestris Triazines T Increased chlorophyll a/b complex 72
Conyza bonariensis Paraquat N Increased ROS detoxification enzymes 73
Kochia scoparia Dicamba ? Increased chalcone synthase expression 74
Eleusine indica Glyphosate T Phosphofructokinase gene amplification 75
Amaranthus palmeri Glyphosate T Numerous co-amplified predicted genes 48
Alopecurusmyosuroides ALS inhibitors N Disease resistance and peroxidases 76
Altered flavonoid metabolism 77
Alopecurus aequalis Mesosulfuron N Salicylic acid-responsive kinases 78
Avena fatua MHR N Numerous differential genes 28
Altered protein and PTM profiles 79
Lolium rigidum MHR N Overexpressed protein kinases 26
Ambrosia trifida Glyphosate ? Altered pathogen response transcripts 31
Eleusine indica Paraquat ? Numerous differentially expressed genes 80
Echinochloa crus-galli Quinclorac ? Photosynthetic and defense proteins 81
Cicer arietinum Imazethapyr ? Transcription factors; core metabolism 82
substantial. In the related species Amaranthus tuberculatus,
an extra ring chromosome containing amplified copies of the
5-enol-pyruvylshikimate 3-phosphate synthase gene appears to
be the result of stress-induced chromosomal alterations.85 Further
detailed investigations of gene amplification mechanisms and
pleiotropic effects in these and other resistant species will no
doubt lead to important insights into NTSR evolution.
Although not yet documented in resistant populations,
herbicide-induced epigenetic effects have been confirmed,
first by low-resolution techniques showing evidence for
glyphosate-induced86 and atrazine-induced87 alterations in
DNA methylation patterns. More recently, detailed investigations
of Arabidopsis plants treated with sublethal glyphosate doses
showed that methylation changes were not uniformly spread
across the genome, but many were clustered in differentially
methylated regions associated with transposons, and the major-
ity were related more specifically to glyphosate injury than to
other abiotic and biotic stresses, suggesting a level of precision in
DNA methylation response.49 Current research will determine if
transposons are in fact activated by these changes, and if their acti-
vation is associated with pleiotropic effects as seen elsewhere88
(Westwood J, personal communication).
5 MULTIPLE HERBICIDE RESISTANT AVENA
FATUA
We recently described the MHR3 and MHR4 populations ofA. fatua
that are resistant to members of all selective (in small grains) her-
bicide families available in North America for A. fatua control.89,90
Specifically they are resistant to the acetyl-CoA carboxylase
inhibitors fenoxaprop-P-ethyl, tralkoxydim, and pinoxaden, the
acetolactate synthase inhibitors imazamethabenz-methyl and
flucarbazone, the growth inhibitor difenzoquat, the photosys-
tem I inhibitor paraquat (MHR3 only), and the very long chain
fatty acid biosynthesis inhibitors triallate and prosulfocarb, with
resistant/susceptible ratios ranging from 1.4 to 57. MHR plants
do not contain known target site mutations for acetyl-CoA car-
boxylase or acetolactate synthase inhibitors, and the cytochrome
P450 monooxygenase inhibitor malathion partially reversed the
resistance phenotype for a subset of herbicides, indicating the
involvement of P450-mediated NTSR mechanisms.90 Resistance
to flucarbazone, imazamethabenz-methyl, and pinoxaden is con-
trolled by three separate but linked nuclear genes.91 Although all
the specific biochemical mechanisms conferring NTSR to this
wide array of herbicides are not yet known, resistance to
fenoxaprop-P-ethyl and imazamethabenz-methyl may be due to
reduced rates of carboxylesterase activity, an enzymatic step nec-
essary for cellular uptake and conversion of these pre-herbicides
into active inhibitors.92 Enhanced herbicide catabolism does not
appear to play a major role in MHR A. fatua, because glutathione
S-transferase specific activities towards fenoxaprop-P-ethyl and
imazamethabenz-methyl were not different between untreated
MHR and susceptible plants34 and only a relatively small number
of constitutively elevated xenobiotic catabolism transcripts and
proteins were detected in MHR plants.28,34 The A. fatua NTSR
phenotype is heritable and stable for multiple generations in the
absence of herbicide treatment.90 Specific herbicide use histories
from the fields where the MHR A. fatua populations were collected
are not available, but regional practices indicate that they evolved
under 40 years of chronological rotations of selective herbicides.90
Additional insights into MHR A. fatua evolution may be gleaned
from comparison with the previously characterized FG93R22 A.
