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

Abstract

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 µm 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 unidentified mechanisms.

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 first 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). Auxin‐type 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 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 confirmed 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 IAA‐dependent epinastic curvature. The possible models of 2,4‐D action against IAA are discussed by comparing the data with those of known IAA antagonists such as a fungal indolic alkaloid (Ditengou and Lapeyrie, 2000).

MATERIALS AND METHODS

Chemicals

IAA, 2,4‐D and other chemicals were purchased from Sigma (St Louis, MO, USA). IAA solutions were prepared by first dissolving IAA in ethanol (1 mm), followed by successive dilutions with hot distilled water (80 °C).

Plant materials and auxin treatment

Seeds of tobacco (Nicotiana tabacum L. ‘Bright Yellow 2’) were sown on agar medium containing MS salts (Murashige and Skoog, 1962) and 2 % sucrose. After germination, tobacco seedlings were transferred to pots (10 cm diameter × 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.

Fig. 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, θ.

Leaf strip curvature

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 (θ) 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 Δ°, reflecting the IAA‐enhanced leaf curvature, was used. The Δ° value was calculated by subtracting the mean degree of curvature in the control leaf strips (θ_C) from that of auxin‐treated leaves (θ_A): Δ° = θ_A – θ_C.

For Lineweaver–Burk kinetic analysis, reciprocals of Δ° (1/Δ°) were plotted as a function of reciprocals of IAA concentration (1/[IAA]). Data points obtained in the absence and presence of 2,4‐D were co‐plotted. The mode of inhibition of IAA activity by 2,4‐D was then analysed graphically.

Leaf strip elongation

Prior to auxin treatments, leaf strips were carefully prepared to be precisely 10 mm long. After 20 h of incubation, the harvested leaf strips were eventually uncurled and the length of strips was measured precisely using a ruler.

RESULTS

Leaf strip curvature

Tobacco leaf strips were incubated in assay media for 20 h in darkness, in the presence or absence of exogenous auxins. In the absence of exogenous auxins, most strips bent upwards slightly (Fig. 2A). Such anti‐epinastic orientation was expressed by negative values for degree of curvature (Fig. 2B).

Fig. 2. Effect of 2,4‐D concentration on the IAA‐induced epinastic curvature response in tobacco leaf strips. Tobacco leaf strips were floated on the incubation media containing 0 or 10 µm IAA, and/or various concentrations of 2,4‐D, for 20 h in darkness. A, Photographs showing typical results from triplicate experiments. B, Effect of 2,4‐D concentration on IAA‐induced leaf curvature. C, Effect of 2,4‐D concentration on IAA‐induced leaf strip elongation. Error bars represent s.d. (n = 20–22).

When leaf strips were incubated with 10 µm IAA (without 2,4‐D), a strong epinastic curvature response (approx. 250–450°) was observed (Fig. 2A and B), as reported previously by Keller and Volkenburgh (1997). Treatment with 2,4‐D in the absence of IAA resulted in a minor epinastic curvature, which was maximal (approx. 50–100°) at a concentration of 3 µm 2,4‐D (Fig. 2A and B). When leaf strips were co‐treated with both 10 µm IAA and various concentrations of 2,4‐D ranging from 0·3 to 30 µm, they showed compromised curvature responses which were significantly lowered compared with responses to IAA alone (Fig. 2A and B). The presence of higher concentrations of 2,4‐D resulted in greater distortion in IAA‐induced epinasty. Therefore, it seems that 2,4‐D inhibits IAA‐induced epinastic curvature in a dose‐dependent manner, although 2,4‐D added alone had a weak IAA‐like effect.

