Enhanced lipoxygenase activity is involved in barley root tip swelling induced by cadmium, auxin or hydrogen peroxide

Accepted Manuscript Title: Enhanced lipoxygenase activity is involved in barley root tip swelling induced by cadmium, auxin or hydrogen peroxide Author: Aster Alemayehu Be´ata Boˇcov´a Veronika Zelinov´a Igor Mistr´ık Ladislav Tam´as PII: DOI: Reference:

S0098-8472(13)00092-0 http://dx.doi.org/doi:10.1016/j.envexpbot.2013.06.004 EEB 2652

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

19-3-2013 16-5-2013 5-6-2013

Please cite this article as: Alemayehu, A., Boˇcov´a, B., Zelinov´a, V., Mistr´ık, I., Tam´as, L., Enhanced lipoxygenase activity is involved in barley root tip swelling induced by cadmium, auxin or hydrogen peroxide, Environmental and Experimental Botany (2013), http://dx.doi.org/10.1016/j.envexpbot.2013.06.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Research Highlights

Cd-, IAA- or H2O2-short-term treatments induced similar morphogenic responses in root LOX inhibitors efficiently inhibited stress-induced LOX activity and root swelling

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scavenger of hydroperoxides severely inhibited the development of root swelling

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Enhanced lipoxygenase activity is involved in barley root tip swelling induced by cadmium, auxin or hydrogen peroxide. Aster Alemayehu, Beáta Bočová, Veronika Zelinová, Igor Mistrík and Ladislav Tamás* Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic.

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*Corresponding author Tel.: +420 2 59426116; Fax: 421 2 54771948; E-mail address: [email protected]

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Abstract Lipoxygenases (EC 1.13.11.12) catalyze the formation of hydroperoxy derivates by oxygenation of polyunsaturated fatty acids. They act as signal molecules, triggering several developmental processes and defence responses under stress conditions. Incubation of CdIAA- or H2O2-short-term treated seedlings in the presence of LOX inhibitors efficiently inhibited both Cd- IAA- or H2O2-induced LOX activity and root swelling in a concentration dependent manner, suggesting a key role of LOX or LOX signalling pathway in radial expansion of root cells. Application of antioxidants (ascorbate or N-acetyl cysteine) to the treated seedlings at low 2 mM concentration did not affect the Cd-, IAA- or H2O2-induced LOX activity and root swelling. At higher, 10 mM concentration antioxidants markedly inhibited root growth, significantly increased the activity of LOX and evoked the radial expansion of root cells leading to root swelling with well developed root hairs already in control roots. By contrast, the lipophilic antioxidant trolox, a scavenger of hydroperoxides, severely inhibited the development of Cd-, IAA- or H2O2-induced root swelling, indicating that not directly LOX, but probably oxylipins, products of LOX pathway, are involved in the induction of root swelling in barley root tip. The results of this study suggest a strong connection between abiotic stress-induced alteration in redox and hormone status caused root growth inhibition and LOX pathway mediated radial expansion of root tip cells.

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Keywords: auxin, cadmium, hydrogen peroxide, lipoxygenase, root growth inhibition, root swelling Abbreviations: IAA – indole-3-acetic acid, ASC – ascorbate, CW – cell wall, LOX – lipoxygenase, NAC – N-acetyl cysteine, PG – propyl gallate, ROS – reactive oxygen species

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1. Introduction

It is well known that reactive oxygen and nitrogen species are not only toxic by-products of aerobic metabolism, but undoubtedly play a crucial role in the regulation of different developmental and physiological processes (Gabbita et al., 2000). However, recently an increasing attention has been directed toward to third group of oxidized reactive compounds derived mainly from unsaturated fatty acids. Fatty acid derived reactive electrophilic species represent the largest group of biologically active compounds (oxylipins) ubiquitously occurring in all higher organisms and they are probably involved as signal molecules in all physiological and developmental processes in plants including stress response (Mueller and Berger, 2009). Non-enzymatic lipid peroxides are continuously formed in organism and probably have an evolutionary ancient signal function both in animals and plants (Mueller, 2004). In addition to non-enzymatically formed hydroperoxides, the overwhelming number of oxylipins is produced through the enzymatic pathways, where lipoxygenases (LOX) catalyze the entry reaction forming fatty acid hydroperoxides (Feussner and Wasternack, 2002).

