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Salicylic acid alleviates cadmium-induced stress responses through the inhibition of Cd-induced auxin-mediated reactive oxygen species production in barley root tips
Journal of Plant Physiology 173 (2015) 1–8
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Physiology
Salicylic acid alleviates cadmium-induced stress responses through the inhibition of Cd-induced auxin-mediated reactive oxygen species production in barley root tips Ladislav Tamás ∗ , Igor Mistrík, Aster Alemayehu, Veronika Zelinová, Beáta Boˇcová, Jana Huttová Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 20 May 2014 Received in revised form 31 July 2014 Accepted 1 August 2014 Available online 22 September 2014 Keywords: Auxin Barley Cadmium Root growth inhibition Root swelling
a b s t r a c t Auxin is a master regulator of root growth by modulating its development under the constantly changing environment. Recently, an antagonistic interaction was suggested between SA and IAA signaling. Therefore, the purpose of this work was to analyze and compare the effect of the indole-3-acetic acid (IAA) signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and salicylic acid (SA) as a potential IAA signaling inhibitor on the root growth, enzyme activity and reactive oxygen species (ROS) production in Cd- and IAA-treated barley root tips. Exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3 h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition and swelling, ROS generation and activation of lipoxygenase or glutathione peroxidase identical to those induced by Cd. Furthermore, both Cd- and IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment. Similarly to PCIB, SA did not affect the IAA content of root tips, suggesting the action of SA on the IAA signaling pathway in barley roots. SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of Cd-induced IAA-mediated ROS generation in roots. © 2014 Elsevier GmbH. All rights reserved.
Introduction A number of previous studies have demonstrated that salicylic acid (SA), a widely distributed phenolic compound in the plant kingdom, modifies a wide range of physiological and developmental processes and stress responses resembling the action of classical plant hormones (Raskin, 1992; Vicente and Plasencia, 2011). The activation of the defense response against pathogens and the induction of systemic acquired resistance are the most studied functions of SA in plants (Durner et al., 1997). In addition to its role in biotic stress responses, SA also participates in the adaptation of plants to various unfavorable environmental conditions, including high salinity (Lee et al., 2010), cold (Kang and Saltveit, 2002), drought
Abbreviations: AAO, ascorbic acid oxidase; DHAR, dehydroascorbate reductase; GPX, glutathione peroxidase; 4-HBA, 4-hydroxybenzoic acid; IAA, indole-3-acetic acid; LOX, lipoxygenase; PCIB, p-chlorophenoxyisobutyric acid; ROS, reactive oxygen species; SA, salicylic acid. ∗ Corresponding author. Tel.: +421 2 59426116; fax: +421 2 54771948. E-mail address: [email protected] (L. Tamás). http://dx.doi.org/10.1016/j.jplph.2014.08.018 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
(Sun et al., 2009) and the excess of heavy metals (Horváth et al., 2007; Hayat et al., 2010). Cadmium (Cd) is one of the most dangerous heavy metals for nearly all organisms due to its high solubility in water and toxicity even at very low concentrations. Numerous biochemical and molecular mechanisms have been suggested to be responsible for Cd toxicity in plants, but the disturbance of reactive oxygen species (ROS) homeostasis undoubtedly plays a key role in the development of Cd toxicity symptoms (Cuypers et al., 2010; Gallego et al., 2012). The root is the first plant organ that encounters Cd toxicity, resulting in enhanced ROS generation and growth inhibition within a few minutes of exposure (Ortega-Villasante et al., 2007). Therefore, the early elimination of ROS is a key component of plant tolerance to Cd. Our previously published paper has demonstrated a strong upregulation of antioxidant enzymes in barley root tip. Glutathione peroxidase (GPX) reduces not only hydrogen peroxide, but also organic and lipid hydroperoxides, maintaining cell function despite the high rate of ROS metabolism in root tips under Cd stress (Zelinová et al., 2013a). Rapid recycling of ascorbate and glutathione as the main antioxidants in plant cells by the enzymes of ascorbate–glutathione cycle also contributes to the enhanced
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tolerance of plants to stress (Zelinová et al., 2011). The treatment of roots with Cd resulted in the greatest changes in dehydroascorbate reductase (DHAR) activity, which was activated within 1 h of Cd treatment along the whole barley root tip (Zelinová et al., 2013b). Several Cd-induced root morphogenic responses, including inhibition of cell elongation, alterations in cell differentiation and reorientation of cell growth mimic the auxin- or polar auxin transport inhibitor-induced morphological changes in root tips, suggesting a role of indole-3-acetic acid (IAA) in these processes (Tamás et al., 2012). In rice primary root tips, a marked IAA accumulation was observed even after 6 h of Cd treatment (Zhao et al., 2013). Similarly, the level of IAA in Arabidopsis roots markedly increased after treatment with Cd or Cu (Sofo et al., 2013). The role of IAA as a modulator of the stress response of roots to Cd excess is also supported by the observation that Cd strongly activated the expression of several auxin-responsive genes (Minglin et al., 2005). Furthermore, ROS act as an intermediate signal in several IAA-mediated processes, such as unilateral inhibition of root elongation during root gravitropic curvature (Joo et al., 2001) and growth retardation caused by auxin-type herbicides (Grossmann et al., 2001). In barley, lipoxygenase-(LOX)-generated oxylipins are probably involved in the reorientation of cell expansion in the root apex after Cd, IAA or hydrogen peroxide treatment (Alemayehu et al., 2013). On the other hand, ascorbic acid oxidase (AAO), as an extracellular electron acceptor generating enzyme of the plasma membrane electron transport system, is strongly inhibited after the exposure of seedlings to Cd, Cu, hydrogen peroxide or IAA (Zelinová et al., 2011). Crosstalk between SA and IAA signaling has been suggested by several studies. Wang et al. (2007) have shown that SA inhibits pathogen growth in plants through the repression of the auxin signaling pathway. In Arabidopsis, SA also activated the expression of GH3 genes encoding auxin-conjugating enzymes, reducing the free active form of IAA (Park et al., 2007). An antagonistic effect of IAA and SA was observed in maize roots, where exogenous IAA increased lateral root growth at the expense of primary root growth, while SA had a stimulating effect on total root biomass (Agtuca et al., 2014). An alleviating effect of SA on Cd-induced toxicity symptoms has been reported in barley (Metwally et al., 2003), soybean (Drazic and Mihailovic, 2005), and rice (Guo et al., 2007). In addition, it has also been reported that Cd exposure increased the free SA content of barley roots (Metwally et al., 2003). Therefore, the purpose of this work was to analyze and compare the effect of the IAA signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and SA as a potential IAA signaling inhibitor on root growth, enzymes activity and ROS production in Cd- and IAA-treated barley root tips.