Leaf strip elongation

As shown in Fig. 2C, treatment with IAA (10 µm) alone induced elongation of leaf strips. Therefore, the IAA‐induced epinastic curvature seems to be associated with an IAA‐enhanced elongation of leaf strips. Similarly, 2,4‐D (0·3–30 µm) induced leaf strip elongation but to a lesser extent than IAA (Fig. 2C). Co‐treatment of leaf strips with IAA and 2,4‐D resulted in compromised elongation (Fig. 2C). As 2,4‐D concentrations in the incubation media increased from 3 to 30 µm, IAA‐dependent elongation of leaf strips was reduced. It is tempting to speculate that inhibition of IAA‐dependent leaf strip curvature by 2,4‐D treatment is tightly linked to 2,4‐D inhibition of IAA‐dependent tissue elongation. It is obvious that the anti‐epinastic action of 2,4‐D is not due to stimulation of elongation growth in tissues on the opposing (abaxial) side of the leaf strips.

Response to increasing IAA concentrations

The epinastic response of leaf strips to increasing concentrations of IAA (ranging from 0·3 to 30 µm) was assessed in the presence or absence of 2,4‐D (0, 3, 10 and 30 µm). When applied alone and at concentrations up to 3 µm, IAA induced curvature of leaf strips in a dose‐dependent manner (Fig. 3A). When 2,4‐D was supplied together with IAA, IAA‐dependent leaf curvature was significantly suppressed for any combination of 2,4‐D and IAA concentrations (Fig. 3A). It is noteworthy that the response induced by the highest concentration of IAA examined here (30 µm) could be significantly lowered in the presence of 3–30 µm 2,4‐D.

Fig. 3. Effect of IAA concentration on the IAA‐induced epinastic curvature response in tobacco leaf strips. A, Leaf curvature and its inhibition by 2,4‐D. B, Tissue elongation and its inhibition by 2,4‐D. Error bars represent s.d.

Elongation of leaf strips was also examined under the same experimental conditions (Fig. 3B). Elongation growth was stimulated by IAA in a dose‐dependent manner up to 3 µm. Leaf strips treated with the optimal or higher IAA concentrations (3, 10 and 30 µm) were approx. 20 % longer than samples at zero time. In the presence of 30 µm 2,4‐D, stimulation of elongation by IAA became hardly detectable (Fig. 3B).

Lineweaver–Burk kinetic analysis

To investigate the mode of 2,4‐D action against IAA‐dependent responses, the leaf curvature induced by various concentrations of IAA was analysed graphically in the presence or absence of 2,4‐D, using the double‐reciprocal plot, the in vivo Lineweaver–Burk kinetic analysis (Fig. 4A).

Fig. 4.In vivo Lineweaver–Burk kinetic analysis assessing the mode of interaction between 2,4‐D and IAA in epinastic leaf curvature of tobacco leaf strips. A, Plots showing all data points, indicative of uncompetitive interaction. B, Plots of data with a limited range of 2,4‐D : IAA ratios (<10), indicative of non‐competitive interaction.

V_max for the IAA‐induced response in the absence of 2,4‐D was calculated to be 397 Δ°. In the presence of 3, 10 and 30 µm 2,4‐D, V_max was reduced to 373, 175 and 117 Δ°, respectively. If interactions are competitive then V_max should not change (Dixon and Webb, 1979). Therefore, the mode of interaction between 2,4‐D and IAA during leaf curvature does not correspond to a characteristic competitive inhibition. Apparent K_m values for IAA in the presence of 0, 3, 10 and 30 µm 2,4‐D were determined to be 0·32, 0·36, 0·21 and 0·15 µM, respectively. Apparent K_m is lowered only in uncompetitive interactions (Dixon and Webb, 1979). K_m for IAA was apparently reduced by high concentrations of 2,4‐D, indicating that the mode of 2,4‐D action against IAA‐induced leaf curvature is uncompetitive inhibition. On the other hand, the possibility that 2,4‐D inhibits IAA action in a non‐competitive manner could not be eliminated since treatment with 3 µm 2,4‐D, which alters V_max, did not lower the apparent K_m for IAA (Fig. 4A). In addition, when data with a limited range of 2,4‐D : IAA ratios (<10) were plotted, no decrease in apparent K_m was observed for IAA (Fig. 4B). If interactions are non‐competitive, apparent K_m should not be altered (Dixon and Webb, 1979). Thus, within a limited range of 2,4‐D : IAA ratios, the mode of interaction is likely to be non‐competitive.