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LOXs are ubiquitously distributed enzymes among eukaryotic organisms, which catalyze the formation of hydroperoxy derivates by oxygenation of polyunsaturated fatty acids (Liavonchanka and Feussner, 2006). In plants multiple isoforms of LOXs exist, which have different temporal and spatial distribution during plant development and stress response (Porta and Rocha-Rosa, 2002). Similarly to reactive oxygen and nitrogen species, reactive electrophilic species act as signal molecules, triggering several developmental processes and defence responses, but they are responsible also for cell damage or even for cell death under severe stress conditions (Mueller and Berger, 2009). Furthermore, enhanced LOX activity was also associated with the production of superoxide in senescing bean leaves (Lynch and Thompson, 1984) or with oxidative burst in Phytophthora sojae-infected soybean roots (Mithöfer et al., 2002). The increased level of lipid peroxides and LOX activity was also associated with high temperature-induced damages both in leaf and root tissues (Ali et al., 2005). Many studies have also suggested that the elevated LOX activity in the presence of excess heavy metals in plant tissues is a causal factor of enhanced lipid peroxidation and membrane damage (Gallego et al., 1996; Aravind and Prasad, 2003; Zhou et al., 2008; Smeets et al., 2009). On the other hand increasing evidence indicates that the appearance of enhanced lipid peroxidation upon exposure of plants to heavy metals does not always result in the oxidative damage of membranes, but may be associated with enhanced LOX activity involved in the initiation of oxylipin pathways. In Arabidopsis and Phaseolus plants, after Cd or copper treatment a rapid but transient increase of jasmonic acid level occurred suggesting the metalinduced activation of LOX pathway (Maksymiec et al., 2005; Maksymiec, 2011; Cuypers et al., 2011). In addition, enhanced transcript level of LOX has been reported in Cd-exposed seedlings without the enhanced level of lipid peroxidation (Opdenakker et al., 2012). In our previous work we have demonstrated that the upregulation of LOX is an important component of stress response in barley roots to toxic Cd and it is probably involved in the morphological stress response of root tips or/and in the alleviation of Cd-induced toxic alterations in plant cell membranes, but it is not responsible for the Cd-induced harmful lipid peroxidation and cell death (Liptáková et al., 2013). Therefore, the purpose of this study was to analyze the effect of LOX inhibitors n-propyl gallate (PG) and naproxen, lipid peroxide scavenger trolox and reactive oxygen species (ROS) scavengers ascorbate (ASC) and Nacetyl cysteine (NAC) on LOX activity and Cd, indole-3-acetic acid (IAA) and H2O2 stressinduced morphogenic response such as root growth inhibition and radial root swelling in barley root tip. 2. Material and methods

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2.1. Plant material and growth conditions Barley seeds (Hordeum vulgare L.) cv. Slaven (Plant Breeding Station – Hordeum Ltd Sládkovičovo-Nový Dvor) were imbibed in distilled water for 15 min followed by germination between two sheets of filter paper (density 110 g.m-2, Papírna Perštejn, Czech Rep.) moistened with distilled water in Petri dishes. The uniformly germinating seeds, 24 h after the onset of seed imbibition, were arranged into row between two sheets of filter paper moistened with distilled water in rectangle trays. Trays were placed into nearly vertical position to enable downward radical growth. Continuous moisture of filter papers was supplied from the reservoir with distilled water through the filter paper wick. Seedlings, with approximately 4 cm long roots, 60 h after the onset of seed imbibition, were used for treatments. 2.2. Short-term treatments