fatuapopulation derived from the same location as MHR with resis-
tance to only triallate and difenzoquat.93 Resistant/susceptible
ratios to these two herbicides are similar in MHR and FG93R22, sug-
gesting commonalities in resistance mechanisms. Triallate resis-
tance in FG93R22 is conferred by reduced rates of sulfoxidase
activity,93,94 a pre-herbicide activation step required for toxicity,
and is controlled by two recessive nuclear genes.95 Difenzoquat
resistance is associated with herbicide efflux from the cytoplasm
and irreversible binding to cell wall components.96 Although it is
not known if MHR plants have the same mechanisms as FG93R22,
it is tempting to speculate that the constitutive elevation of xeno-
biotic transporter transcripts in MHR plants28 is related to difenzo-
quat resistance.
Potential fitness costs associated with the MHR A. fatua pheno-
type are unclear. Under non-competitive conditions in the green-
house, photosynthetic and relative growth rates were not different
between susceptible and MHR plants, but MHR plants produced
fewer tillers and seeds than susceptible plants.89 However, a sec-
ond greenhouse study showed that there were no differences in
competitive abilities between susceptible and MHR plants under
gradients of biotic (competition withTriticumaestivum) and abiotic
(limiting nitrogen) stresses.97 Fitness comparisons have not been
conducted under field conditions.
6 PLEIOTROPIC FEATURES IN RESISTANT
WEEDS
As shown in Table 1, all resistant populations characterized to date
exhibit various pleiotropic effects at the whole plant, biochemical,
or molecular level. Of these, MHR A. fatua contains more docu-
mented molecular changes than other species, as well as several
unusual resistance mechanisms, and so it has been used as a model
to investigate the primary stress effects associated with the evo-
lution of NTSR.28,34 Initial efforts to describe these have focused
on constitutive differences between MHR and susceptible popu-
lations, because they are most likely to represent changes that
would lead to pleiotropic effects. Transcriptome and proteome
analyses showed that MHR plants have constitutively higher lev-
els of transcripts and proteins with functions in stress response,
disease resistance, redox maintenance, xenobiotic transport and
catabolism, transcriptional regulation, and signal transduction.28
For example, transcripts for three protein kinases including the
lectin S-receptor-like serine/threonine-protein kinase LecRK2, a
well-characterized SAR receptor/signal transducer, were constitu-
tively elevated in MHR4 plants. By contrast, levels of heat shock
protein (HSP) transcripts and proteins were constitutively lower
than in susceptible plants.28 In the first phosphoproteome and
redox proteome comparisons between resistant and susceptible
plants of any species, PTMs of proteins with functions in core cel-
lular processes were shown to be constitutively reduced in MHR
A. fatua plants, perhaps in association with certain fitness costs.79
PTMs of proteins involved in xenobiotic and stress response, ROS
detoxification/redox maintenance, heat shock response, and intra-
cellular signaling were more abundant in MHR than susceptible
plants. Overall, this suite of constitutive changes in MHR A. fatua
populations could represent pleiotropic effects of NTSR, or it is
more likely that they are an evolutionary signature of ongoing
stress responses like systemic acquired acclimation or SAR, of
which NTSR is only one aspect.
7 NTSR: A ROLE FOR HEAT SHOCK
PROTEINS?
