Auxin-like effects of the natural coumarin scopoletin on Arabidopsis cell structure and morphology

Abstract

The mode of action and phytotoxic potential of scopoletin, a natural compound belonging to the group of coumarins, has been evaluated in detail. Analysis conducted by light and electron transmission microscopy showed strong cell and tissue abnormalities on treated roots, such as cell wall malformations, multi-nucleated cells, abnormal nuclei and tissue disorganization. Scopoletin compromised root development by inducing wrong microtubule assembling, mitochondrial membrane depolarization and ultimate cell death, in a way similar to auxin herbicides. The structural similarities of the natural compound scopoletin and the auxin herbicide 2,4-D, as well as the ability of scopoletin to fit into the auxin-binding site TIR1, were analyzed, suggesting that the phytotoxic activity of scopoletin matches with that exhibited by auxinic herbicides.

Introduction

Due to the increasing side effects of synthetic herbicides (weed resistance, environmental toxicity, human diseases, etc.), many natural compounds (mainly plant secondary metabolites) with phytotoxic potential have been already identified, and their regulatory capacity on plant growth has been studied to use them as eco-friendly bioherbicides (Duke et al., 2009, Duke, 2015, Koul et al., 2008, Oerke, 2006).

Coumarins are an important group of plant secondary metabolites with strong phytotoxic activity (Araniti et al., 2015, Avers and Goodwin, 1956, Lupini et al., 2010, Rice, 1984). Coumarins are usually related to plant defense, as their biosynthesis is usually induced under stressful conditions (Kai et al., 2006) and their exudation has been involved in iron chelation in conditions of iron deficiency (Tsai and Schmidt, 2017a, Tsai and Schmidt, 2017b). The phytotoxic activity of these compounds can result in photophosphorylation inhibition by blocking the electron transport in chloroplasts, effects on mitochondrial respiration, and growth inhibition (Dayan and Duke, 2006). As well, a recent study (Araniti et al., 2017) has also related coumarin to oxidative stress (increase of H₂O₂ content and lipid peroxidation and inhibition of antioxidant enzymes) and alteration of water status (reduction of relative water content and increase of leaf osmotic potential) on Arabidopsis adult plants.

Scopoletin (7-hydroxy-6-methoxy-2H-chromen-2-one) is a phenolic coumarin classified into the group of phytoalexins (Tal and Robenson, 1986), which have been specifically related to microbial attack and other stress conditions, like mechanical injury or dehydration (Tanaka et al., 1983). Scopoletin can be found in different plant species like tobacco (Andreae and Andreae, 1949), the poisonous plant henbane bell (Scopolia carniolica; Rollinger et al., 2004), in bael, a medicinal tree from India (Aegle marmelos; Panda and Kar, 2006); in Avena sativa (Goodwing and Kavanagh, 1949); plum tree (Prunus domestica; Hillis and Swain, 1959); sunflower (Helianthus annuus; Tal and Robenson 1986); or sweet potato (Ipomoea batatas; Imbert and Wilson, 1970). In 1963, scopoletin was described as the most common coumarin in higher plants (Robinson, 1963), and was found in 50 species of plants, including both mono- and dicotyledonous species (Winkler, 1967).

The phytotoxic potential of scopoletin was firstly described by Goodwing and Taves, 1950 in Avena sativa in 1950. Two years later, Andreae (1952) discovered that root growth of potato plants was inhibited by high concentrations (50 ppm) of scopoletin while stimulated by low concentrations (1 ppm). He connected these results to the ones found by Thimann (1937), who found the same effects after auxin application, suggesting that scopoletin could be inducing indoleacetic acid accumulation that will be stronger depending on the tested concentration.

This was corroborated by Imbert and Wilson (1970) that found inhibition of IAA oxidase activity in sweet potato when treated with scopoletin concentrations stronger than 13 μM, and stimulation when the tested concentrations were lower. Moreover, they described scopoletin as ‘the most potent naturally occurring stimulator of IAA oxidase activity so far reported’ to that date. One year later, growth and photosynthetic rate inhibition of tobacco, sunflower and pigweed seedlings after scopoletin treatment was positively related to scopoletin-caused stomatal closure (Einhellig and Kuan, 1971). No more studies on the phytotoxic potential of scopoletin were done until 1999, when it was described to cause root growth inhibition on germinating wheat seeds (Shukla et al., 1999).

Some authors also suggested that scopoletin is converted to its glycoside, scopolin (7-O-glucoside) into the plants, which was confirmed by Taguchi and collaborators in 2000, who investigated scopoletin accumulation in tobacco cells and found that the molecule is stored as scopolin, the glycoconjugated form of scopoletin, into the vacuoles.

However, despite these evidences, no deep studies were done on the mode of action of scopoletin on plant metabolism. Therefore, the goal of this study is in-depth analysis of the mode of action of scopoletin in the model species Arabidopsis thaliana, to elucidate the mechanisms behind its strong phytotoxic potential, and especially its similarities with auxinic herbicides.

Auxinic herbicides mimic the biphasic effects of IAA by interacting with specific cellular receptors as TIR1 (Transport Inhibitor-Response 1) and inducing a chain of effects similar to those of auxin. Among the different auxin herbicides, the synthetic IAA analogue 2,4-D (2,4-dichlorophenoxyacetic acid) acts on actin cytoskeleton structures leading to alteration of the mobility of peroxisomes and mitochondria, affecting their metabolism and inducing severe oxidative stress (Rodríguez-Serrano et al., 2014). Due to the loss of cell wall structure, ROS are able to penetrate deep into the plasma membrane where they can interact with phospholipids promoting: (i) unsaturation of plasma membrane lipids, (ii) leakage of the cytosol, and (iii) cell death (Christoffoleti et al., 2015).

Previous studies have provided highly relevant molecular information about the structural requirements of IAA analogues (Song, 2014). The crystallographic studies of substrate-binding of the auxin receptor Arabidopsis TIR1 have been reported (Kepinski and Layser, 2005). A structural explanation, of how TIR1 perceive and is activated by auxin, has helped to elucidate the structure–activity relationships of auxins analogues.

Through a carboxyl group, IAA analogues are tethered to the floor of the TIR1 pocket. The dichlorophenyl ring of the 2,4-D is accommodated by the overall shape and by hydrophobic properties of the TIR1 cavity. In summary, the auxin-binding site of TIR1 is defined by two highly selective polar residues (Arg 403 and Ser 438) spatially coupled to a less selective hydrophobic cavity with a fixed shape (Tan et al., 2007). This partially promiscuous hormone-binding site in TIR1 explains how the auxin receptor can potentially bind a variety of compounds. Actually, there are different compounds found to interact with TIR1 in a similar way to auxin (Hayashi et al., 2012, Oono et al., 2003, Peer, 2013, Takanashi et al., 2011).