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Roots of barley seedlings were immersed into distilled water (dw - control) or into 15 µM CdCl2, 10 µM IAA (from 1 mM stock in ethanol) or 1 mM H2O2 for 30 min. After washing in distilled water for 5 min the seedlings were incubated between two sheets of filter paper moistened with distilled water or with solutions containing 1 or 1.5 mM trolox; 0.25, 0.5 or 1 mM naproxen; 1, 1.5 or 2 mM n-propyl gallate; 2 or 10 mM N-acetyl cysteine and 2 or 10 mM sodium ascorbate as described above. After 6 h of incubation the 4 mm long root tips were used for analysis.

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2.3. Root length measurement

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For the determination of root length increment the positions of root tips following the treatments were marked on the filter paper. After 6 h, roots were excised at the position of marks and the length increment was measured after recording with stereomicroscope (STMPRO BELPhotonics, Italy) using BEL micro image analyzer. For the localization of root swelling roots were stained with 0.005 % Toluidine blue for 5 min and after washing with distilled water were photographed with stereomicroscope.

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2.4. Protein extraction and enzyme assays

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The root segments were homogenized in a pre-cooled mortar with 0.1 M potassium phosphate extraction buffer (pH 7.8) containing 1 mM EDTA. After centrifugation at 12 000 × g for 10 min, proteins were quantified with bovine serum albumin as the calibration standard by the method of Bradford (1976). Lipoxygenase (Linoleate:oxygen oxidoreductase; EC 1.13.11.12) activity was measured using the colorimetric method according to Anthon and Barrett (2001). The reaction mixture contained in a final volume of 110 μL 5 mM 3-dimethylaminobenzoic acid in 0.25 M sodium phosphate buffer (pH 6.0), 0.5 mM linoleic acid (from 25 mM stock solution dissolved in Tween 20) and 2.5 µg of proteins from the root extract. The mixture was incubated at 30 °C for 15 min then the mix of 20 μL of 1 mM 3-methyl-2-benzothiazolinone and 20 μL of hemoglobin (500 μg/mL) was added. After 5 min incubation at room temperature the absorbance was measured at 598 nm. To test the possible effects of peroxidases on reaction 1 mM azide (from 10 mM water stock solution) was added into the reaction mixture to inhibit non-specific peroxidase activity. 2.5. Statistical analyses

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The experiments were carried out in five independent series with three replicates (30 root tips per replicate). The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. 3. Results and discussion 3.1. Effect of LOX inhibitors on Cd-induced morphogenic responses and LOX activity Inhibition of root growth is the most widely observed symptom of various abiotic stresses, including heavy metals. In addition to root growth inhibition evoked by the short-term treatment of roots with 15 M Cd (Fig. 1A), radial expansion of cortical cells and accelerated root hair development in the elongation zone (during treatment) of root tips was detected appearing as a visible root swelling with long root hairs 6 h after the short-term treatment (Fig. 1C). Short-term Cd treatment of barley roots also led to the induction of LOX,

