ROLE OF REACTIVE OXYGEN SPECIES
IN AUXIN HERBICIDE PHYTOTOXICITY:
CURRENT INFORMATION AND HORMONAL
Iva McCarthy-Suárez, PhD
Role of reactive oxygen species in auxin herbicide
phytotoxicity: current information and hormonal implications
Instituto de Biologia Molecular y Celular de Plantas. Consejo Superior de
Investigaciones Científicas (C.S.I.C.)-Universidad Politécnica de Valencia. Ingeniero
Fausto Elio s/n, 46022, Valencia, Spain
Running head: Role of ROS in auxin herbicide toxicity
Correspondence: Dr. Iva McCarthy-Suárez, Instituto de Biologia Molecular y Celular
de Plantas, Ingeniero Fausto Elio s/n, 46022, Valencia, Spain. Tel: (+34) 96 387 78 86;
Fax: (+34) 96 387 78 59; E-mail: email@example.com
Word count = 10.804
Reactive oxygen species (ROS) have been suggested as participants in the injury
and the death of sensitive plants treated with auxin herbicides. However, their precise
role in the phytotoxicity of these compounds has not been completely elucidated. Not
only ROS might be essential to induce plant senescence and tumors, but they might be
crucial to the overproduction of ethylene, abscisic acid, and jasmonic acid known to be
triggered upon application of these compounds. Also, it has not been clarified which are
the main sources of ROS overproduction and their subcellular location in auxin
herbicide-treated plants. Recent studies have suggested a role for xanthine oxidase
(XOD), a superoxide radical (O2.-)-producing and nucleic acid catabolism-related
activity, as well as for leaf peroxisomes, in the oxidative stress and the senescence
induced by auxin herbicides in sensitive plants. However, confirmatory studies at the
molecular level are still needed, as well as studies on the involvement of gibberellins,
cytokinins, and polyamines, and their corresponding hormone-ROS crosstalk, in auxin
herbicide mode of action. The results of these studies might be relevant not only to
elucidate the phytotoxic mechanism of auxin herbicides, but to design ecologically-safer
herbicides or auxin herbicide-resistant plants for agriculture.
Keywords: Auxin herbicides, ROS, Senescence, Apoptosis, Plant tumors.
Overproduction of reactive oxygen species (ROS) and associated oxidative stress have
been shown to play a key role in the phytotoxicity of bipyridinium, diphenyl ether,
cyclic imide, oxadiazole, lutidine and urea herbicides (Arora et al., 2002). The
photosynthesis inhibition and the lipid membrane destruction caused by these herbicides
have been correlated to the formation of oxygen-derived free radicals.
Recent studies have suggested that ROS might also be essential to the
phytotoxicity of auxin herbicides (Grossmann et al., 2001; McCarthy-Suárez, 2004;
Romero-Puertas et al., 2004; Šimonovičová et al., 2004; Sunohara & Matsumoto, 2004,
2008), agrochemicals used to control broad-leaved and dicotyledonous weeds in cereal
crops which mimic the effect of high auxin concentrations in plants. Thus, in Galium
aparine L. (cleaver) and Pisum sativum L. (pea) plants, the tissue damage and the death
caused by application, respectively, of the auxin herbicides quinclorac (3,7-dichloro-8-
quinolinecarboxilic acid; quinolinecarboxylic acid group) and 2,4-D (2,4-
dichlorophenoxyacetic acid; phenoxycarboxylic acid group) were correlated to an
overproduction of H2O2 in the leaves (Grossmann et al., 2001; Romero-Puertas et al.,
2004). Also, cell death in 2,4-D-exposed Hordeum vulgare L. (barley) (Šimonovičová
et al., 2004) and quinclorac-treated Zea mays L. (maize) plants (Sunohara &
Matsumoto, 2008) was linked to an excessive generation of superoxide radicals (O2.-)
and H2O2 in the roots.
That auxin herbicides induce oxidative stress in the leaves and the roots of
sensitive plants has also been demonstrated by the up-regulation of ROS-scavenging
enzymes and by the changes in the oxidative stress parameters. Thus, in the leaves of
2,4-D-treated P. sativum, as well as in the leaves and roots of quinclorac-treated
Echinocloa orizycola Vasing (barnyard grass) plants, an induction of the antioxidant
activities catalase (CAT), superoxide dismutase (SOD), guaiacole peroxidase (GPOX),
glutathione reductase (GR) and glutathione S-transferase (GST), as well as an increase
of lipid peroxidation and protein carbonyls, along with a decrease of total and protein
thiols, have been reported (McCarthy-Suárez, 2004; Romero-Puertas et al., 2004;
Sunohara & Matsumoto, 2004, 2008).
In yeast and animal systems, auxin herbicide toxicity also involves induction of
oxidative stress. In yeast, 2,4-D induces a dose-dependent overproduction of hydroxyl
radicals (·OH) capable of damaging DNA, lipid membranes and proteins (Texeira et al.,
2004). Moreover, yeasts adapted to 2,4-D are those able to up-regulate their antioxidant
defenses (e.g. CAT, SOD, glutaredoxin, alkyl hydroperoxide reductase) and to increase
their membrane fatty acid-saturation degree (Viegas et al., 2005). A pro-oxidative mode
of action for 2,4-D in yeast has also been confirmed by microarray analysis. Exposure
of yeast to 2,4-D immediately induces the genes of the fatty acid beta-oxidation and the
mitochondrial oxidative phosphorylation (Texeira et al., 2007), metabolic pathways
involved in ROS production.