Of all the changes associated with MHR A. fatua discussed above,
perhaps the most intriguing is the constitutive reduction of HSP
transcripts and proteins28 and altered HSP PTM patterns.92 In all
eukaryotes, HSPs are typically induced and subjected to PTMs in
response to heat shock as well as a wide variety of biotic and abi-
otic stresses,98 and they are implicated in hormetically induced
epigenetic effects on phenotype.99 HSPs primarily function as
molecular chaperones that repair misfolded client proteins dur-
ing stress, although a subset regulate transcription factors for a
diverse set of genes.100 For example, several HSPs are involved
in adaptation of the yeast Saccharomyces cerevisiae to the aux-
inic herbicide 2,4-D.101 In the same species102 and related fungi,103
the Ssa1p HSP70 binds to and represses the Pdr3p transcription
factor controlling expression of an ATP-binding cassette trans-
porter that confers multiple drug resistance. Thus, a reduced
level of Ssa1p derepresses transporter expression and leads to
enhanced resistance in fungi. In MHR A. fatua, constitutively
reduced HSP transcripts and proteins, and elevated xenobiotic
transporter transcripts28 may indicate the presence of an orthol-
ogous regulatory system in higher plants that contributes to her-
bicide resistance.
Constitutively reduced HSP profiles have also been reported in
stress-tolerant plant biotypes. For example, a comparison of nat-
ural Chenopodium album biotypes showed that those adapted
to stressful environments contained lower levels of certain HSPs
than those from moderate habitats, both constitutively and in
response to thermal stress.104 Comparable ecological studies of
Drosophila105 and Arabidopsis106 reported similar findings, sug-
gesting that this is an evolutionarily conserved strategy for stress
acclimation. In a series of papers starting in 1998, it was proposed
that HSP90 functions as a global regulator for stress adaptation by
acting as a capacitor for phenotypic variation.107,108 Under stress,
HSP90 levels are typically induced but under some conditions
cannot keep pace with demands for client protein re-folding and
repair of regulatory pathway components. Limiting HSP90 levels
thus allows normally buffered polymorphisms to persist, providing
novel sources of variation upon which selection can act, and the
polymorphisms that are adaptive in the new stressed environment
become fixed. Experimental evidence shows that pharmacologic
reductions of HSP90 levels inDrosophila108 and Arabidopsis107 pro-
duced a wide variety of novel phenotypes, and the same treat-
ment in S. cerevisiae and Candida albicans led to the evolution of
resistance to two classes of fungicides.109,110 The role of HSP90 as
a global regulator of diverse stress-induced signaling pathways is
now well accepted.98 The results from MHR A. fatua plants indicate
that a very different stress, i.e. sublethal herbicide damage, may
have caused them to evolve a similar strategy during herbicide
selection, leading to a heritable downregulation of certain HSPs
and the consequent evolution of NTSR.
It is important to point out that the phenotype just described is
much more complex than the acute induction of HSPs in response
to a single stress event, which is well documented in eukaryotes.98
However, neither acute HSP induction nor the constitutive HSP
reductions seen in MHR A. fatua have been investigated in resis-
tant populations of other species. It is hoped that other transcrip-
tome and proteome datasets will be re-examined for these and
other constitutive changes that may inform a better understand-
ing of NTSR evolution. Similarly, it will be of interest to determine
whether herbicide-resistant populations are also more tolerant to
other biotic and abiotic stresses, as is well documented for sys-
temic acquired acclimation and SAR. For example, a coral (Acropora
formosa) population adapted to a polluted environment was more
tolerant to glyphosate and elevated temperature than one from a
pristine environment.111 For MHR A. fatua, research is underway to
determine levels of resistance to other abiotic and biotic stresses.
8 IS MHR AVENA FATUA AN OUTLIER OR
HARBINGER?
As noted above, the number and variety of constitutive transcript
and protein alterations in MHR A. fatua appears to be larger (or
better documented) than in other resistant populations. Also, A.
fatua and the MHR phenotype have several characteristics that are
not typical of other species. First, unlike L. rigidum, A. myosuroides,
and A. tuberculatus, A. fatua is a predominantly self-pollinating
species with limited seed dispersal and substantial levels of seed
dormancy,112 features that tend to delay the appearance and
spread of novel traits. Second, A. fatua is an allohexaploid, in
contrast to the diploid species just mentioned. Polyploidy provides
additional standing genetic variation upon which selection can
act, but at the same time may buffer novel resistance mutations
through expression of homoeologous sensitive alleles, slowing
HR evolution.113 Third, more than 40 years of annual herbicide
applications were required before the MHR populations became
noticeable,90 a considerably longer time frame than is typical. Thus,
the MHR A. fatua phenotype may be an outlier when compared
with other species.
