ARTICLE ADDENDUM

Plant Signaling & Behavior 7:3, 425427; March 2012;

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2012 Landes Bioscience

Insights into the toxicity mechanism of and cell response to the herbicide 2,4-D in plants
Departamento de Bioqumica; Biologa Celular y Molecular de Plantas; Estacin Experimental del Zaidn; CSIC; Granada, Spain

lthough structurally similar to the natural plant hormone indol-3acetic acid, auxin herbicides were developed for purposes other than growth, and have been successfully used in agriculture for the last 60 years. Concerted efforts are being made to understand and decipher the precise mechanism of action of IAA and synthetic auxins. Innumerable results need to be interconnected to resolve the puzzle of auxin biology and action mode of auxin herbicides. To date, different breakthroughs are providing more insights into the process of plant-herbicide interactions. Here we highlight some of the latest findings on how the 2,4dichlorophenoxyacetic acid damages susceptible broadleaf plants, emphasizing the role of ROS as a downstream component of the auxin signal transduction under herbicide treatment.

dose dependent, and its effect also depends on tissue sensitivity and species. A few hours after application, some symptoms can be discerned, such as growth distortions throughout the plant, giving rise leaf epinasty and stem curvature, and finally senescence and death.2 Chloroplasts have been considered the initial target of 2,4-D, since the chemical is mainly accumulated and subsequently metabolized in these organelles. Carbon assimilation and photochemical reactions may be strengthened by low concentrations of 2,4-D, whereas higher concentrations prove inhibitory.3 By inhibiting the Hill reaction, 2,4-D blocks electronic transport in photosystem II.4 The chlorophyll and carotenoid content, as well as the chlorophyll/benzoquinone relation, are reduced by 2,4-D, while the xanthophyll/ carotene relation increases due to the herbicide, these effects triggering senescence of the photosynthetic machinery.5 Role of Reactive Oxygen Species and NO in 2,4-D Toxicity During the last decade it was demonstrated that reactive oxygen species (ROS) play a central role in the toxicity of 2,4-D. ROS regulation requires a homeostasis between production and scavenging, generating a baseline from which ROS can act as signal in different cellular processes6 such as growth and development, and also can synchronize responses to the environmental cues.6 In processes such as gravitropism, involving the redistribution of auxin, ROS production appears to be necessary for phosphorylation and the activation of proteins involved in this process.7 An overdose of

Keywords: dichlorophenoxyacetic acid, ethylene, jasmonic acid, nitric oxide, oxidative stress, reactive oxygen species, salicylic acid, stress related proteins Submitted: 12/16/11 Accepted: 12/20/11 http://dx.doi.org/10.4161/psb.19124
*Correspondence to: Luisa M. Sandalio; Email: luisamaria.sandalio@eez.csic.es Addendum to: Pazmio DM, RodrguezSerrano M, Romero-Puertas MC, Archilla-Ruiz A, Del Ro LA, Sandalio LM. Differential response of young and adult leaves to herbicide 2,4-dichlorophenoxyacetic acid in pea plants: role of reactive oxygen species. Plant Cell Environ 2011; 34:187489; PMID:21707656; http://dx.doi. org/10.1111/j.1365-3040.2011.02383.x

Toxicity of 2,4-Dichlorophenoxyacetic Acid The main role of natural auxins is the growth, but this is not the case for synthetic auxins, also called auxinic herbicides, which perform the opposite task of their biological functions. Little is known about the molecular mode of action of natural auxins and that of their synthetic homolog. Natural auxins such as IAA are usually inactivated very quickly through conjugation and degradation in the plant,1 while synthetic auxins such as 2,4dichlorophenoxiacetic acid (2,4-D) persist for long periods of time within the plant, and therefore are more effective and lethal. The action mode of auxinic herbicides is

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Diana M. Pazmio, Mara C. Romero-Puertas and Luisa M. Sandalio*

Figure 1. Scheme showing 2,4-D effect on leaf epinasty and cell death. 2,4-D induces ROS production. This ROS triggers leaf epinasty and oxidative processes giving rise senescence and cell death, but also participates as signal molecule activating defense cell response. ROS accumulation and gene expression are also regulated by changes in the hormone balance.