Directly oxidative and pro-oxidative abilities of auxin herbicides -such as 2,4-D
and MCPA (4-chloro-2-methylphenoxyacetic acid; phenoxycarboxylic acid group)- and
of their metabolites have also been described in fish, chicken, rats and humans
(Bukowska, 2003, 2006; Martínez-Tabche et al., 2004; Bongiovanni et al., 2007; Ferri
et al., 2007; Mi et al., 2007; Bukowska et al., 2008). Moreover, in animal cells, 2,4-D
and 2,4,5-T (2,4-trichlorophenoxyacetic acid; phenoxycarboxylic acid group) have been
shown to deplete the glutathione (GSH) and the protein thiols, to cause
lipoperoxidation, and to induce the genes of the H2O2-producing fatty acid beta-
oxidation and the genes of the stress response (Palmeira et al., 1995; Bukowska, 2003,
2004, 2006; Oruc et al., 2004; Bharadwaj et al., 2005; Bongiovanni et al., 2007).
Although an excessive production of ROS seems to be responsible -at least in
part- for the death of susceptible plants treated with auxin herbicides, it has not been
clarified yet whether the ROS overproduction is an upstream event in the toxic action of
these compounds or it is a consequence of stress.
Exposure of sensitive plants to auxin herbicides, or to high doses of the natural
auxin 3-indoleacetic acid (IAA), sequentially induces in shoots (leaves + stems) an
overproduction of ethylene (ET), abscisic acid (ABA) and jasmonic acid (JA) (Hansen
& Grossmann, 2000; Grossmann et al., 2004; Kraft et al., 2007), phytohormones
implicated in plant growth inhibition, senescence and tumor formation (Smart, 1994;
Swiatek et al., 2003; Veselov et al., 2003) (Figs. 1 and 2).
High levels of ET, ABA and JA stimulate ROS production in plants (Mori &
Schroeder, 2004; Browse, 2005; Castagna et al., 2007) but high levels of ROS can also
stimulate ET, ABA and JA generation (Wu & Ge, 2004; Xing et al., 2004; Kim et al.,
2008). This means that the interplay between the ET-ABA-JA production and oxidative
stress in auxin herbicide-treated plants deserves further investigation. Given the
importance of ROS in ET, ABA and JA biosynthesis (Wu & Ge, 2004; Xing et al.,
2004; Kim et al., 2008) and signalling (Vandenabeele et al., 2003; Liu et al., 2008), as
well as the contribution of ROS not only to cell damage, senescence and death
(Bhattacharjee, 2005) but also to tumor-like growth (Laurent et al., 2005; Lee et al.,
2009), this review will discuss, in the context of the up-to-date knowledge, the role
played by ROS -as well as the possible involvement of gibberellins, cytokinins, and
polyamines - in the phytotoxicity of auxin herbicides.
Oxidative stress might be essential to the formation of ET, ABA
and JA in auxin herbicide-treated plants
Oxidative stress might be of key importance to the sequential overproduction of ET,
ABA and JA in auxin herbicide-treated plants. The ROS, essential to ET biosynthesis
(Arora et al., 2002), have been shown to mediate the auxin-induced ET production in
Vigna radiata (mung bean) hypocotyls (Song et al., 2007). Furthermore, conversion of
the ET precursor ACC (1-amino-1-cyclopropane carboxylic acid) to ET is inhibited by
free radical scavengers, as shown in Triticum aestivum (wheat) coleoptiles
(Macháčková & Zmrhal, 1981). In particular, H2O2 and O2.- induce ET biosynthesis by
up-regulating ACC synthase and ACC oxidase gene expression (Watanabe et al., 2001;
Song et al., 2007; Kim et al., 2008). Superoxide radicals can also stimulate ACC
synthase activity by increasing the enzyme affinity to its substrate (Ke et al., 2007).
Participation of ·OH radicals in the synthesis of ET from ACC has also been
demonstrated chemically (Legge et al., 1982). On the other hand, ROS are also
important for the biosynthesis of stress-induced ABA and JA. Stressed plants do not
accumulate ABA if pretreated with ROS scavengers (Zhao et al., 2001; Xing et al.,
2004). In fact, the conversion of the ABA precursor, abscisic aldehyde, to ABA is
mediated by a O2.- radical-generating enzyme, the aldehide oxidase (AO) activity
(Yesbergenová et al., 2005). Also, the overproduction of H2O2 and O2.- is an upstream
event in the generation of JA (Wu & Ge, 2004).