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representing a twofold increase in the activity at 6 h after treatment in comparison with the control root tips (Fig. 1B). The peroxidase inhibitor KCN had no significant effect on enzymatic reaction (controls: without KCN – 0.512 ± 0.048 or with KCN 0.521 ± 0.061 and Cd – without KCN 0.932 ± 0.032 or with KCN 0.911 ± 0.069; similar results were observed in the case of IAA or H2O2 – data not shown) suggesting that it was catalyzed by LOX but not by non-specific peroxidases. In addition, our previous studies have shown that two of three LOX isozymes are strongly stimulated by Cd and were insensitive to KCN (Liptáková et al., 2013). Several observations suggest that LOXs are involved in various developmental processes and in responses to stress conditions. Incubation of Cd-short-term treated seedlings at the presence of LOX inhibitor naproxen efficiently inhibited both Cd-induced LOX activity and root swelling in a concentration dependent manner (Fig. 1), suggesting a key role of LOX or LOX signalling pathway in radial expansion of root cells. In contrast to root swelling, in the case of root growth a synergistic effect of Cd and naproxen action was observed, resulting in a more severe root growth inhibition than in Cd or naproxen treatment alone (Fig. 1A). In control seedlings, exposed to distilled water for 30 min and subsequently incubated in the presence of naproxen, a significant decrease of LOX activity and a marked root growth inhibition was observed especially at the higher concentrations of naproxen. In spite of a slight decrease in the LOX activity, root growth was not affected by 0.25 mM naproxen in control roots. Furthermore, naproxen at higher concentration completely inhibited the development of root hairs. These findings are consistent with results that high LOX activity is generally associated with young, rapidly growing tissues (Eiben and Slusarenko, 1994; Hilbers et al., 1995; Berger et al., 2001; Bailly et al., 2002). Therefore, its inhibition in root tissues resulted in inhibition of root and root hair growth, which are the most rapidly growing parts of the root tip. Under Cd stress root swelling also represents a rapidly growing tissue of root tips with enhanced LOX activity. Therefore it is not surprising that the inhibition of LOX activity in our experiments strongly affects the radial enlargement of root cells. Function of LOX has also been described during the nodule development in soybean roots, where it is probably involved in the growth and development of specific cells within the root and nodules (Hayashi et al., 2008). On the other hand, nodule associated LOX in bean was abundant in growing tissues and highly activated by wounding, cold and drought and by treatment with jasmonic acid or abscisic acid in the mature region of seedlings (Porta et al., 1999). The involvement of LOX in growth processes is supported also by observation that new isoform appeared before the growth resumption of pea seedlings during germination (Chateigner et al., 1999) suggesting its role in the remodelling of membrane composition during growth. Similarly to Cd-induced root swelling in barley, an increased level of LOX mRNA positively correlated with tuber initiation and growth in potato, while its suppression by naproxen evoked reduced tuber yield, decreased tuber size and disruption of tuber formation suggesting its role in the regulation of tuber formation (Kolomiets et al., 2001). PG another widely used inhibitor of LOX (Fornaroli et al., 1999), eliminated the development of Cd-induced radial expansion of cells in barley root tip (Fig. 2). In contrast to naproxen, PG at 1 mM concentration had a beneficial effect on root growth of Cd-treated plants in spite of the considerable inhibition of root swelling (Fig. 2C). Exposure of Cdtreated plants to 1.5 mM PG fully blocked the development of root swelling, without negative effect on root growth. However, at higher PG concentration similarly to naproxen, a synergistic inhibitory effect of Cd and PG on root growth was observed. Inhibition of gamma irradiation-induced radial expansion of Arabidopsis root cells by PG was reported by Nagata et al. (2004). Authors suggest that the inhibition of root swelling was associated with the antioxidant properties of PG. The antioxidant activity of PG has been reported in tobacco cell culture, where it sufficiently prevents cryptogein-induced ROS production and cell death

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(Hirasawa et al., 2005). In contrast to naproxen, in Cd-treated plants PG at 1 mM concentration has a beneficial effect on root growth, in spite of that the inhibition of root swelling was considerable reduced. In our experiments, this antioxidant activity of PG (at 1 mM concentration) is probably responsible for the alleviation of Cd-induced root growth inhibition, similarly to NAC, but not for the inhibition of radial expansion of root cells. Our results indicate that this PG-mediated inhibition of Cd-induced root swelling is probably evoked through the inhibition of LOX activity.