2,4-D Induced Hormone Imbalance Synthetic auxins such as 2,4-D, have an important consequence on the endogenous auxin balance; either inhibiting its transport and promoting its oxidation, or competing for substrate, thereby prompting a reduction of the auxin complex needed during regular plant growth.12 Additionally, 2,4-D and auxinic herbicides interact with other hormones and can disrupt their balance. An overdose of 2,4D as well as of IAA, activate de novo

synthesis of 1-aminocyclopropane-1carboxylic acid (ACC) synthase, which is a key enzyme in ethylene biosynthesis.13 However, the role of ET on 2,4-D toxicity is controversial and several authors have demonstrated that it does not participate in 2,4-D-dependent leaf epinasty,9,14,15 although it can participate in the 2,4-Dinduced plant senescence.8,9 Another consequence of ET overproduction is the formation of cyanide, an inhibitor of plant enzymes and proteins such as cytochrome-c oxidase, RuBisCo, nitrate/ nitrite reductase, catalase, and peroxidase.13 As a response to the ET burst, the biosynthesis of abscisic acid (ABA) has been reported to be induced; through the overexpression of genes such as NCED1, which encodes a key regulatory enzyme of ABA biosynthesis.16 ABA accumulation causes stomatal closure, which limits biomass production and can induce overproduction of reactive oxygen species (ROS), the main factor responsible for the oxidative damage and cell death.9,10 Other hormones described to act during stress conditions such as jasmonic acid (JA) and salicylic acid (SA), can also be disturbed by 2,4-D,17 although the mechanism has not been well established. Raghavan et al.18 have observed in Arabidopsis plants treated with 2,4-D an induction of OPR2, involved in jasmonic acid biosynthesis. Auxin is interconnected

with SA, and both share transcription factors.19,20 Induction of General Stress Response by 2,4-D Auxinic herbicides have been shown to upregulate genes encoding related stress and defense proteins such as LTI6A, LTI78 (LTI, low temperature induced protein), HSP70,18 PRP4-A, HSP 71.1,9 and SCL14 (proteins involved in the detoxification of harmful xenobiotics.)20 How 2,4-D is perceived by the cell and how it activates defense- or stress-related gene expression remains to be elucidated, but unpublished results from our laboratory point to ROS as the earliest effectors to 2,4-D. Auxin, 2,4-D, and SA induced the transcriptional activation of defenseand stress-related genes, probably by changes in the redox state of transcription factors TGAs.20 Arabidopsis mutants defective in antioxidative capacity showed altered auxin homeostasis and development,21 and recently ROS-derived changes in the expression of auxin signaling genes have also been reported.22 Conclusions ROS overproduction is key in the effect of 2,4-D and may have differential roles in epinasty development, senescence, cell

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2,4-D exerts a pro-oxidant effect, inducing harmful ROS concentrations, and a failure in the detoxifying cell mechanisms, causing oxidative damage, nuclear-DNA fragmentation, and cell death.8 The increase of ROS production induced by 2,4-D is a direct consequence of the activation of specific enzymes such as xanthine oxidoreductase (XOD) involved in ureide metabolism, acyl-CoA oxidase (ACX) involved in fatty acid -oxidation and jasmonic acid biosynthesis, and lipoxygenase (LOX).9 NADPH oxidases from the plasma membrane have been considered one of the main sources of ROS induced by auxinic compounds, although in pea plants this activity has been found not to change in response to 2,4-D.9 ROS are also involved in 2,4-D-induced epinasty by promoting cell expansion and vasculartissue proliferation,9,10 also acting as signal molecules to induce the cellular response against stress conditions.6 In addition, these oxidants can participate in the onset of specific gene expression in response to 2,4-D, although the gene network involved in the cell response to 2,4-D governed by ROS has not yet been defined and thus is still under study (RomeroPuertas et al., unpublished results). In addition to ROS, nitric oxide (NO) appears to be key in 2,4-D response. A reduction of this molecule has been reported in pea leaves treated with 2,4-D as well as an induction of nitrosoglutathione reductase activity, and changes in the S-nitrosylation of peroxisomal proteins.11 The role of NO under these conditions is not clear but it can participate with ROS in the regulation of different process such as stomatal movement or gene regulation.

death, and gene regulation. Disturbances in hormone balance can also contribute to ROS accumulation and regulation of cell response to 2,4-D ( Fig. 1 ). In addition to transcriptional regulation, posttranscriptional protein modification such
References
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as S-nitrosylation or phosphorylation/ dephosphorylation add more complexity to the signaling network regulation of the cell response to 2,4-D11 (Pazmio et al., unpublished results) and further studies are required to unravel this system.
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Acknowledgments

This work supported by ERDF-cofinanced grant BIO2008-04067 from HICINN, Spain.

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