Materials and methods Plant material and growth conditions Barley seeds (Hordeum vulgare L.) cv. Slaven (Plant Breeding Station – Hordeum Ltd., Sládkoviˇcovo-Novy´ Dvor) were imbibed in distilled water for 15 min followed by germination between two sheets of filter paper (density 110 g/m2 , Papírna Perˇstejn) moistened with distilled water in Petri dishes at 25 ◦ C in darkness. The uniformly germinating seeds, 24 h after the onset of seed imbibition, were arranged into rows between two sheets of filter paper moistened with distilled water in rectangular trays. The 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.
Short-term treatments Roots of seedlings were pre-treated by immersion into distilled water (dw – control) or into 15 M CdCl2 or 1 M IAA (from 10 mM stock in ethanol) for 30 min. After brief washing in distilled water, the roots were immersed into 50 or 100 M PCIB (from 10 mM stock in ethanol) or into 0.25 or 0.5 mM SA or 0.25 mM 4-HBA (from stock in DMSO) for 10 min. After treatments, the seedlings were incubated between two sheets of filter paper moistened with distilled water as described above. After 3 or 6 h of incubation the root tips (3 mm in length) were used for analysis. Root length measurement For the determination of root length changes, the positions of root tips following the treatments were marked on the filter paper. After 6 h, the roots were excised at the position of marks and the length increment was measured after recording with stereomicroscope using micro image analyzer. For localization of root swelling, the roots were stained with 0.05% Toluidine blue for 10 min and after washing with distilled water were photographed with a stereomicroscope. Protein extraction and sample preparation The root tips were homogenized in a pre-cooled mortar with 100 mM potassium phosphate extraction buffer pH 7.8 containing 1 mM EDTA 6 h after the treatments. 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). Enzyme assays Lipoxygenase (LOX, EC 1.13.11.12) activity was measured using the colorimetric method according to Anthon and Barrett (2001). The reaction mixture contained (160 L in a final volume) 5 mM 3-dimethylaminobenzoic acid in 25 mM sodium phosphate buffer (pH 6.0), 1 mM KCN, 0.5 mM linoleic acid (from 25 mM stock solution dissolved in Tween 20) and 2 g of proteins from the root extract. The mixture was incubated at 30 ◦ C for 15 min, then the mix of 20 L of 1 mM 3-methyl-2-benzothiazolinone and 20 L of hemoglobin (500 g/mL) was added. After 5 min incubation at room temperature the absorbance was measured at 598 nm. Specific LOX activities were expressed as A598 mg−1 min−1 . Ascorbic acid oxidase (AAO, EC 1.10.3.3) activity was determined by measuring the decrease in absorbance at 265 nm reflecting the ascorbate oxidation (Arrigoni et al., 1981). The reaction mixture (250 L) contained 50 mM potassium phosphate buffer pH 7.0, 0.15 mM ascorbate, 0.5 mM EDTA and 2 g of proteins from root extract. The reaction mixture was incubated at 30 ◦ C for 30 min. Specific activities were expressed as A265 mg−1 min−1 . Glutathione peroxidase (GPX, EC 1.11.1.9) activity was measured by a spectrophotometric method according to Drotar et al. (1985) with some modifications using microplate reader (Synergy HT, BIO-TEK, USA). The reaction mixture (200 L) contained 2 mM glutathione, 0.5 mM NADPH, 1 mM EDTA, 2 mM t-butyl hydroperoxide and 0.5 U of glutathione reductase in 100 mM sodium phosphate buffer, pH 7.0 and 20 g of extracted proteins. The rate of NADPH oxidation was measured at 340 nm over a time period of 20 min at 30 ◦ C. Specific GPX activities were expressed as A340 mg−1 min−1 . Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was assayed by measuring the rate of ascorbate formation at 265 nm in a reaction mixture containing 100 mM potassium phosphate buffer pH 7.0, 1 mM reduced glutathione, 0.5 mM dehydroascorbate and
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10 g of proteins from root extract. The reaction mixture (250 L) was incubated at 30 ◦ C for 20 min (Arrigoni et al., 1981). Specific activities were expressed as A265 mg−1 min−1 . Localization of ROS production Intact roots (3 h after treatments) were immersed into the solution of 10 M 2,7-dichlorodihydrofluorescein diacetate (from 20 mM stock in DMSO) in 20 mM sodium phosphate buffer pH 6.0 for 30 min at 25 ◦ C in darkness. After brief washing with distilled water fluorescence of whole roots was observed immediately (for no more than 5 min) using fluorescence stereomicroscope (excitation 500 ± 20 nm; emission 535 ± 30 nm). IAA quantification The root tips (3 h after treatments) were homogenized in a pre-cooled mortar with ice-cold 80% methanol containing 1 mM butylated hydroxytoluene (80 root tips/mL). After centrifugation at 12,000 × g for 10 min, samples were passed through a C18 column (Chromabond, Macherey-Nagel) preconditioned with 80% methanol at 4 ◦ C. After evaporation of methanol, samples were methylated using trimethylsilyldiazomethane in hexane (2 L/500 L) at 42 ◦ C for 30 min and diluted in 25 mM sodium phosphate buffer (pH 7.2) containing 15 mM NaCl. Quantification of IAA was performed by competitive enzyme-linked immunosorbent assay (IAA immunoassay kit Olchemin). Statistical analyses The experiments were carried out in three independent series with three replicates (20 root tips for root length measurement and ROS analysis, 40 root tips for enzyme analysis and 80 root tips for IAA analysis per replicate). The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Short-term exposure of barley roots to 15 M Cd caused a marked reduction in root growth (Fig. 1A), which