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3.2. Effect of antioxidants on Cd-induced morphogenic responses and LOX activity

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Although Cd is a non-redox active heavy metal unable to directly generate ROS, a great deal of results indicates that the Cd-induced accumulation of ROS in root tissues is responsible for most of the Cd-induced adaptation and toxicity symptoms in roots (Benavides et al., 2005; Sharma and Dietz, 2009). Therefore, we have examined the effect of antioxidants on the Cdinduced responses. Incubation of control roots in the presence of low 2 mM concentration of antioxidants (ASC or NAC), which did not significantly reduce root growth, did not lead to LOX activation and to the development of root swelling (Fig. 3). While NAC at this concentration did not affect the Cd-induced LOX activity and root swelling, ASC had an additive effect on the activity of LOX (Fig. 4). In contrast, the application of NAC or ASC at concentration of 10 mM to control roots markedly inhibited root growth, significantly increased the activity of LOX and evoked the radial expansion of root cells leading to root swelling with well developed root hairs (Fig. 3). This observation indicates that not only increased ROS generation (Tamás et al., 2012), but also their down-regulation may evoke similar morphogenic responses in barley root tip. Trolox, a lipophilic antioxidant at 1.5 mM concentration, despite the marked inhibition of root and root hair growth, had no effect on LOX activity and did not induce the swelling of control root tips (Fig. 3). As well, it severely inhibited the development of Cd-induced root swelling and root hair without modulating the Cd-induced LOX activity (Fig. 4). This indicates that not directly LOX, but probably oxylipins, products of LOX pathway, are involved in the induction of root swelling in barley root tip exposed to Cd treatment. In addition to the inhibition of radial cell expansion, trolox alleviated the Cd-induced inhibition of root growth, which is probably associated with the antioxidant properties of trolox against Cd-induced ROS. Similarly to trolox, this alleviation of Cd-induced root growth inhibition was observed also after the application of NAC. However co-treatment of control roots with trolox and NAC strongly affected root growth and LOX activity similarly to high NAC or ASC concentrations, it did not contribute to the radial expansion of root cells (Fig. 4C). On the other hand, their application to the short-term Cdtreated roots had a synergistic effect on the alleviation of Cd-induced root growth inhibition. These results indicate that the co-treatment of roots with trolox and NAC markedly disturbs the redox homeostasis of control root tips leading to the inhibition of root growth and to the activation of LOX pathway, but due to the presence of oxylipin scavenger trolox, it does not activate the radial expansion of cells in root tip. In Arabidopsis, exogenously applied oxylipins evoked numerous changes in root development such as root waving, loss of apical dominance and decreased root growth (Vellosillo et al., 2007), which are also characteristic features of roots growing under some stress conditions. The role of oxylipins in Cd-induced responses of roots is also supported by the observation that a mutant of the Arabidopsis LOX1 gene showing altered signalling and oxidative stress related responses after cadmium exposure failed to develop stress response similar to wild-type seedlings (Remans et al., 2012; Keunen et al., 2013).

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3.3. Effect of antioxidants and LOX inhibitors on IAA-induced morphogenic responses and LOX activity

