RESEARCH PAPER Regulation of epinasty induced by 2,4-dichlorophenoxyacetic acid in pea and Arabidopsis plants D. M. Pazmi ~ no, M. Rodr  ıguez-Serrano, M. Sanz, M. C. Romero-Puertas & L. M. Sandalio Departamento de Bioqu  ımica, Biolog  ıa Celular y Molecular de Plantas, Estaci  on Experimental del Zaid  ın, CSIC, Granada, Spain Keywords 2,4-D; epinasty; ethylene; phosphorylation; reactive oxygen species; signalling; xanthine oxidoreductase. Correspondence L. M. Sandalio, Departamento de Bioqu  ımica, Biolog  ıa Celular y Molecular de Plantas, Estaci  on Experimental del Zaid  ın, CSIC, Apartado 419, 18080 Granada, Spain. E-mail: luisamaria.sandalio@eez.csic.es Editor G. Noctor Received: 5 August 2013; Accepted: 4 October 2013 doi:10.1111/plb.12128 ABSTRACT The herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) causes uncontrolled cell division and malformed growth in plants, giving rise to leaf epinasty and stem curvature. In this study, mechanisms involved in the regulation of leaf epinasty induced by 2,4-D were studied using different chemicals involved in reactive oxygen species (ROS) accu- mulation (diphenyleniodonium, butylated hydroxyanisole, EDTA, allopurinol), calcium channels (LaCl 3 ), protein phosphorylation (cantharidin, wortmannin) and ethylene emission/perception (aminoethoxyvinyl glycine, AgNO 3 ). The effect of these compounds on the epinasty induced by 2,4-D was analysed in shoots and leaf strips from pea plants. For further insight into the effect of 2,4-D, studies were also made in Arabidopsis mutants deficient in ROS production (rbohD, rbohF, xdh), ethylene (ein 3-1, ctr 1-1, etr 1-1), abscisic acid (aba 3.1), and jasmonic acid (coi 1.1, jar 1.1, opr 3) pathways. The results suggest that ROS production, mainly ·OH, is essential in the development of epinasty triggered by 2,4-D. Epinasty was also found to be regulated by Ca 2+ , protein phosphorylation and ethylene, although all these factors act down- stream of ROS production. The use of Arabidopsis mutants appears to indicate that abscisic and jasmonic acid are not involved in regulating epinasty, although they could be involved in other symptoms induced by 2,4-D. INTRODUCTION The selective control of broadleaf weeds in cereal crops using auxinic herbicides has made this group of chemicals one of the most widely used herbicides. These herbicides are thought to act as hormone mimics and, although their exact action mech- anism is not fully known, it involves the disruption of auxin (IAA) responses (Grossmann 2000). These herbicides inhibit cell growth in meristematic regions, causing rapid uncontrolled cell division and malformed growth in other regions (Gross- mann 2000; Pazmi~ no et al. 2011, 2012). One of the most char- acteristic symptoms of auxinic herbicides is leaf epinasty, which consists in the downward bending of leaves as a result of higher expansion of adaxial cells, as compared to the abaxial surface cells of leaves (Kang & Burg 1972; Kang 1979). The disruption of growth coordination in epidermal, palisade mesophyll and vascular tissues also contribute to epinasty (Paz- mi~ no et al. 2011). Plant hormones such as ethylene (ET), absci- sic acid (ABA) and brassinosteroids have also been demonstrated to regulate epinasty (Haubrick & Assamann 2005; Lee et al. 2008). In addition, Kuhn et al. (2011) reported that flavonoids can regulate auxin transport and epinasty. There is evidence that auxins can act in either an ET-inde- pendent or ET-dependent manner (Abeles et al. 1992; Wei et al. 2000). It has recently been proposed that auxin-induced shoot growth inhibition in certain species is mediated by both ET and ABA (Grossmann 2000). The application of growth inhibiting levels of auxin stimulates ET production, in turn raising levels of ABA, which is considered to be the endogenous growth inhibitor (Grossmann 2000). However, other authors hold that auxin-induced shoot growth inhibition and herbicide damage are independent of ET because the inhibitor of ET syn- thesis, aminoethoxyvinyl glycine (AVG), does not prevent leaf epinasty (Keller & Van Volkenburgh 1997; Valenzuela-Valen- zuela et al. 2002). Plants are exposed to a wide range of environmental stress throughout their lives and have developed different mecha- nisms to increase their tolerance through molecular changes in response to the onset of stress. The first step in this response is to perceive the stress and then switch on a signal transduction pathway to regulate gene expression, thereby altering metabolic pathways. Most abiotic stresses raise free calcium levels in the cytosol and alter phosphatase as well as kinase activities (White & Broadley 2003; Pitzschke & Hirt 2006). Reactive oxygen spe- cies (ROS) also play an important role in signalling by control- ling the response to biotic and abiotic stress; in addition, they regulate processes such as growth and development, and par- ticipate in programmed cell death (del R  ıo et al. 2006). ROS- mediated signalling is controlled through a balance between production and scavenging by antioxidants, and subcellular sources of ROS production can be key in the cellular transduc- tion of ROS signals, giving rise to different physiological, bio- chemical and molecular responses (Gadjev et al. 2006; Mittler et al. 2011). Various ROS sources have been identified in chlo- roplasts, plasma membranes, mitochondria and peroxisomes (Sandalio et al. 2009). Apparently, the expression of at least 1– 2% of Arabidopsis genes depends on H 2 O 2 , and some of these genes encode antioxidants while others encode proteins involved in signalling, such as calmodulin, protein kinases and transcription factors (Desikan et al. 2001; Neill et al. 2002). Plant Biology 16 (2014) 809–818 © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands 809 Plant Biology ISSN 1435-8603