was accompanied by considerable radial expansion of root tips (Fig. 1B). Post-treatment of Cd-stressed plants with 50 M PCIB or 0.25 mM SA (concentrations were selected in order to obtain the greatest alleviating effect on the Cd-induced root growth inhibition) markedly alleviated the Cd-induced inhibition of root growth and radial expansion of root tips (Fig. 1). At higher concentrations, both PCIB and SA inhibited root growth in a concentration-dependent manner in controls as well as in Cd-treated roots (data not shown). Both Cd-induced LOX and GPX activity were reduced by the posttreatment of plants with PCIB or SA (Fig. 2A and B). On the contrary, PCIB or SA post-treatment did not alleviate the Cd-induced changes in DHAR (DHAR activity was further stimulated by PCIB in Cdtreated roots) or AAO activity (Fig. 2C and D). The analysis of IAA content in the root apex revealed a considerable accumulation of IAA 3 h after the Cd treatment (Fig. 3). However, neither PCIB nor SA affected this Cd-induced accumulation of IAA in the root apex. Treatment of seedlings with 1 M IAA inhibited root growth and evoked radial expansion of root tips in a similar extent as was observed in seedlings after the exposure to Cd (Fig. 4). In addition, the alleviating effect of PCIB or SA (however, in IAAtreated seedlings higher concentrations of PCIB or SA were required to obtain the greatest alleviating effect in comparison with Cdtreated seedlings) on root growth inhibition (Fig. 4A) and swelling (Fig. 4B) was also observed in IAA-treated seedlings. PCIB and SA
Fig. 1. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
post-treatment also reduced the IAA-induced LOX and GPX activity (Fig. 5A and B). However, the stimulation of DHAR and the inhibition of AAO activity by IAA treatment were less profound in comparison with the changes induced by Cd stress (Fig. 5C and D). 4-HBA a biologically inactive analog of SA, did not alleviate the Cd-induced root growth inhibition (Fig. 6A) or root swelling (Fig. 6B) and had no effect on the activity of analyzed enzymes in the control or in the Cd-treated seedlings (Fig. 7). The analysis of ROS generation revealed that Cd and IAA had a similar effect on ROS generation in the root apex (Fig. 8). While in control roots ROS were observed in the differentiation zone (3 mm behind root apex) and decreased gradually in the direction of elongation zone, in Cd- and IAA-treated seedlings, the ROS generation was markedly increased at the end of elongation and the beginning of differentiation zone. PCIB and SA treatment markedly inhibited this ROS generation in control, Cd- or IAA-treated roots. Discussion The exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3 h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition, swelling, ROS generation and activation of LOX and GPX identical with those induced by Cd. Previous studies have shown that SA also participates in the adaptation of
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Fig. 2. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
plants to various unfavorable environmental conditions, including the excess of heavy metals (see Introduction section). However, an antagonistic effect has been reported between IAA and SA action on root architecture in the root response to biotic stress (Agtuca et al., 2014). This effect of SA was also supported by the observation that accumulation of SA in Arabidopsis roots caused the repression of auxin-related genes and insensitivity to exogenously applied auxin (Wang et al., 2007). Therefore, in this study we compared
Fig. 3. IAA content of root tips 3 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
the effect of PCIB as an IAA signaling inhibitor and SA as a potential IAA signaling inhibitor on root morphology, enzyme activity and ROS production in Cd- and IAA-treated barley root tips. Both Cdand IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment, indicating that SA reversed the Cd-induced responses through the inhibition of IAA action. In addition, similarly to PCIB, SA did not affect the IAA content of Cd-treated root tips, suggesting the action of SA on the IAA signaling in barley roots. It is well known that, at low concentrations, IAA is necessary for proper root growth, but with increasing concentrations it strongly inhibits root elongation in a dose-dependent manner (Tanimoto, 2005). In accordance with these results, we observed that PCIB at higher concentrations inhibited root growth in control seedlings treated only with distilled water (data not shown). On the contrary, at lower concentrations, it had a promoting effect, suggesting that the inhibition of IAA signaling may, to some degree, also accelerate root elongation under unstressed conditions probably due to the inhibition of IAA-mediated differentiation of root cells. On the other hand, the handling of seedlings during the experiment and the immersion of control roots into distilled water may represent a certain degree of stress activating IAA signaling-mediated inhibition of root growth. This inhibition may be eliminated by the application of PCIB at low concentrations. Earlier studies have clearly demonstrated the role of ROS in IAA-mediated root growth inhibition (Joo et al., 2001; Grossmann et al., 2001). Therefore, the Cd-induced IAA accumulation is likely responsible for the enhanced ROS generation, causing a marked reduction of cell elongation. On the other hand, it was shown that Cd-stimulated rice root growth is also associated with the alterations in IAA and ROS homeostasis (Zhao et al., 2011) suggesting a central role of IAA and ROS in the Cd-induced responses in roots.