In spite of all these data connecting ROS to the biosynthesis of ET, ABA and
JA, no studies have assessed whether ROS might be the initial triggers of auxin
herbicide phytotoxicity. Interestingly, an upstream event in the auxin signal
transduction pathway is the extracellular production of ROS catalyzed by cell wall
peroxidases and plasma membrane NAD(P)H oxidases (Kawano, 2003). In addition, it
is known that auxin-induced ET production is mediated by ROS (Song et al., 2007) and
that plant susceptibility to auxin herbicides is inversely correlated to the antioxidant
capacity of the plant (Sunohara & Matsumoto, 2004). However, the implication of ROS
in the sequential induction of ET, ABA and JA biosynthesis in sensitive plants treated
with auxin herbicides has not been investigated yet. In that sense, studies using either
free radical scavengers or transgenic plants over-expressing ROS-scavenging enzymes
(such as CAT or SOD) prior to auxin herbicide application could be useful to clarify the
relevance of ROS in the ET, ABA and JA overproduction induced by auxin herbicides
in sensitive plants.
Sources of oxidative stress in sensitive plants treated with
auxin herbicides. Contribution of leaf peroxisomes.
The principal mechanism responsible for triggering oxidative stress in sensitive plants
treated with auxin herbicides still remains unknown. Up to now, only a few sources of
ROS overproduction have been described in the roots and the leaves of susceptible
plants treated with these compounds. Also, it has not been elucidated yet the relative
contribution of the different cell organelles to the oxidative stress induced by these
Thus, in the roots of 2,4-D-treated H. vulgare and quinclorac-exposed E.
orizycola, an NAD(P)H oxidase activity was linked to the H2O2 and O2.- overproduction
induced by these herbicides (Šimonovičová et al., 2004; Sunohara & Matsumoto, 2004).
Also, in the leaves of quinclorac-treated G. aparine, the photosynthesis inhibition -due
to ABA-induced stomata closure- was claimed as responsible for the H2O2
accumulation observed (Grossmann et al., 2001). However, in the leaves of 2,4-D
foliarly-treated pea, where H2O2 also accumulated, a dramatic increase (up to 2500%
versus the control) in the O2.--producing xanthine oxidase (XOD) activity, as well as a
milder increase in the H2O2-producing activities of acylCoA-oxidase (ACOX), of the
fatty acid beta oxidation, and SOD, were also reported (McCarthy-Suárez, 2004;
Romero-Puertas et al., 2004). Moreover, the huge increase of XOD activity was also
observed in the leaves of pea plants treated with 2,4-D by roots, in which no stomata
closure but an increase of stomata conductance had been detected (McCarthy-Suárez,
XOD activity is involved in the catabolism of nucleic acids (Corpas et al., 1997),
a process stimulated by auxin herbicides in the leaves and the roots of sensitive plants
(Cárdenas et al., 1968; Grossmann et al., 2001). As XOD-derived O2.- radicals quickly
dismutate to H2O2, either spontaneously or through the SOD activity, the 2,4-D-induced
XOD activity might be a potential source for the high H2O2 levels found in the leaves of
auxin herbicide-treated plants (Fig. 1). Moreover, the massive induction of XOD in the
leaves of auxin herbicide-treated plants could trigger the senescence of these plants. Up-
regulation of XOD activity is known to induce the senescence-associated lipoxygenase
activity (Leshem et al., 1981) and to be essential for senescence onset and apoptosis
(Kumagai et al., 2002; Tewari et al., 2009). In addition, as XOD-derived O2.- radicals
can react with ABA-generated nitric oxide (NO.) (Dat et al., 2000) they might lead to
the strong oxidant peroxinitrite (ONOO-) in the leaves of auxin herbicide-treated plants.
However, the most damaging role of XOD-derived O2.- radicals in these plants could be
to become precursors, through metal-catalyzed Haber Weiss reactions with H2O2, of
hydroxyl radicals (.OH), the most dangerous oxidants known (Halliwell & Gutteridge,
2000). Able to alter DNA replication, transcription and repair, and to promote strands
breakage, mutations and chromatin structure modifications, once formed, OH radicals
can react with any cell compound, destroying nucleic acids, proteins and membranes
(Nunoshiba et al., 1999; Halliwell & Gutteridge, 2000). Although these data point to a
prominent role of XOD in the oxidative stress promotion in sensitive plants treated with
auxin herbicides, no studies, however, have assessed the implication of this enzyme in
the damage and death of the plants exposed to these compounds.
XOD activity, encoded by the xanthine oxidoreductase (XOR) gene, also coding
for xanthine dehydrogenase (XDH), exists in thermodynamic equilibrium with XDH
activity. Xanthine dehydrogenase can be converted to XOD either irreversibly by
proteolysis or reversibly by oxidation of the sulfhydryl groups of two conserved Cys to
disulfide bonds (Nishino et al., 2008). Interestingly, XDH activity, which also generates
O2.- radicals, is up-regulated by ABA in stressed plants (Yesbergenová et al., 2005). In
addition, XDH protein levels increase as leaves age, supporting a role for this enzyme in
leaf senescence (Nakagawa et al., 2007). As stress (i.e. H2O2) increases ABA levels
(Tuteja, 2007) and promotes the conversion of XDH into XOD (Haberland et al., 1992),
and given that senescence increases both ABA and XDH protein levels (Hung & Kao,
2004; Nakagawa et al., 2007), it is tempting to speculate that the induction of XOD in
auxin herbicide-treated plants might be directly linked to the stress-induced ABA
production and the senescence promotion (Fig. 1).