However, the environment, herbicide spectrum, and chronology
of herbicide use that selected for MHR A. fatua populations are
not unusual, and so other species in similar circumstances may
be following a parallel evolutionary path. Certainly, MHR is not
unusual, since its incidence is rapidly increasing worldwide espe-
cially in monocots.114 MHR populations of self-pollinating poly-
ploid species in weedy genera like Eleusine and Echinochloa have
been reported, and if these traits contribute to a delay in NTSR evo-
lution as argued here for A. fatua, additional resistant populations
may continue to appear. Further, transcriptome and proteome sur-
veys of many HR and MHR populations of other species have also
detected SAR-like constitutive alterations,24,26 as is shown here for
MHR A. fatua. By far the largest category of elevated transcripts
before and after herbicide treatment of NTSR Apera spica-venti
populations was for nucleotide binding site-leucine-rich repeat
proteins,115 a large family of receptors involved in SAR to plant
diseases.116 Although it is possible that such constitutive changes
are unrelated to NTSR, it seems more likely that both they and NTSR
are part of a general stress response. The commonality of such
changes among resistant populations of many species indicates
that the MHR A. fatua phenotype may indeed be a harbinger of
additional, similar cases in the future.
Figure 2 shows a proposed model for some of the key cellular
players in recognition, uptake, signal transduction, and subse-
quent induced responses to sublethal herbicide doses. Actively
absorbed herbicides117 are transported by membrane-bound
receptors, of which (except for auxinic herbicides118) very little
is known. If they are similar to receptor-like kinases involved
in systemic acquired acclimation and SAR, an HSP90-mediated
signal transduction cascade is activated, leading to transcriptional
remodeling, PTMs, and possibly epigenetic events. These changes
in turn lead to the SAHR phenotype through translation or modi-
fication of stress-related proteins with functions in defense, repair,
ROS management, xenobiotic inactivation, and likely NTSR. This
Figure 2. A model for sublethal herbicide-induced stress and plant
responses. Actively absorbed herbicides are transported by cell-surface
receptors, which may be similar to receptor-like kinases involved in sys-
temic acquired acclimation and systemic acquired resistance (SAR) that
activate a heat shock protein 90 (HSP90)-mediated signal transduction
cascade, leading to transcriptional remodeling, post-translational modi-
fications (PTMs), and possibly epigenetic events. These changes in turn
cause translation or modification of stress-related proteins with functions
in defense, repair, reactive oxygen species (ROS) management, xenobiotic
inactivation, and likely non-target site resistance (NTSR). RLKs, receptor-like
kinases; CH3, DNA methylation; nc RNAs, non-coding RNAs; TFs, transcrip-
tion factors.
review proposes that an experimental focus on the components
of these processes will provide valuable insights into NTSR evo-
lution. Much has been learned about SAR and other plant stress
responses by characterizing their receptors, the ensuing signal
transduction, and induced effects.
9 FINDING AND STUDYING NASCENT NTSR
POPULATIONS
Valuable insights into NTSR and its evolution can be obtained by
changing the way that we sample for resistance. Because most
if not all field-selected NTSR populations originated from pro-
ducer complaints of herbicide non-performance, it is likely that the
university or company researchers visiting these fields collected
only the most robust and therefore most resistant plants/seeds
for further study, and the barely surviving weeds under the crop
canopy (survivors) were ignored. However, these survivors may
have been in the early stages of NTSR evolution, and a conscious
effort to sample and study them could provide valuable informa-
tion. For example, survivor and robust NTSR populations of the
same species could be compared to distinguish between two pos-
sible chronologies of NTSR evolution. In the traditionally accepted
evolutionary chronology, herbicides initially select for direct NTSR
mechanisms such as enhanced metabolism or transport.36 How-
ever, high fitness costs correlated with these alleles limit popula-
tion sizes of survivors, so their appearance is usually overlooked.