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Fig. 4. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 1 M IAA for 30 min and subsequent treatment with dw or 100 M PCIB or 0.5 mM SA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
Fig. 5. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) or 1 M IAA for 30 min and subsequent treatment with dw or 100 M PCIB or 0.5 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
In this study, we demonstrated that the alleviating effect of both PCIB and SA either on Cd- or IAA-induced responses was associated with decreased ROS generation in root tissues. Similarly to our results, previous studies have shown that exogenous application of SA alleviates Cd or Hg toxicity by reducing ROS accumulation in roots (Zhou et al., 2009; Zhang et al., 2011a,b). While a shorter
SA pre-treatment decreased the Cd-induced H2 O2 production leading to attenuation of Cd-induced root growth inhibition, during the prolonged exposure of rice roots to SA, an increase in H2 O2 production was observed (Guo et al., 2007). In addition, exogenous application of SA promotes Arabidopsis seed germination by reducing ROS production under high salinity stress (Lee and Park, 2010). Today, it is undisputed that ROS are ubiquitous signaling molecules involved in the regulation of different developmental and physiological processes. Previous studies have shown that LOX inhibitors efficiently inhibited both Cd-, IAA- and H2 O2 -induced root swelling in a concentration-dependent manner, suggesting a key role of LOX or the LOX signaling pathway in radial expansion of root cells (Alemayehu et al., 2013). In agreement with these results, the IAA signaling inhibitor PCIB and SA reversed both Cd- and IAAinduced LOX activity, resulting in a considerable inhibition of radial cell expansion. Several studies have indicated that SA mitigates the Cd toxicity by activation of non-enzymatic and enzymatic antioxidants, leading to reduction of Cd-induced ROS generation in plants (Guo et al., 2009; Popova et al., 2009; Zhang et al., 2011a,b). By contrast, the PCIB or SA post-treatments considerably reduced the Cd- or IAAinduced GPX activity, indicating the role of IAA in the activation of antioxidant response in barley roots. Metwally et al. (2003) have reported that SA alleviated Cd toxicity in barley root through root growth promotion and reduction of lipid peroxidation. However, it also suppressed the Cd-induced upregulation of numerous antioxidant enzymes, including GPX. These and our results indicate that SA attenuates ROS generation in Cd-treated roots by other mechanisms than the activation of antioxidant enzymes. On the contrary, SA just attenuates the IAA-mediated generation of ROS, which function as signal molecules responsible for the activation of some components of the defense system (such as GPX) in root tips against the potential oxidative stress during the prolonged Cd stress. These results are also supported by previous observations that both Cdinduced enhanced LOX and GPX activity were not associated with harmful oxidative stress such as lipid peroxidation or cell death in barley root tips (Liptaková et al., 2013). On the other hand, our results also showed that SA, similarly to PCIB, had a different effect on the analyzed enzymes. While LOX and GPX were strongly downregulated, DHAR and AAO were not affected by the SA or PCIB post-treatment, suggesting the participation of different signaling pathways in their activation or inhibition during Cd stress. While both LOX and GPX activity were strongly
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Fig. 6. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 0.25 mM SA or 0.25 mM 4HBA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
stimulated, DHAR was not activated by exogenously applied H2 O2 (Zelinová et al., 2013b). Therefore, inhibition of IAA-mediated ROS generation by PCIB or SA cannot affect DHAR activity in these experiments. On the other hand, exogenous IAA significantly stimulated DHAR activity, suggesting that ROS are involved in the activation of DHAR neither by Cd, nor by IAA in barley root tip. It has previously been reported that ROS strongly inhibited AAO activity in barley roots (Zelinová et al., 2011). However, despite a marked reduction of ROS generation by PCIB and SA, the inhibition of AAO in Cdtreated roots was not eliminated, suggesting that apart from the ROS-mediated inhibition of AAO activity, it is also inhibited through other mechanisms in Cd-treated roots.
Taken together, the results of this study indicate that IAAmediated ROS generation plays a key signaling role in the response of roots to stress conditions, through the activation of the defense systems, including morphogenic changes such as root growth inhibition or swelling and through the induction of some antioxidant enzymes. The Arabidopsis IAA signaling mutant exhibits a higher rate of root elongation than the wild type seedlings under both oxidative and salinity stress (Iglesias et al., 2010). By contrast, auxin-overproducing mutants show a lower rate of root elongation than wild type seedlings under Al stress (Zhu et al., 2013). In this work, we have shown that inhibition of IAA signaling by PCIB or SA resulted in the alleviation of Cd- or IAA-induced morphogenic
Fig. 7. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 0.25 mM SA or 0.25 mM 4HBA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
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Acknowledgements We wish to thank Margita Vaˇsková for excellent technical assistance. This work was supported by the Grant Agency VEGA, project No. 2/0019/13. References
Fig. 8. ROS production 3 h after the treatment of roots with distilled water (dw), 15 M Cd or 1 M IAA for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA in the case of Cd treated seedlings and 100 M PCIB or 0.5 mM SA in the case of IAA-treated seedlings for 10 min. S – swollen root part. The white arrows indicate the area of enhanced ROS production in comparison with seedlings treated with dw (controls).
responses and also prevented the activation of the antioxidant enzyme GPX. Even though PCIB had this marked alleviating effect on root growth inhibition, it did not influence the uptake of Cd in barley roots (Tamás et al., 2012). Furthermore, in spite of the SA- or PCIB-mediated elimination of Cd-induced root growth inhibition or antioxidative enzyme activation, the presence of toxic Cd in roots is revealed by a strong stimulation of DHAR and inhibition of AAO activity. These results suggest that some responses (and to a certain degree, also the inhibition of root growth) under mild stress should not be perceived as an unavoidable symptom of Cd toxicity, but rather as an active component of the defense response to the presence of toxic metals. Therefore, it is tempting to speculate that root growth inhibition in many cases does not indicate the toxicity degree of stresses as has been described in numerous previous works. Thus, SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of the IAA signaling pathway.
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Zelinová V, Boˇcová B, Huttová J, Mistrík I, Tamás L. Impact of cadmium and hydrogen peroxide on ascorbate–glutathione recycling enzymes in barley root. Plant Soil Environ 2013b;59:62–7. Zhang F, Zhang H, Xia Y, Wang G, Xu L, Shen Z. Exogenous application of salicylic acid alleviates cadmium toxicity and reduces hydrogen peroxide accumulation in root apoplast of Phaseolus aureus and Vicia sativa. Plant Cell Rep 2011b;30:1475–83. Zhao FY, Hu F, Han MM, Zhang SY, Liu T. Superoxide radical and auxin are implicated in redistribution of root growth and the expression of auxin and cell-cycle genes in cadmium-stressed rice. Russ J Plant Physiol 2011;58: 851–63. Zhao FY, Hu F, Zhang SY, Wang K, Zhang CR, Liu T. MAPKs regulate root growth by influencing auxin signaling and cell cycle-related gene expression in cadmiumstressed rice. Environ Sci Pollut Res 2013;20:5449–60. Zhou ZS, Guo K, Elbaz AA, Yang ZM. Salicylic acid alleviates mercury toxicity by preventing oxidative stress in roots of Medicago sativa. Environ Exp Bot 2009;65:27–34. Zhu XF, Lei GJ, Wang ZW, Shi YZ, Braam J, Li GX, et al. Coordination between apoplastic and symplastic detoxification confers plant aluminum resistance. Plant Physiol 2013;162:1947–55.