DISCUSSION

Classical works on plant hormones have revealed that the epinastic growth response can be induced by the gaseous plant hormone, ethylene (Abeles, 1972), and by environmental stresses that induce ethylene production, e.g. salt stress (El‐Iklil et al., 2000). It is known that application of IAA increases endogenous ethylene production in many plant species and in a wide range of tissues (Morgan, 1976). The action of IAA on epinastic curvature has been shown to depend on induction of ethylene production in many plant materials, including tomato petioles (Stewart and Freebairn, 1969; Amrhein and Schneebeck, 1980), sunflower petioles and leaves (Palmer, 1985), and etiolated plumular hooks of Phaseolus vulgaris L. and P. mungo L. (Klein et al., 1957; Kang and Ray, 1969; Gee, 1977). Synthetic auxins, such as NAA and 2,4‐D, appear to induce epinasty in the same way, i.e. by stimulating endogenous ethylene production (Morgan and Hall, 1962; Abeles and Rubinstein, 1964). However, the effect of 2,4‐D on epinasty in the absence of ethylene action has not been assessed to date. Since the epinastic curvature response in tobacco leaf strips incubated in auxin solutions was shown to be independent of ethylene biosynthesis (Keller and Von Volkenburgh, 1997), and the leaf strips were incubated in darkness, both ethylene‐induced epinasty and phototropic curvature responses were avoided here. Therefore, it is possible to address directly the mode of interaction between the two auxins without interference from the photo‐ or ethylene‐signal transduction pathways.

IAA‐induced epinastic curvature was analysed by means of in vivo Lineweaver–Burk kinetics, originally designed to analyse enzyme reactions (Dixon and Webb, 1979). In vivo Lineweaver–Burk analysis has frequently been applied to studies of interactions among different plant growth substances in living plant material, e.g. to analyse interactions between ethylene and CO₂ (Burg and Burg, 1967), ethylene precursor and its analogues (Zhong et al., 2001), and cytokinin and abscisic acid (Christianson, 2000). Traditionally, this analysis has been employed to assess the mode of action of putative inhibitors of auxin activity, and most known auxin inhibitors, such as p‐chlorophenoxyisobutyric acid (PCIB), have been assessed in this way (McRae and Bonner, 1964).

In this work it has been demonstrated that 2,4‐D acts against the IAA‐induced leaf curvature response in a complex manner; it was not possible to determine whether the mode of action was non‐competitive or uncompetitive. However, data strongly indicate that the mode of interaction between IAA and 2,4‐D in the leaf curvature response is not competitive interaction.

To the best of our knowledge, there is no previous report of 2,4‐D acting against IAA’s physiological activities. As discussed above, epinastic curvature in tobacco leaf strips is a non‐ethylene‐mediated phenomenon, while early studies using different systems have shown that the epinasty induced by IAA and 2,4‐D depends solely on the action of ethylene. Therefore, action of 2,4‐D on leaf epinasty in the absence of ethylene influence was analysed for the first time in the present study. A clue towards elucidating the anti‐epinastic action of 2,4‐D was found in early studies: in the recovery phase after epinasty had developed fully in 2,4‐D‐treated sunflower, leaves became hyponastic and were more erect than usual (Palmer, 1985). Palmer (1985) explained this as being the result of transient 2,4‐D‐induced ethylene production (as reported for cotton plants; Morgan and Hall, 1962). Thus, over time, the plants gradually recovered from the ethylene‐mediated epinasty. Palmer (1985) suggested that an explanation for hyponasty in the post‐epinastic phase is obtained if the lower half of the sunflower petiole is able to respond to 2,4‐D by elongation growth in the absence of ethylene production. However, the anti‐epinastic action of 2,4‐D in the tobacco leaf strips examined was not attributable to stimulation of ventral tissue growth (Figs 2C and 3B). In addition, 2,4‐D treatment of tobacco leaf strips did not result in hyponastic curvature; instead a novel phenomenon was observed in which 2,4‐D acts against IAA‐induced epinasty.