Over time, additional stress responses evolve that compensate for
the initial fitness deficits, allowing populations to recover, prosper,
and be noticed by producers. The genetic basis of this chronol-
ogy for fungicide resistance in Aspergillus nidulans was recently
described.119 If this is also true for herbicide selection, then the-
ory predicts that survivor populations would display high fitness
costs,53 while costs would have been purged or compensated for
in robust populations. At the molecular level, constitutive changes
in survivor populations would include the downregulation of tran-
scripts/proteins with housekeeping functions related to fitness.
A second possible chronology of NTSR evolution is more
aligned with the scenario proposed in this review: general stress
responses are first induced by sublethal herbicide doses, and
these can simultaneously include both NTSR and any number
of pleiotropic effects. If pleiotropic fitness costs are high, limited
population growth may eventually be ameliorated through out-
crossing and/or compensatory adaptation. However, if fitness
costs are moderate, NTSR populations will rapidly predominate
under continuing herbicide use. In this scenario, both survivor
and robust populations would be expected to display SAR-related
constitutive changes in transcripts and proteins, as is seen for
the species listed in Table 1 and the MHR A. fatua populations
described here.
Obtaining evidence for these two possible chronologies of NTSR
evolution may be difficult, but both will add to our basic knowl-
edge about plant responses to stress and will also inform improved
resistant weed management strategies. If pleiotropic effects are
related to primary stress effects as argued above, then a bet-
ter characterization of pleiotropy in well-characterized NTSR pop-
ulations of L. rigidum, A. myosuroides, and species with strong
genetic/genomic resources will be informative. Further, because
it is established that low-dose impacts cannot be predicted from
high dose experiments,120 further research on the role of initial
sublethal herbicide effects on NTSR evolution is clearly warranted.
10 CONCLUSION
This review proposes that, in addition to the evolution of her-
bicide resistance through selection on standing genetic varia-
tion, it also evolves in response to stress induced by sublethal
herbicide exposure. In the latter case, NTSR appears to be only
one of many possible stress-induced responses, and together,
these responses can lead to the SAHR phenotype and a num-
ber of unpredictable pleiotropic effects. This view is supported by
phenological changes associated with resistance, and by transcrip-
tome and proteome surveys showing that current resistant pop-
ulations exhibit a number of constitutive alterations apparently
unrelated to NTSR mechanisms. If designed correctly, such surveys
generate vast amounts of unbiased data, and then the subsequent
challenge often centers on deciding which differences between
resistant and susceptible populations to verify and pursue. In the
NTSR surveys published to date, this choice was based on pre-
vious knowledge about established herbicide resistance mecha-
nisms like enhanced metabolism, and so the results reinforce what
is already known. A more informative approach is proposed here
that includes a focus on candidates for the primary regulators and
physiological markers of stress, with the idea that such features
can provide deep insights into the fundamental mechanisms of
NTSR evolution and its pleiotropic effects. Resistance due to gene
amplification can also present unique opportunities, by provid-
ing a window into the potential pleiotropic effects of genomic
rearrangements and additional genes that are co-amplified along
with herbicide target site genes. The co-occurrence of plants with
both target site resistance and NTSR in the same population indi-
cates that current definitions of these terms may be unneces-
sarily restrictive from a research standpoint121 and this author
believes that a shifted focus on the primary stress effects of sub-
lethal herbicide exposure will inform both types of resistance.
And finally, as both the appearance of NTSR populations and the
worldwide occurrence of environmental stress are expected to
increase, expanded research on NTSR evolution and its potential
for pleiotropic effects should be a high priority.
ACKNOWLEDGEMENTS
Sincere appreciation is extended to Jonny Gressel for the invita-
tion to write this review, to René Feyereisen, William Molin, Regina
Belz, and James Westwood for discussion and sharing unpub-
lished work, to Tyler Maxwell and Katie Steward for graphics, and
to seven anonymous reviewers for their constructive comments.
Work in the author’s laboratory is supported by USDA-NIFA-AFRI
grant 2016-2067 013-24 888, US EPA Strategic Agricultural Initia-
tive grant X8–97873401-0, Bayer CropScience, the Montana Nox-
ious Weed Trust Fund, the Montana Wheat and Barley Committee,
and the Montana Agricultural Experiment Station.
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