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Physiology
Salicylic acid alleviates cadmium-induced stress responses through the inhibition of Cd-induced auxin-mediated reactive oxygen species production in barley root tips Ladislav Tamás ∗ , Igor Mistrík, Aster Alemayehu, Veronika Zelinová, Beáta Boˇcová, Jana Huttová Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic
a r t i c l e
i n f o
Article history: Received 20 May 2014 Received in revised form 31 July 2014 Accepted 1 August 2014 Available online 22 September 2014 Keywords: Auxin Barley Cadmium Root growth inhibition Root swelling
a b s t r a c t Auxin is a master regulator of root growth by modulating its development under the constantly changing environment. Recently, an antagonistic interaction was suggested between SA and IAA signaling. Therefore, the purpose of this work was to analyze and compare the effect of the indole-3-acetic acid (IAA) signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and salicylic acid (SA) as a potential IAA signaling inhibitor on the root growth, enzyme activity and reactive oxygen species (ROS) production in Cd- and IAA-treated barley root tips. Exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3 h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition and swelling, ROS generation and activation of lipoxygenase or glutathione peroxidase identical to those induced by Cd. Furthermore, both Cd- and IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment. Similarly to PCIB, SA did not affect the IAA content of root tips, suggesting the action of SA on the IAA signaling pathway in barley roots. SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of Cd-induced IAA-mediated ROS generation in roots. © 2014 Elsevier GmbH. All rights reserved.
Introduction A number of previous studies have demonstrated that salicylic acid (SA), a widely distributed phenolic compound in the plant kingdom, modifies a wide range of physiological and developmental processes and stress responses resembling the action of classical plant hormones (Raskin, 1992; Vicente and Plasencia, 2011). The activation of the defense response against pathogens and the induction of systemic acquired resistance are the most studied functions of SA in plants (Durner et al., 1997). In addition to its role in biotic stress responses, SA also participates in the adaptation of plants to various unfavorable environmental conditions, including high salinity (Lee et al., 2010), cold (Kang and Saltveit, 2002), drought
Abbreviations: AAO, ascorbic acid oxidase; DHAR, dehydroascorbate reductase; GPX, glutathione peroxidase; 4-HBA, 4-hydroxybenzoic acid; IAA, indole-3-acetic acid; LOX, lipoxygenase; PCIB, p-chlorophenoxyisobutyric acid; ROS, reactive oxygen species; SA, salicylic acid. ∗ Corresponding author. Tel.: +421 2 59426116; fax: +421 2 54771948. E-mail address: [email protected] (L. Tamás). http://dx.doi.org/10.1016/j.jplph.2014.08.018 0176-1617/© 2014 Elsevier GmbH. All rights reserved.
(Sun et al., 2009) and the excess of heavy metals (Horváth et al., 2007; Hayat et al., 2010). Cadmium (Cd) is one of the most dangerous heavy metals for nearly all organisms due to its high solubility in water and toxicity even at very low concentrations. Numerous biochemical and molecular mechanisms have been suggested to be responsible for Cd toxicity in plants, but the disturbance of reactive oxygen species (ROS) homeostasis undoubtedly plays a key role in the development of Cd toxicity symptoms (Cuypers et al., 2010; Gallego et al., 2012). The root is the first plant organ that encounters Cd toxicity, resulting in enhanced ROS generation and growth inhibition within a few minutes of exposure (Ortega-Villasante et al., 2007). Therefore, the early elimination of ROS is a key component of plant tolerance to Cd. Our previously published paper has demonstrated a strong upregulation of antioxidant enzymes in barley root tip. Glutathione peroxidase (GPX) reduces not only hydrogen peroxide, but also organic and lipid hydroperoxides, maintaining cell function despite the high rate of ROS metabolism in root tips under Cd stress (Zelinová et al., 2013a). Rapid recycling of ascorbate and glutathione as the main antioxidants in plant cells by the enzymes of ascorbate–glutathione cycle also contributes to the enhanced
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tolerance of plants to stress (Zelinová et al., 2011). The treatment of roots with Cd resulted in the greatest changes in dehydroascorbate reductase (DHAR) activity, which was activated within 1 h of Cd treatment along the whole barley root tip (Zelinová et al., 2013b). Several Cd-induced root morphogenic responses, including inhibition of cell elongation, alterations in cell differentiation and reorientation of cell growth mimic the auxin- or polar auxin transport inhibitor-induced morphological changes in root tips, suggesting a role of indole-3-acetic acid (IAA) in these processes (Tamás et al., 2012). In rice primary root tips, a marked IAA accumulation was observed even after 6 h of Cd treatment (Zhao et al., 2013). Similarly, the level of IAA in Arabidopsis roots markedly increased after treatment with Cd or Cu (Sofo et al., 2013). The role of IAA as a modulator of the stress response of roots to Cd excess is also supported by the observation that Cd strongly activated the expression of several auxin-responsive genes (Minglin et al., 2005). Furthermore, ROS act as an intermediate signal in several IAA-mediated processes, such as unilateral inhibition of root elongation during root gravitropic curvature (Joo et al., 2001) and growth retardation caused by auxin-type herbicides (Grossmann et al., 2001). In barley, lipoxygenase-(LOX)-generated oxylipins are probably involved in the reorientation of cell expansion in the root apex after Cd, IAA or hydrogen peroxide treatment (Alemayehu et al., 2013). On the other hand, ascorbic acid oxidase (AAO), as an extracellular electron acceptor generating enzyme of the plasma membrane electron transport system, is strongly inhibited after the exposure of seedlings to Cd, Cu, hydrogen peroxide or IAA (Zelinová et al., 2011). Crosstalk between SA and IAA signaling has been suggested by several studies. Wang et al. (2007) have shown that SA inhibits pathogen growth in plants through the repression of the auxin signaling pathway. In Arabidopsis, SA also activated the expression of GH3 genes encoding auxin-conjugating enzymes, reducing the free active form of IAA (Park et al., 2007). An antagonistic effect of IAA and SA was observed in maize roots, where exogenous IAA increased lateral root growth at the expense of primary root growth, while SA had a stimulating effect on total root biomass (Agtuca et al., 2014). An alleviating effect of SA on Cd-induced toxicity symptoms has been reported in barley (Metwally et al., 2003), soybean (Drazic and Mihailovic, 2005), and rice (Guo et al., 2007). In addition, it has also been reported that Cd exposure increased the free SA content of barley roots (Metwally et al., 2003). Therefore, the purpose of this work was to analyze and compare the effect of the IAA signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) and SA as a potential IAA signaling inhibitor on root growth, enzymes activity and ROS production in Cd- and IAA-treated barley root tips.