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Increasing evidence indicates the role of plant hormones, especially IAA, in response of root to stress conditions (Tognetti et al., 2012). The function of IAA in Cd toxicity was supported by the observation that Cd strongly activated the expression of auxin-responsive genes (Minglin et al., 2005). Beside the IAA-induced inhibition of root growth, it has long been known that the radial expansion of cortical cells is a characteristic phenomenon of IAAtreated roots (Svensson, 1972). Previous studies have shown that similarly to Cd at low concentration, externally applied IAA or IAA transport inhibitor induce similar morphological changes such as root growth inhibition or radial swelling (Tamás et al., 2012). In the present study we have shown that similarly to Cd-induced LOX activity in barley roots, IAA-induced LOX activity was considerable decreased after the application of LOX inhibitor naproxen or PG, which was also accompanied by the inhibition of radial expansion of root tips (Fig. 5). Antioxidants ASC or NAC had no significant effect on IAA-induced LOX activity and root swelling. In contrast, trolox effectively suppressed the development of IAAmediated root swelling. Trolox significantly affected IAA-induced LOX activity and further increased the negative effect of IAA on root growth. This negative effect on root growth was also observed after the incubation of short-term IAA-treated roots in the presence of ASC suggesting some differences between IAA- and Cd-induced responses. It was reported that jasmonate and methyl jasmonate, the well characterized oxylipins in plants, strongly inhibited root growth and activated several defence genes in Arabidopsis (Staswick et al., 1992). In addition, similar physiological responses were observed to jasmonic acid and heavy metals suggesting the involvement of jasmonic acid signal pathway in the metal-induced responses of plants (Maksymiec and Krupa, 2002). High level of jasmonic acid in potato tuber buds is correlated with the radial expansion of meristematic cells in the buds during their transformation into sprouts (Castro et al., 1999). It has been previously shown that IAA activated the synthesis of jasmonic acid by the induction of expression of genes involved in jasmonic acid synthesis including LOX (Liu et al., 1991; Tiryaky and Staswick, 2002). Furthermore, there is a strong positive feedback regulation because jasmonic acid also activated the expression of genes in LOX pathway leading to the considerable amplification of IAA induced signal transduction. 3.4. Effect of antioxidants and LOX inhibitors on H2O2-induced morphogenic responses and LOX activity

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The role of H2O2 is well known in the Cd- or IAA-mediated root growth inhibition either as a toxic factor (Grossmann et al., 2001; Benavides et al., 2005) or as a signal molecule (Joo et al., 2001; Sharma and Dietz, 2009). In our previous work we showed that auxin signalling inhibitor alleviates or even blocks the Cd-induced root growth inhibition and radial root expansion, which was accompanied with the reduction of Cd-generated H2O2 (Tamás et al., 2012). These results suggest the possible role of H2O2 as a key signal molecule in the induction of root swelling. Similarly to what was observed for Cd and IAA short-term treatments, the enhanced activity of LOX, the inhibition of root growth and the induction of root swelling with well developed root hairs are the characteristic symptoms also of roots exposed to H2O2 for 30 min (Fig. 6). Furthermore, both LOX inhibitors naproxen and PG or the lipid hydroperoxide scavenger trolox suppressed, while antioxidants NAC or ASC did not affect the appearance of root swelling after H2O2 short-term treatment. In soybean it was reported that H2O2 at physiological concentration is a potent activator of LOX activity (Kulkarni et al., 1990). In addition it has been reported that H2O2 is able to activate LOX

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directly by oxidizing iron centre to catalytically active ferric state (Skrzypczak-Jankun et al., 2001). In Arabidopsis, H2O2 plays a key signalling role in the activation of LOX during the transition of seedlings from vegetative growth to flowering (Bañuelos et al., 2008). However, our results showed that either excess or deficiency of H2O2 is capable of triggering LOX activation. Similar results were observed in the regulation of glutathione S-transferase gene expression in rice roots, where both H2O2 and antioxidant treatments strongly up-regulated its expression (Moons, 2003). These results suggest that redox perturbation, either decreases or increases of ROS, may participate in the stress responses of roots by activating the LOX signalling pathway. The observation that scavenging of H2O2 also induced LOX activity and stimulated root swelling suggests that H2O2 is not a general signal for the induction of LOX mediated radial root expansion in barley root tip. One common feature of all Cd, IAA, H2O2 scavengers and H2O2 treatments is the marked inhibition of root growth, which is probably associated with changes in cell wall (CW) structure and physiology. It is well known that any disturbance in CW structure caused by irregular CW synthesis in cellulose synthesis mutants or by inhibition of cellulose synthesis evoked root growth inhibition as well as radial root swelling and induction of defence-related genes, probably as a consequence of constitutive active jasmonate and ethylene signalling pathways (Ellis and Turner, 2001). The role of CW and membranes in the LOX regulation was also supported by the observation that touch treatment induces a strong increase in LOX transcript levels (Mauch et al., 1997). In addition it was suggested that 9-LOX pathway operates mainly through the modification of CW as well as the production of ROS (Vellosillo et al., 2007). These and our results indicate a strong connection between abiotic stress-induced alteration in redox and hormone status-mediated root growth inhibition and LOX-mediated radial expansion of root tip cells.