To clarify this question, studies employing plants underexpressing XOR or
plants pretreated with the XOD inhibitor allopurinol prior to the auxin herbicide
application might be useful to estimate the importance of XOD induction in the
oxidative stress, senescence, and death, promoted by these herbicides in sensitive plants.
Alternatively, pretreatment of plants with NAD(P)H oxidase inhibitors, such as
diphenylene iodonium (DPI), could help understand the role of NAD(P)H oxidases in
auxin herbicide toxicity.
Apart from the stimulation of the ROS-producing activities (NAD(P)H oxidase,
peroxidase, XOD, ACOX) and the photosynthesis inhibition mentioned earlier, other
possible sources of ROS in auxin herbicide-treated plants could be the auxin herbicides
themselves. Auxin herbicides have been shown to directly generate ROS in yeast and
animal cells (Teixeira et al., 2004; Bukowska et al., 2008). In turn, the 2,4-D-derived
plant metabolite 2,4-dichlorophenol has been demonstrated as a very potent oxidant in
strawberry plants (Chkanikov et al., 1977). Auxin properties of auxin herbicides might
also contribute to the ROS generation in the leaves and the roots of susceptible plants
treated with these compounds. ROS production is an upstream and downstream event in
the auxin signal transduction pathway (Morré et al., 1999; Schopfer et al., 2002; Joo et
al., 2005). Furthermore, auxins induce growth by stimulating the production of O2.-,
H2O2 and ·OH by plasma membrane NAD(P)H oxidases and cell wall peroxidases
(Schopfer et al., 2002; Joo et al., 2005; Carol & Dolan, 2006).
Other sources of ROS in sensitive plants treated with auxin herbicides could be
the overproduced phytohormones ET, ABA and JA. ET stimulates H2O2 production in
plant cells (De et al., 2002). In addition, the ET biosynthesis subproduct, cyanide, is a
source of oxidative stress both in plant and animal cells (Kamendulis et al., 2002; Oracz
et al., 2009). ABA is also known as a ROS production-inducer in plants (Hu et al.,
2006; Zhang et al., 2009). In fact, both the NO· generation and the stomata closure
induced by ABA depend on ABA-stimulated H2O2 synthesis (Bright et al., 2006). The
ROS, in turn, act both upstream and downstream in ABA signaling (Guan et al., 2000;
Pei et al., 2000; Zhang et al., 2001; Zhao et al., 2001). Other studies have also
documented the ability of JA of inducing H2O2 production (Browse, 2005). In spite of
these reports on the ROS-producing abilities of ET, ABA and JA, no studies have
determined whether the ROS overproduction and the associated oxidative stress in
sensitive plants treated with auxin herbicides is dependent upon ET or due to the
oxidative ability of these synthetic compounds. A possible approximation could be
using ET biosynthesis inhibitors or ET-insensitive mutants prior to auxin herbicide
Adverse physiological changes induced by auxin herbicides in the leaves and the
roots of treated plants might also contribute to the observed ROS overproduction.
Photosynthesis inhibition and reduced CO2 fixation, described in auxin herbicide-
treated plants, could promote ROS generation in leaves (Grossmann et al., 2001). In
addition, the water stress symptoms reported in auxin herbicide-treated plants, as
deduced from the root water absorption inhibition (Kozinka, 1966; Cárdenas et al.,
1968), the decreased water content in leaves (Brown, 1946), and the increased levels of
Na+ and Cl- in leaves and roots (Wolf et al., 1950; Cooke, 1957; Johnson, 1982;
McCarthy-Suárez, 2004), could also contribute to the oxidative stress observed, as
drought induces ROS production (Cruz de Carvalho & Contour-Ansel, 2008).
The accelerated leaf senescence triggered by auxin herbicides in sensitive plants
might also contribute to the overproduction of ROS. During leaf senescence, the levels
of the promoting factors ET and JA, of lipoxygenase activity, and of ROS increase to
support the oxidative deterioration which ends up in cell death (Smart, 1994; Jabs,
Apart from the accelerated senescence, the decrease of non-enzymatic
antioxidants in the leaves and roots of auxin herbicide-treated plants might also worsen
the oxidative stress situation. Low levels of ascorbate and thiols have been reported in
the leaves of auxin herbicide-treated plants (Key & Wold, 1961; Key, 1962; Beevers et
al., 1963). On the other hand, the decrease of xanthophylls -lipid antioxidants against
singlet oxygen (O2↑)- due to their conversion to ABA in the leaves of auxin herbicide-
treated plants might also contribute to the oxidative stress observed (Kleudgen, 1979).
The subcellular location of the ROS-overproducing systems in auxin-herbicide-
treated plants has also not been investigated in depth yet. Works in the nineties showed
that the chloroplasts and the plasma membrane could be important sites for the ROS
overproduction taking place in the leaves and the roots of sensitive plants treated with
these compounds (Segura-Aguilar et al. 1995; Morré et al. 1999). However, such works
were not followed by additional studies on the enzymatic systems involved or by studies
assessing the relevance of the different cell compartments as sources of ROS in the
oxidative stress induced by auxin herbicides.