Materials and methods Plant material and growth conditions Barley seeds (Hordeum vulgare L.) cv. Slaven (Plant Breeding Station – Hordeum Ltd., Sládkoviˇcovo-Novy´ Dvor) were imbibed in distilled water for 15 min followed by germination between two sheets of filter paper (density 110 g/m2 , Papírna Perˇstejn) moistened with distilled water in Petri dishes at 25 ◦ C in darkness. The uniformly germinating seeds, 24 h after the onset of seed imbibition, were arranged into rows between two sheets of filter paper moistened with distilled water in rectangular trays. The 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.
Short-term treatments Roots of seedlings were pre-treated by immersion into distilled water (dw – control) or into 15 M CdCl2 or 1 M IAA (from 10 mM stock in ethanol) for 30 min. After brief washing in distilled water, the roots were immersed into 50 or 100 M PCIB (from 10 mM stock in ethanol) or into 0.25 or 0.5 mM SA or 0.25 mM 4-HBA (from stock in DMSO) for 10 min. After treatments, the seedlings were incubated between two sheets of filter paper moistened with distilled water as described above. After 3 or 6 h of incubation the root tips (3 mm in length) were used for analysis. Root length measurement For the determination of root length changes, the positions of root tips following the treatments were marked on the filter paper. After 6 h, the roots were excised at the position of marks and the length increment was measured after recording with stereomicroscope using micro image analyzer. For localization of root swelling, the roots were stained with 0.05% Toluidine blue for 10 min and after washing with distilled water were photographed with a stereomicroscope. Protein extraction and sample preparation The root tips were homogenized in a pre-cooled mortar with 100 mM potassium phosphate extraction buffer pH 7.8 containing 1 mM EDTA 6 h after the treatments. 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). Enzyme assays Lipoxygenase (LOX, EC 1.13.11.12) activity was measured using the colorimetric method according to Anthon and Barrett (2001). The reaction mixture contained (160 L in a final volume) 5 mM 3-dimethylaminobenzoic acid in 25 mM sodium phosphate buffer (pH 6.0), 1 mM KCN, 0.5 mM linoleic acid (from 25 mM stock solution dissolved in Tween 20) and 2 g of proteins from the root extract. The mixture was incubated at 30 ◦ C for 15 min, then the mix of 20 L of 1 mM 3-methyl-2-benzothiazolinone and 20 L of hemoglobin (500 g/mL) was added. After 5 min incubation at room temperature the absorbance was measured at 598 nm. Specific LOX activities were expressed as A598 mg−1 min−1 . Ascorbic acid oxidase (AAO, EC 1.10.3.3) activity was determined by measuring the decrease in absorbance at 265 nm reflecting the ascorbate oxidation (Arrigoni et al., 1981). The reaction mixture (250 L) contained 50 mM potassium phosphate buffer pH 7.0, 0.15 mM ascorbate, 0.5 mM EDTA and 2 g of proteins from root extract. The reaction mixture was incubated at 30 ◦ C for 30 min. Specific activities were expressed as A265 mg−1 min−1 . Glutathione peroxidase (GPX, EC 1.11.1.9) activity was measured by a spectrophotometric method according to Drotar et al. (1985) with some modifications using microplate reader (Synergy HT, BIO-TEK, USA). The reaction mixture (200 L) contained 2 mM glutathione, 0.5 mM NADPH, 1 mM EDTA, 2 mM t-butyl hydroperoxide and 0.5 U of glutathione reductase in 100 mM sodium phosphate buffer, pH 7.0 and 20 g of extracted proteins. The rate of NADPH oxidation was measured at 340 nm over a time period of 20 min at 30 ◦ C. Specific GPX activities were expressed as A340 mg−1 min−1 . Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity was assayed by measuring the rate of ascorbate formation at 265 nm in a reaction mixture containing 100 mM potassium phosphate buffer pH 7.0, 1 mM reduced glutathione, 0.5 mM dehydroascorbate and
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10 g of proteins from root extract. The reaction mixture (250 L) was incubated at 30 ◦ C for 20 min (Arrigoni et al., 1981). Specific activities were expressed as A265 mg−1 min−1 . Localization of ROS production Intact roots (3 h after treatments) were immersed into the solution of 10 M 2,7-dichlorodihydrofluorescein diacetate (from 20 mM stock in DMSO) in 20 mM sodium phosphate buffer pH 6.0 for 30 min at 25 ◦ C in darkness. After brief washing with distilled water fluorescence of whole roots was observed immediately (for no more than 5 min) using fluorescence stereomicroscope (excitation 500 ± 20 nm; emission 535 ± 30 nm). IAA quantification The root tips (3 h after treatments) were homogenized in a pre-cooled mortar with ice-cold 80% methanol containing 1 mM butylated hydroxytoluene (80 root tips/mL). After centrifugation at 12,000 × g for 10 min, samples were passed through a C18 column (Chromabond, Macherey-Nagel) preconditioned with 80% methanol at 4 ◦ C. After evaporation of methanol, samples were methylated using trimethylsilyldiazomethane in hexane (2 L/500 L) at 42 ◦ C for 30 min and diluted in 25 mM sodium phosphate buffer (pH 7.2) containing 15 mM NaCl. Quantification of IAA was performed by competitive enzyme-linked immunosorbent assay (IAA immunoassay kit Olchemin). Statistical analyses The experiments were carried out in three independent series with three replicates (20 root tips for root length measurement and ROS analysis, 40 root tips for enzyme analysis and 80 root tips for IAA analysis per replicate). The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Short-term exposure of barley roots to 15 M Cd caused a marked reduction in root growth (Fig. 1A), which was accompanied by considerable radial expansion of root