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4. Conclusion

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Incubation of Cd-, IAA- or H2O2-short-term treated seedlings in the presence of LOX inhibitors efficiently inhibited both Cd- IAA- or H2O2-induced LOX activity and root swelling in a concentration dependent manner, suggesting a key role of LOX or LOX signalling pathway in radial expansion of root cells. Whereas, the presence of antioxidants ASC or NAC did not affect the Cd-, IAA- or H2O2-induced LOX activity and root swelling, the lipophilic antioxidant trolox, a scavenger of hydroperoxides, severely inhibited the development of Cd-, IAA- or H2O2-induced root swelling, indicating that not directly LOX, but probably oxylipins, products of LOX pathway, are involved in the induction of root swelling in barley root tip. Taken together, the results of this study indicate a strong connection between abiotic stress-induced alteration in redox and hormone status caused root growth inhibition and LOX pathway mediated radial expansion of root tip cells. Acknowledgements We wish to thank Margita Vašková for excellent technical assistance. This work was supported by the Grant agency VEGA, project No. 2/0019/13. References Ali, M.B., Hahn, E-J., Paek, K-Y., 2005. Effects of temperature on oxidative stress defence systems, lipid peroxidation and lipoxygenase activity in Phalaenopsis. Plant Physiol. Biochem. 43, 213-223. Anthon, G.E., Barrett, D.M., 2001. Colorimetric method for the determination of lipoxygenase activity. J. Agric. Food. Chem. 49, 32-37.

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Figure legends Fig. 1. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) or 15 µM Cd for 30 min and subsequently incubated in the presence of 0, 0.25, 0.5 and 1 mM naproxen (N) for 6 h. The black arrows show the starting position of new root growth after the short-term treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05).

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Fig. 2. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) or 15 µM Cd for 30 min and subsequently incubated in the presence of 0, 1, 1.5 and 2 mM propyl gallate (PG) for 6 h. The black arrows show the starting position of new root growth after the short-term treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05).

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Fig. 3. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) 30 min and subsequently incubated in the presence of dw, 2 and 10 mM N-acetyl cysteine (NAC), 2 and 10 mM ascorbate (ASC) or 1 and 1.5 mM trolox for 6 h. The black arrows show the starting position of new root growth after the shortterm treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05).

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Fig. 4. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) or 15 µM Cd for 30 min and subsequently incubated in the presence of dw, 2 mM ascorbate (ASC), 2 mM N-acetyl cysteine (NAC), 1 mM trolox (T) or 2 mM N-acetyl cysteine and 1 mM trolox (NAC+T) for 6 h. The black arrows show the starting position of new root growth after the short-term treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05).

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Fig. 5. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) or 10 µM IAA for 30 min and subsequently incubated in the presence of dw, 0.5 mM naproxene (N), 1.5 mM propyl gallate (PG), 2 mM ascorbate (ASC), 2 mM N-acetyl cysteine (NAC) or 1 mM trolox (T) for 6 h. The black arrows show the starting position of new root growth after the short-term treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05). Fig. 6. Root length increments (A), LOX activity (B) and root swelling (C) of short-term treated roots with distilled water (dw) or 1 mM hydrogen peroxide (HP) for 30 min and subsequently incubated in the presence of dw, 0.5 mM naproxen (N), 1.5 mM propyl gallate (PG), 2 mM ascorbate (ASC), 2 mM N-acetyl cysteine (NAC) or 1 mM trolox (T) for 6 h. The black arrows show the starting position of new root growth after the short-term treatments. S – swollen root part. Mean values ± SD (n = 5). Different letters indicate statistical significance according to Tukey’s test (P0.05).

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