Recently, a role for leaf peroxisomes in the oxidative injury induced by auxin
herbicides in sensitive plants has been proposed (McCarthy-Suárez et al. 2011a). In pea
plants treated with 2,4-D, peroxisomes contributed to the oxidative stress of leaves by
increasing their membrane and matrix production of O2.- radicals and by enhancing their
matrix production of H2O2. Given that O2.- radicals easily dismutate to the lipid-soluble
H2O2, overproduction of O2.- radicals by the membranes and the matrices of leaf
peroxisomes of pea plants likely resulted in high levels of peroxisomal H2O2 poured
into the cytosol. Moreover, Haber-Weiss reactions between O2.- radicals and H2O2 at the
matrix or the membrane of leaf peroxisomes of pea plants might also have lead to the
formation of deleterious .OH radicals, able to destroy proteins, lipids and nucleic acids.
Apart from contributing to oxidative stress, leaf peroxisomes could also have a
role in the JA overproduction induced by auxin herbicides in sensitive plants. The last
steps of JA biosynthesis, involving the fatty acid beta oxidation pathway, are known to
take place in peroxisomes (Delker et al., 2007; Schilmiller et al., 2007), and an increase
in the activity of this pathway was observed in the leaf peroxisomes of pea plants after
treatment with 2,4-D (McCarthy-Suárez et al., 2011a).
Role of ROS in the injury and death of sensitive plants treated
with auxin herbicides: induction of senescence in leaves and
tumor-like growth in stems.
ROS might be essential to the damage and the death of sensitive plants treated with
auxin herbicides. In susceptible plants, foliarly applied-auxin herbicides inhibit
photosynthesis, growth, transpiration, stomata conductance, as well as water and
mineral absorption, and promote leaf senescence (Brown, 1946; Wildon et al., 1957;
Cárdenas et al., 1968; Grossmann et al., 2001), but these events can also be triggered in
plants by high levels of ROS (McAinsh et al., 1996; Woo et al., 2004; Krelavski et al.,
2007; Singh et al., 2009; Tamás et al., 2009).
Thus, the chlorophyll loss and the cell damage induced by quinclorac in leaves
of E. oryzicola could be suppressed by treatment with the free radical scavenger α-
tocopherol (Sunohara & Matsumoto, 2004). Moreover, the photosynthesis/growth
inhibition and the senescence triggered by the overproduced ET, ABA and JA in the
leaves and roots of auxin herbicide-treated plants (Grossmann et al., 2004; Kraft et al.,
2007) might have been mediated by ROS. In rice seedlings, an overproduction of ROS
mediates the photosynthesis inhibition and the induction of necrosis triggered by high
doses of JA (Rakwal & Komatsu, 2001). In fact, an overproduction of ROS triggers the
methyl jasmonate (MeJA)-activated PCD in stressed plants (Zhang & Xing, 2008).
Likewise, an H2O2 overproduction is known to mediate the senescence process induced
by ABA (Hung & Kao, 2004).
That oxidative stress is essential to auxin herbicide phytotoxicity is illustrated by
the fact that susceptibility of plants to quinclorac is determined by their antioxidative
capability (Sunohara & Matsumoto, 2004). However, a demonstration of the necessity
of ROS in the mechanism of lethality of auxin herbicides is still needed. Whether ROS
trigger the death of auxin herbicide-treated plants could be tested by using the
antioxidant NAC (N-acetyl-L-cysteine). This compound neutralizes oxidative stress
both by sequestering ROS and by replenishing glutathione (GSH) levels, and could be
used as a means to study whether abolishing the effect of a ROS overproduction can
prevent the death of plants treated with auxin herbicides. Auxin herbicides deplete
cellular GSH (Palmeira et al., 1995), and GSH depletion has been demonstrated to
trigger ROS imbalance and cell death (Mytilineou et al., 2002). The role of oxidative
stress in the phytotoxicity of auxin herbicides could also be assessed by using CAT-
overexpressing plants, or plants treated with the XOR inhibitor allopurinol prior to the
auxin herbicide treatment. Plants under-expressing XOR could also be used to estimate
the importance of XOR-derived ROS in auxin herbicide phytotoxicity. In addition, the
role of ET in the oxidative burst occurring in auxin herbicide-treated plants could be
analyzed by using ET biosynthesis inhibitors or ET-insensitive mutants prior to auxin
In particular, ROS might be crucial to the membrane, protein and nucleic acid
damage induced by auxin herbicides. Indeed, auxin herbicides damage the membranes
of sensitive plants in a dose-dependent manner (Bradberry et al., 2004). Breakage of the
plasmalemma, tonoplast and endoplasmic reticulum, and of the mitochondrial and
nuclear membranes, as well as distortion and disintegration of the chloroplast tylacoids,
have been reported in the leaves of Phaseolus vulgaris, Nicotiana tabacum, Pinus
radiata, Eucaliptus viminalis, Picea abies, Galium aparine and Pisum sativum plants
treated with 2,4-D, 2,4,5-T, quinclorac or picloram (4-Amino-3,5,6-trichloro-2-
pyridinecarboxylic acid; pyridinecarboxylic acid group) (Hallam, 1970; White &
Hemphyll, 1972; Anderson & Thomson, 1973; Ayling, 1976; Segura-Aguilar et al.,
1995; Grossmann et al., 2001; McCarthy-Suárez, 2004). Damage to the peroxisomal
membrane has also been observed in P. sativum plants treated with 2,4-D by leaves
(McCarthy-Suárez, 2004). In that sense, the ROS, especially the .OH radicals, are
deleterious to cell membranes. Moreover, the breakdown of membranes and the
biosynthesis of ET in plants - which seem to be linked (Arora et al., 2002) - are known
to involve free radical production.
