Inhibition of the Indole‐3‐acetic acid‐induced Epinastic Curvature in Tobacco Leaf Strips by 2,4‐Dichlorophenoxyacetic Acid

Annals of Botany 91: 465±471, 2003 doi:10.1093/aob/mcg043, available online at www.aob.oupjournals.org Inhibition of the Indole-3-acetic acid-induced Epinastic Curvature in Tobacco Leaf Strips by 2,4-Dichlorophenoxyacetic Acid N A K A K O K A W A N O 1 , T O M O N O R I K A W A N O 1 , 2 and FR E D E R I C L A P EY R I E 1 , * Mixte de Recherche INRA-UHP Interactions Arbres/Micro-organismes, Institut National de la Recherche Agronomique, F-54280 Champenoux, France and 2Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Japan 1Unite  Received: 4 September 2002 Returned for revision: 18 October 2002 Accepted: 27 November 2002 It has been reported that auxin induces an epinastic growth response in plant leaf tissues. Leaf strips of tobacco (Nicotiana tabacum L. `Bright Yellow 2') were used to study the effects of indole-3-acetic acid (IAA), the principal form of auxin in higher plants, and a synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), on epinastic leaf curvature. Incubation of leaf strips with 10 mM IAA resulted in a marked epinastic curvature response. Unexpectedly, 2,4-D showed only a weak IAA-like activity in inducing epinasty. Interestingly, the presence of 2,4-D resulted in inhibition of the IAA-dependent epinastic curvature. In vivo Lineweaver±Burk kinetic analysis clearly indicated that the interaction between IAA and 2,4-D reported here is not a result of competitive inhibition. Using kinetic analysis, it was not possible to determine whether the mode of interaction between IAA and 2,4-D was non-competitive or uncompetitive. 2,4-D inhibits the IAA-dependent epinasty via complex and as yet unidenti®ed mechanisms. ã 2003 Annals of Botany Company Key words: Auxin, bioassay, 2,4-D, epinasty, IAA, inhibition, leaf curvature, Nicotiana tabacum, tobacco. INTRODUCTION Auxins regulate several fundamental cellular processes, including division, elongation and differentiation, and represent one of the most important classes of signalling molecules described in plants (Bennett et al., 1998). Auxin has been implicated as the major signal-mediating tropic stimuli and, as early as the 1920s, the Cholodny±Went hypothesis was formulated to explain the gravitropic response of plant roots and shoots (Palme and Galweiler, 1999). Indole-3-acetic acid (IAA), the principal form of auxin in higher plants, is ®rst synthesized within young apical tissues, then conveyed to its basal target tissues by a specialized delivery system termed polar auxin transport. Studies of the organic chemistry and biology of auxins have contributed greatly to the development of agrochemistry. Agricultural use of herbicides began in the early 1950s and 60s with auxin-type compounds, followed by inhibitors of cell division and photosynthesis (Loos, 1975). Auxintype herbicides, including 2,4-dichlorophenoxyacetic acid (2,4-D), interfere with plant hormone regulation, but their modes of action at the level of molecular perception and cellular signal transduction are still unclear (Fedtke, 1982). Recently, Keller and Von Volkenburgh (1997, 1998) have shown that auxins, including IAA and naphthaleneacetic acid (NAA), induce an epinastic growth response when applied to excised leaf strips of tobacco (Nicotiana tabacum L.). It is notable that the epinastic response in * For correspondence. Fax +33 383 394069, e-mail lapeyrie@nancy. inra.fr tobacco leaf strips was shown to be independent of endogenous ethylene production (Keller and Von Volkenburgh, 1997) despite many earlier studies that indicated ethylene action (Palmer, 1985). The response was greatest in intercostal or non-veinal leaf tissues. Although auxin was found to induce growth of all tissues across the leaf, the epinasty resulted from relatively greater auxin-induced growth of the adaxial (dorsal) epidermis and underlying palisade mesophyll, than of the abaxial (ventral) epidermis (Keller and Von Volkenburgh, 1997). Epinastic sensitivity to auxin in tobacco leaves is strongly developmentally regulated, with responsiveness correlating with the cell-expansion phase of growth. This phenomenon has been con®rmed in leaf strips of tobacco plants overexpressing the auxin-binding protein (Jones et al., 1998). The classical studies conducted between the 1950s and 70s were indicative of induction of epinasty by 2,4-D, one of the most frequently used synthetic auxins (Yamamoto and Yamamoto, 1999) in many systems, and it was believed that production of ethylene mediates the epinasty-inducing action of 2,4-D (Palmer, 1985). However, the effect of 2,4-D on the epinastic response has not been examined in leaf strips of tobacco, the only known bioassay system for assessing the non-ethylene-mediated epinastic response. In this study, we examined and compared the effect of various concentrations of 2,4-D and IAA on epinastic leaf curvature in tobacco leaf strips. Unexpectedly, the epinastic response was only weakly induced by 2,4-D. Instead, the presence of 2,4-D resulted in inhibition of the IAAdependent epinastic curvature. The possible models of 2,4-D action against IAA are discussed by comparing the ã 2003 Annals of Botany Company 466 Kawano et al. Ð 2,4-D Inhibits Action of IAA in Epinasty (10 cm diameter 3 15 cm) containing soil and were allowed to grow under a 12 : 12 h light : dark regimen, at 22 °C. Tobacco leaves approx. 20 cm long were harvested and used immediately for experiments. Leaf strips (10 mm long, 1´5 mm wide) were excised from the central portion of leaves (Fig. 1A). Between 15 and 20 strips were placed in plastic Petri dishes and were incubated at room temperature for 20 h in 7 ml of incubation medium (0´5 mM Tris-HCl buffer, pH 6´0; 10 mM KCl; 10 mM sucrose) eventually supplemented with varying concentrations of IAA and/or 2,4-D. Dishes were covered with aluminium foil to prevent exposure to light. Leaf strip curvature F I G . 1. Preparation of tobacco leaf strips for analysis of epinastic leaf curvature. A, Strips are excised from the middle of tobacco leaves and soaked in auxin-containing media for 20 h in darkness. B, Epinastic and anti-epinastic (hyponastic) orientation of leaf curvature. C, Geometrical analysis of leaf curvature, q. After 20 h of incubation with and without auxins, leaf strips were harvested from the incubation media, positioned on plastic dishes (top: adaxial epidermis, greener surface with numerous hairs; bottom: abaxial epidermis, whiter surface, fewer hairs) and photographed digitally. Epinastic (downward bending) or anti-epinastic (upward bending) leaf curvature was assessed on images (Fig. 1B). The degree of curvature (q) was determined geometrically as illustrated in Fig. 1C. Thus, an unbent leaf strip has zero curvature, an epinastically bent leaf strip was attributed a positive value, and an anti-epinastically (hyponastically) bent leaf strip a negative value. For kinetic analysis, the value D°, re¯ecting the IAAenhanced leaf curvature, was use (...truncated)