tips (Fig. 1B). Post-treatment of Cd-stressed plants with 50 M PCIB or 0.25 mM SA (concentrations were selected in order to obtain the greatest alleviating effect on the Cd-induced root growth inhibition) markedly alleviated the Cd-induced inhibition of root growth and radial expansion of root tips (Fig. 1). At higher concentrations, both PCIB and SA inhibited root growth in a concentration-dependent manner in controls as well as in Cd-treated roots (data not shown). Both Cd-induced LOX and GPX activity were reduced by the posttreatment of plants with PCIB or SA (Fig. 2A and B). On the contrary, PCIB or SA post-treatment did not alleviate the Cd-induced changes in DHAR (DHAR activity was further stimulated by PCIB in Cdtreated roots) or AAO activity (Fig. 2C and D). The analysis of IAA content in the root apex revealed a considerable accumulation of IAA 3 h after the Cd treatment (Fig. 3). However, neither PCIB nor SA affected this Cd-induced accumulation of IAA in the root apex. Treatment of seedlings with 1 M IAA inhibited root growth and evoked radial expansion of root tips in a similar extent as was observed in seedlings after the exposure to Cd (Fig. 4). In addition, the alleviating effect of PCIB or SA (however, in IAAtreated seedlings higher concentrations of PCIB or SA were required to obtain the greatest alleviating effect in comparison with Cdtreated seedlings) on root growth inhibition (Fig. 4A) and swelling (Fig. 4B) was also observed in IAA-treated seedlings. PCIB and SA
Fig. 1. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
post-treatment also reduced the IAA-induced LOX and GPX activity (Fig. 5A and B). However, the stimulation of DHAR and the inhibition of AAO activity by IAA treatment were less profound in comparison with the changes induced by Cd stress (Fig. 5C and D). 4-HBA a biologically inactive analog of SA, did not alleviate the Cd-induced root growth inhibition (Fig. 6A) or root swelling (Fig. 6B) and had no effect on the activity of analyzed enzymes in the control or in the Cd-treated seedlings (Fig. 7). The analysis of ROS generation revealed that Cd and IAA had a similar effect on ROS generation in the root apex (Fig. 8). While in control roots ROS were observed in the differentiation zone (3 mm behind root apex) and decreased gradually in the direction of elongation zone, in Cd- and IAA-treated seedlings, the ROS generation was markedly increased at the end of elongation and the beginning of differentiation zone. PCIB and SA treatment markedly inhibited this ROS generation in control, Cd- or IAA-treated roots. Discussion The exposure of plants to Cd resulted in a more than threefold increase of IAA content in the root apex even 3 h after the treatment. In addition, exogenously applied IAA evoked root responses such as root growth inhibition, swelling, ROS generation and activation of LOX and GPX identical with those induced by Cd. Previous studies have shown that SA also participates in the adaptation of
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Fig. 2. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
plants to various unfavorable environmental conditions, including the excess of heavy metals (see Introduction section). However, an antagonistic effect has been reported between IAA and SA action on root architecture in the root response to biotic stress (Agtuca et al., 2014). This effect of SA was also supported by the observation that accumulation of SA in Arabidopsis roots caused the repression of auxin-related genes and insensitivity to exogenously applied auxin (Wang et al., 2007). Therefore, in this study we compared
Fig. 3. IAA content of root tips 3 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
the effect of PCIB as an IAA signaling inhibitor and SA as a potential IAA signaling inhibitor on root morphology, enzyme activity and ROS production in Cd- and IAA-treated barley root tips. Both Cdand IAA-induced stress responses were markedly reversed by PCIB or SA post-treatment, indicating that SA reversed the Cd-induced responses through the inhibition of IAA action. In addition, similarly to PCIB, SA did not affect the IAA content of Cd-treated root tips, suggesting the action of SA on the IAA signaling in barley roots. It is well known that, at low concentrations, IAA is necessary for proper root growth, but with increasing concentrations it strongly inhibits root elongation in a dose-dependent manner (Tanimoto, 2005). In accordance with these results, we observed that PCIB at higher concentrations inhibited root growth in control seedlings treated only with distilled water (data not shown). On the contrary, at lower concentrations, it had a promoting effect, suggesting that the inhibition of IAA signaling may, to some degree, also accelerate root elongation under unstressed conditions probably due to the inhibition of IAA-mediated differentiation of root cells. On the other hand, the handling of seedlings during the experiment and the immersion of control roots into distilled water may represent a certain degree of stress activating IAA signaling-mediated inhibition of root growth. This inhibition may be eliminated by the application of PCIB at low concentrations. Earlier studies have clearly demonstrated the role of ROS in IAA-mediated root growth inhibition (Joo et al., 2001; Grossmann et al., 2001). Therefore, the Cd-induced IAA accumulation is likely responsible for the enhanced ROS generation, causing a marked reduction of cell elongation. On the other hand, it was shown that Cd-stimulated rice root growth is also associated with the alterations in IAA and ROS homeostasis (Zhao et al., 2011) suggesting a central role of IAA and ROS in the Cd-induced responses in roots.