Apart from having a damaging effect on cell membranes, ROS are also
deleterious to nucleic acids and proteins (Halliwell & Gutteridge, 2000). Once formed
by Fenton reactions between O2.- radicals and H2O2, the .OH radicals can impair DNA
replication, transcription and repair, and promote DNA mutations and breakage
(Nunoshiba et al., 1999; Halliwell & Gutteridge, 2000). Interestingly, the genotoxicity
of auxin herbicides has been widely demonstrated both in animals and plants (Kumari &
Vaidyanath, 1989; González et al., 2007). Thus, the high levels of O2.- radicals and H2O2
in the leaves and the roots of sensitive plants treated with auxin herbicides could imply
high levels of .OH radicals, which, in turn, might account for the diverse injuries
The ROS-induced cell membrane and nucleic acid damage in the leaves and
roots of auxin herbicide-treated plants could also bring about plant senescence or
apoptotic-like PCD (Fig. 1). Plants treated with auxin herbicides are known to display
accelerated leaf senescence symptoms (Hansen & Grossmann, 2000; Grossmann et al.,
2001; McCarthy-Suárez, 2004; Romero-Puertas et al., 2004), such as photosynthesis
decay, increased protein-, lipid- and nucleic acid-degradation, enhanced plastoglobuli
accumulation in chloroplasts, and breakage of the tonoplast, endoplasmic reticulum and
plasmalemma membranes (Shaw & Manocha, 1965; Butler, 1967; Thompson & Platt-
Aloia, 1987; Smart, 1994; Noodén et al., 1997; Fischer et al., 1998). In addition, it is
known that the membrane damage caused by the up-regulation of ROS-dependent
lipoxygenases, lipid degradation-associated enzymes, triggers PCD in tobacco (Cacas et
al., 2005). Also, it is known that DNA damage induces senescence by stimulating an
NADPH-oxidase-dependent ROS production (Probin et al., 2007).
Thus, the NADPH oxidase and XOD-derived oxidative stress in susceptible
plants treated with auxin herbicides could largely contribute to their senescence and
death. Moreover, the huge stimulation of leaf XOD activity observed in plants treated
with 2,4-D (McCarthy-Suárez, 2004; Romero-Puertas et al., 2004) with be in tune with
the nuclease-induced DNA laddering, the massive rRNA degradation, and the
stimulation of nitrogen mobilization-enzymes observed in plants during leaf senescence
(Noodén et al., 1997). In fact, it is known that XOD up-regulates the senescence-
associated lipoxygenase activity (Leshem et al., 1981) and that auxin herbicides induce
the DNAse and lipoxygenase activities in leaves of susceptible plants (Grossmann et al.,
2001; Pazmiño et al., 2011).
Although the presence of apoptosis in the leaves of susceptible plants treated
with auxin herbicides has not been investigated yet, induction of apoptotic-like death as
part of the toxicity mechanism of these compounds cannot be ruled out.
The apoptosis, characterized by chromatin condensation, cell shrinking, nuclei
and cytoplasm partitioning into apoptotic bodies, and DNA fragmentation (Noodén et
al., 1997; Ateeq et al., 2006), has been shown to be induced by auxin herbicides in
human lymphocytes (Kaioumova et al., 2001), Hep G2 cells (Tuschl & Schwab, 2003),
cerebellar granule cells (DeMoliner et al., 2002) and in the fish Clarias batrachus
(Ateeq et al., 2006). Cell shrinking, cytoplasm partitioning, and apoptotic-like bodies
were also observed in leaves of pea plants treated with 2,4-D (McCarthy-Suárez, 2004).
High levels of ROS, auxins, ET, ABA and JA have also been shown to induce
PCD in plants. In tobacco, the accumulation of H2O2 triggered an apoptotic-like cell
death (Dat et al., 2003). Also dependent on O2.- radical accumulation were the cell death
of Arabidopsis plants (Jabs et al., 1996) and the ethylene-induced PCD of O3-sensitive
plants (Overmyer et al., 2000). In that sense, the O2.- radical-producing XOD activity,
highly induced in the leaves of 2,4-D-treated plants (McCarthy-Suárez, 2004; Romero-
Puertas et al., 2004), is known to play an important role in the apoptosis of animal cells
(Kumagai et al., 2002).
High levels of auxin and ET have also been implicated in the apoptotic cell
death of interspecific hybrids of Nicotiana glutinosa L. x N. Repanda expressing
temperature-sensitive lethality (Yamada et al., 2001). In barley pericarp, PCD is also
controlled by ET and JA and mediated by different proteases (Sreenivasulu et al.,
2006). Treatment of Arabidopsis with high concentrations of Me-JA has also been
shown to trigger PCD by a mechanism dependent on ROS production (Zhang & Xing,
2008). In addition, high levels of JA induce ROS production and PCD in Nicotiana
tabacum plants (Mur et al., 2006) as well as apoptosis in human cancer cells (Fingrut &
Flescher, 2002), whereas ABA-induced NO. activates PCD in BY-2 tobacco cells (Neill
et al., 2003).