We have recently demonstrated that an alkaloid, hypaphorine, from an ectomycorrhizal fungus, Pisolithus tinctorius, acts specifically against IAA in eucalyptus roots (Ditengou and Lapeyrie, 2000; Ditengou et al., 2000; Kawano et al., 2001b) as well as in an in vitro enzyme assay system (Kawano et al., 2001a, 2002). Hypaphorine has been shown to be the major indolic compound present in free‐living hyphae of Pisolithus tinctorius (it occurs at concentrations 1000‐fold higher than those of IAA; Beguiristain et al., 1995), and its synthesis in the hyphae can be stimulated by interaction with its host plants (Beguiristain and Lapeyrie, 1997). Due to its structural similarity to IAA, hypaphorine secreted from P. tinctorius controls the development of root hairs in a host plant by counteracting the action of IAA (Ditengou et al., 2000). Since hypaphorine inhibited all measurable morphological effects of IAA in eucalyptus seedlings, a putative role has been proposed for hypaphorine as an anti‐auxin in plant–microbe interactions (Ditengou and Lapeyrie, 2000). The mode of interaction between IAA and hypaphorine has been shown to be competitive inhibition, both in in vitro enzyme assays (Kawano et al., 2001a, 2002) and in in vivo assays using eucalyptus roots (Kawano et al., 2001b). It is likely that this alkaloid and IAA target the same auxin receptor or the auxin‐binding domain on the same protein, since hypaphorine is structurally similar to IAA commonly sharing the indolic moiety (Martin et al., 1997). In contrast, the present study showed that 2,4‐D acts against IAA‐induced epinasty in a non‐competitive and/or uncompetitive manner. Two hypotheses are therefore suggested: (1) IAA activity in epinasty may involve a receptor that is different from that involved in 2,4‐D activity; or (2) the binding site on a putative auxin receptor or auxin‐binding protein involved in leaf curvature may be different for 2,4‐D and IAA.

These hypotheses are supported by earlier data showing that hypaphorine, the competitive inhibitor of IAA (thus possibly binding to an IAA‐binding site), shows no inhibitory activity against 2,4‐D in roots of eucalyptus seedlings (Ditengou and Lapeyrie, 2000). Since the activities of auxins are thought to be mediated by auxin‐binding proteins (Claussen et al., 1996; Jones et al., 1998), there may be at least two different auxin‐binding proteins (or receptors) involved in perception of IAA and 2,4‐D, respectively. Sugaya et al. (2000) have isolated a 2,4‐D‐binding protein from peach shoot extract that has high affinity for 2,4‐D but not for IAA. The identification of such proteins, which may behave as receptors for 2,4‐D rather than IAA, could provide further information. Although 2,4‐D has been shown to differ from IAA with respect to its affinity for auxin influx carriers (Yamamoto and Yamamoto, 1998, 1999; Marchant et al., 1999; Friml and Palme, 2002), this difference could not explain the novel effects of 2,4‐D on leaf epinasty.

Another view is that plants possess unknown mechanisms for 2,4‐D perception and/or signal transduction, with different degrees of affinity for 2,4‐D, since treatment with moderate concentrations of 2,4‐D (3 µm) led to a weak IAA‐like epinastic response, whereas concentrations over 3 µm were inhibitory (Fig. 2B and C). Results are indicative of the presence of such mechanisms in which a large excess of 2,4‐D over IAA results in uncompetitive inhibition of the IAA‐induced epinasty, while less abundant 2,4‐D leads to a non‐competitive mode of inhibition (Fig. 4). One mechanism may have a higher affinity for 2,4‐D, and the other a lower affinity, and these may be involved in the positive and negative responses of leaf curvature induction, respectively. Thus, the mechanism with a low affinity for 2,4‐D that requires high concentrations of 2,4‐D may act against the epinasty‐inducing activity of 2,4‐D itself and of IAA.

ACKNOWLEDGEMENTS

T.K. was supported by a fellowship from the Région Lorraine (France). This research was supported by the European Commission INCO‐DC programme (contract number: ERBIC18CT‐98319).

Received: 4 September 2002; Returned for revision: 18 October 2002; Accepted: 27 November 2002