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Fig. 4. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 1 M IAA for 30 min and subsequent treatment with dw or 100 M PCIB or 0.5 mM SA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
Fig. 5. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) or 1 M IAA for 30 min and subsequent treatment with dw or 100 M PCIB or 0.5 mM SA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
In this study, we demonstrated that the alleviating effect of both PCIB and SA either on Cd- or IAA-induced responses was associated with decreased ROS generation in root tissues. Similarly to our results, previous studies have shown that exogenous application of SA alleviates Cd or Hg toxicity by reducing ROS accumulation in roots (Zhou et al., 2009; Zhang et al., 2011a,b). While a shorter
SA pre-treatment decreased the Cd-induced H2 O2 production leading to attenuation of Cd-induced root growth inhibition, during the prolonged exposure of rice roots to SA, an increase in H2 O2 production was observed (Guo et al., 2007). In addition, exogenous application of SA promotes Arabidopsis seed germination by reducing ROS production under high salinity stress (Lee and Park, 2010). Today, it is undisputed that ROS are ubiquitous signaling molecules involved in the regulation of different developmental and physiological processes. Previous studies have shown that LOX inhibitors efficiently inhibited both Cd-, IAA- and H2 O2 -induced root swelling in a concentration-dependent manner, suggesting a key role of LOX or the LOX signaling pathway in radial expansion of root cells (Alemayehu et al., 2013). In agreement with these results, the IAA signaling inhibitor PCIB and SA reversed both Cd- and IAAinduced LOX activity, resulting in a considerable inhibition of radial cell expansion. Several studies have indicated that SA mitigates the Cd toxicity by activation of non-enzymatic and enzymatic antioxidants, leading to reduction of Cd-induced ROS generation in plants (Guo et al., 2009; Popova et al., 2009; Zhang et al., 2011a,b). By contrast, the PCIB or SA post-treatments considerably reduced the Cd- or IAAinduced GPX activity, indicating the role of IAA in the activation of antioxidant response in barley roots. Metwally et al. (2003) have reported that SA alleviated Cd toxicity in barley root through root growth promotion and reduction of lipid peroxidation. However, it also suppressed the Cd-induced upregulation of numerous antioxidant enzymes, including GPX. These and our results indicate that SA attenuates ROS generation in Cd-treated roots by other mechanisms than the activation of antioxidant enzymes. On the contrary, SA just attenuates the IAA-mediated generation of ROS, which function as signal molecules responsible for the activation of some components of the defense system (such as GPX) in root tips against the potential oxidative stress during the prolonged Cd stress. These results are also supported by previous observations that both Cdinduced enhanced LOX and GPX activity were not associated with harmful oxidative stress such as lipid peroxidation or cell death in barley root tips (Liptaková et al., 2013). On the other hand, our results also showed that SA, similarly to PCIB, had a different effect on the analyzed enzymes. While LOX and GPX were strongly downregulated, DHAR and AAO were not affected by the SA or PCIB post-treatment, suggesting the participation of different signaling pathways in their activation or inhibition during Cd stress. While both LOX and GPX activity were strongly
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Fig. 6. Root length increments (A) and root morphology (B) 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 0.25 mM SA or 0.25 mM 4HBA for 10 min. S – swollen root part. The black triangles show the starting position of new root growth after the treatments. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
stimulated, DHAR was not activated by exogenously applied H2 O2 (Zelinová et al., 2013b). Therefore, inhibition of IAA-mediated ROS generation by PCIB or SA cannot affect DHAR activity in these experiments. On the other hand, exogenous IAA significantly stimulated DHAR activity, suggesting that ROS are involved in the activation of DHAR neither by Cd, nor by IAA in barley root tip. It has previously been reported that ROS strongly inhibited AAO activity in barley roots (Zelinová et al., 2011). However, despite a marked reduction of ROS generation by PCIB and SA, the inhibition of AAO in Cdtreated roots was not eliminated, suggesting that apart from the ROS-mediated inhibition of AAO activity, it is also inhibited through other mechanisms in Cd-treated roots.
Taken together, the results of this study indicate that IAAmediated ROS generation plays a key signaling role in the response of roots to stress conditions, through the activation of the defense systems, including morphogenic changes such as root growth inhibition or swelling and through the induction of some antioxidant enzymes. The Arabidopsis IAA signaling mutant exhibits a higher rate of root elongation than the wild type seedlings under both oxidative and salinity stress (Iglesias et al., 2010). By contrast, auxin-overproducing mutants show a lower rate of root elongation than wild type seedlings under Al stress (Zhu et al., 2013). In this work, we have shown that inhibition of IAA signaling by PCIB or SA resulted in the alleviation of Cd- or IAA-induced morphogenic
Fig. 7. LOX (A), GPX (B), DHAR (C), AAO (D) activity 6 h after the treatment of roots with distilled water (dw) or 15 M Cd for 30 min and subsequent treatment with dw or 0.25 mM SA or 0.25 mM 4HBA for 10 min. Mean values ± SD (n = 3). Different letters indicate statistical significance according to Tukey’s test (P < 0.05).
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Acknowledgements We wish to thank Margita Vaˇsková for excellent technical assistance. This work was supported by the Grant Agency VEGA, project No. 2/0019/13. References
Fig. 8. ROS production 3 h after the treatment of roots with distilled water (dw), 15 M Cd or 1 M IAA for 30 min and subsequent treatment with dw or 50 M PCIB or 0.25 mM SA in the case of Cd treated seedlings and 100 M PCIB or 0.5 mM SA in the case of IAA-treated seedlings for 10 min. S – swollen root part. The white arrows indicate the area of enhanced ROS production in comparison with seedlings treated with dw (controls).
responses and also prevented the activation of the antioxidant enzyme GPX. Even though PCIB had this marked alleviating effect on root growth inhibition, it did not influence the uptake of Cd in barley roots (Tamás et al., 2012). Furthermore, in spite of the SA- or PCIB-mediated elimination of Cd-induced root growth inhibition or antioxidative enzyme activation, the presence of toxic Cd in roots is revealed by a strong stimulation of DHAR and inhibition of AAO activity. These results suggest that some responses (and to a certain degree, also the inhibition of root growth) under mild stress should not be perceived as an unavoidable symptom of Cd toxicity, but rather as an active component of the defense response to the presence of toxic metals. Therefore, it is tempting to speculate that root growth inhibition in many cases does not indicate the toxicity degree of stresses as has been described in numerous previous works. Thus, SA probably does not alleviate the Cd toxicity in roots, but rather prevents or partially inhibits the root defense response to the presence of Cd through the inhibition of the IAA signaling pathway.
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