Thus, the high levels of ROS, auxin, ET, JA and ABA (and probably NO.) in the
leaves of sensitive plants treated with auxin herbicides could promote an apoptotic-like
cell death. As leaves of sensitive plants treated with auxin herbicides undergo oxidative
stress, as mentioned earlier, and as ROS overproduction can determine the onset of
apoptosis (De Pinto et al., 2006), it would be worth analyzing specific apoptosis
parameters in the leaves of auxin herbicide-treated plants (i.e. citochrome c release from
mitochondria, caspase activation, etc) to confirm the induction of apoptosis as a death-
contributing factor. Moreover, given that auxin induces gibberellic acid (GA)
biosynthesis (Ross et al., 2000; Wolbang et al., 2004), a phytohormone that triggers
PCD in barley aleurone (Bethke et al., 1999), and given that the effective 2,4-D
concentration to kill water hyacinth can be diminished 10 times by applying a low dose
of GA at the same time (Pieterse et al., 1980), a possible role for GA in auxin herbicide
mode of action, that would have to be assessed, could be hypothesized (Fig.1).
The likely decrease of cytokinins (CKs) and polyamines (PAs) in the leaves of
auxin herbicide-treated plants might also contribute to the senescence/apoptosis induced
in these organs (Fig.1). Auxins inhibit the biosynthesis of CKs (Nordström et al., 2004),
hormones that scavenge ROS, delay senescence, promote nucleic acid/protein
biosynthesis, and induce cell division (Miller et al., 1955; Dyer & Osborne, 1971;
Leshem et al., 1981; Basra & Basra, 1997; Song et al., 2006). Moreover, kinetin and
analogues inhibit XOD activity (Sheu et al., 1996) and counteract the XOD-induced up-
regulation of the senescence-associated lipoxygenase activity (Leshem et al., 1981).
Cytokinins also stimulate the production of PAs (Legocka & Żarnowska, 2002),
antioxidant compounds which inhibit the lipoxygenase activity and the lipid
peroxidation (Borrell et al., 1997). In fact, polyamine levels decrease as leaves age
(Kaur-Sawhney et al., 1982).
Recently, a role for ROS in the induction of abnormal overgrowths in the stems
of auxin herbicide-treated plants has also been suggested (McCarthy-Suárez et al.,
2011b). As high auxin concentrations do (Haber, 1962), auxin herbicides induce a callus
or tumour-like overgrowth in the stems of sensitive plants (Eames, 1950; Cárdenas et
al., 1968; Sen et al., 1983) which could be favoured by the low levels of ROS found in
these organs after the auxin herbicide treatment (McCarthy-Suárez et al., 2011b). In
fact, low levels of ROS mediate the auxin-induced expansion and proliferation of plant
cells (Pasternak et al., 2005; Fehér et al. 2008) whereas high levels of ROS have a role
in plant growth inhibition, stress, senescence, and PCD (Gadjev et al., 2008). Thus, in
plants treated with auxin herbicides, a differential distribution of ROS might take place
among the different plant organs. While high levels of ROS in the leaves and roots of
these plants would induce senescence, controlled levels of ROS in the stems, such as
those arising as a result of the increased ACOX activity (McCarthy et al., 2011b), might
contribute to the tumor-like growth reported in these organs (Fig. 2). In fact, in 2,4-D
treated pea plants, the massive induction of the O2.- radical-producing XOD activity in
leaves was accompanied by the oxidative stress and the senescence of these organs,
whereas the inhibition of this activity in stems was not accompanied by oxidative stress
or senescence but by a stem protein increase (McCarthy-Suárez et al., 2011b).
Interestingly, XOD activity is inhibited by CKs, hormones of antioxidant and
senescence-delaying properties. Thus, the massive induction of XOD activity in the
leaves of auxin herbicide-treated plants might correlate to the senescence process and
the likely low levels of CKs and PAs in these organs.
High levels of auxins and CKs, followed by high levels of ET, ABA and JA,
have also been implicated in the formation and vascularization of stem tumors (Ullrich
& Aloni, 2000; Veselov et al., 2003). Moreover, an auxin/citokinin imbalance in stems,
resulting in high cytokinin levels, has been proposed to promote auxin herbicide effects
on plant growth and development, triggering tumor formation (Van Overbeek, 1964;
Ullrich & Aloni, 2000). Such imbalance would specifically increase the biosynthesis of
proteins specialized in inducing cell division but not cell differentiation, abolishing
meristem size control (Kung, 1981; Sen et al., 1983; Beemster & Baskin, 2000).
Although there are no reports on the levels of CKs in the stems of auxin
herbicide-treated plants, the fact that high concentrations of kinetin and 2,4-D promote
callus growth, that CKs induce antioxidant activities, biosynthesis of PAs, and radial
cell divisions in plant stems, and the fact that CKs scavenge ROS and prevent plant
senescence (Miller et al., 1955; Leshem et al., 1981; Basra & Basra, 1997; Synkova et
al., 2006; Stoparić & Maksimović, 2008), might support the idea of non-negligible
amounts of CKs in the stems of auxin herbicide-treated plants (Fig. 2). Moreover, as
CKs inhibit XOD activity (Sheu et al., 1996) and induce the proliferation and inhibit the
differentiation of shoot meristem-stem cells (Su et al., 2011), it is likely that the
inhibition of XOD activity in the stems of 2,4-D-treated pea plants, which was
accompanied by the absence of symptoms of oxidative stress or senescence in these
organs (McCarthy-Suárez et al., 2011b), might have been due to CKs action.
Furthermore, as CKs are part of certain tRNA, allowing them to regulate cell division
and growth through protein biosynthesis (Galston & Davies, 1969), the high levels of
nitrogen, aminoacids and protein in the stems of auxin herbicide-treated plants (Sell et
al., 1949; Cárdenas et al., 1968; McCarthy-Suárez et al., 2011b) would rule out the
possibility of low CK levels in stems. Also, as CKs are mainly transported from roots to
leaves, where they delay senescence, the obstruction of phloem transport in auxin
herbicide-treated plants, due to stem tissue proliferation –which could be regarded as a
pseudo-mechanical stress-, would reduce leaf CKs levels (Fig. 1), thereby accumulating
them in stems (Fig. 2). This phloem flux interruption might also prevent auxin
herbicides to flux from leaves to stems under foliar applications, and from roots to
stems under root herbicidal applications, contributing to balance auxins/CKs in stems.
Premises supporting this hypothesis would be the decreased auxin & auxin herbicide
concentrations in the stems of auxin herbicide-treated plants (Hay, 1956; Hay &
Thimann, 1956; Klems et al., 1998), the inhibitory effect of CKs on polar auxin
transport (Shkolnik-Inbar & Bar-Zvi, 2010), the suggested accumulation of CKs and the
abnormal radial growth in the stems of plants undergoing ET-induced polar auxin
transport inhibition (PAT) or mechanical stress (Basra & Basra, 1997), and the
development of tumors or abnormal stem tissue in CK-treated or CK-overexpressing
plants (Kung, 1989; Li et al., 1992).
Moreover, as CKs induce GAs catabolism (Jasinsky et al., 2005) the likely high
levels of CKs in stems might correlate to the possibly low stem levels of GAs (Fig. 2).
Conversely, the possibly low levels of CKs in leaves might be in tune with
hypothetically high leaf levels of GAs (Fig. 1). Interestingly, mechanical stress in plants
-in which ethylene plays an important role- induces a radial thickening and a reduction
of GAs levels in stems (Takano et al., 1995) as well as a local and systemic increase of
ACOX activity (Castillo et al., 2004).
Thus, possibly high levels of antioxidant CKs and PAs in the stems - and low
levels in the leaves - of susceptible plants treated with auxin herbicides might be
hypothesized. However, molecular studies on the role of these plant growth regulators
in auxin herbicide mode of action, as well as on their relationship with the different
levels of ROS in leaves and stems of auxin herbicide-treated plants are still needed. The
results of these studies on hormone-ROS crosstalk might be relevant not only to
elucidate the phytotoxic mechanism of auxin herbicides, but to design ecologically-safer
herbicides or auxin herbicide-resistant plants for agriculture.
The author wishes to acknowledge Dr. Luis Alfonso del Río (Estación Experimental del
Zaidín, CSIC, Granada, Spain) for supporting the writing of this paper and to the JAE-
Doc postdoctoral fellowship (C.S.I.C., Spain) for financial support.
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Model proposed to explain the oxidative metabolism-and hormone-related action of
auxin herbicides in the leaves of sensitive plants. In leaves, auxin herbicides might
trigger senescence / apoptosis not only by increasing the levels of the senescence-
promoting hormones ethylene (ET), abscisic acid (ABA) and jasmonic acid (JA), but
also by inducing a ROS overproduction through the up-regulation of the O2.--producing
and nucleic acid catabolism-related xanthine oxidase (XOD) activity, and of other ROS-
generating activities, such as acyl-coA oxidase (ACOX) and superoxide dismutase
(SOD). Possible changes in the levels of cytokinins, polyamines and gibberellins in
leaves might also contribute to this situation.
Model proposed to explain the oxidative metabolism-and hormone-related action of
auxin herbicides in the stems of sensitive plants. In stems, auxin herbicides might
induce a tumor-like growth not only by generating specific hormonal changes resulting
from ethylene (ET)-induced polar auxin transport (PAT) inhibition, but also by inducing
controlled levels of ROS in these organs through the inhibition of xanthine oxidase
(XOD) activity and the stimulation of acyl-coA oxidase (ACOX) activity. Possible
changes in the levels of cytokinins, polyamines and gibberellins -opposite to those
likely occurring in leaves- could also contribute to this situation.
Hypoxanthine Uric Acid
↑DNAse Protein Oxidation
? Oxidative Stress
Senescence / Apoptosis
(↑ET, ↑ABA, ↑JA)
↓Gibberellins - ↑RNA