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Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip
Journal of Plant Physiology 169 (2012) 1375–1381
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Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip Ladislav Tamás ∗ , Beáta Boˇcová, Jana Huttová, L’ubica Liptáková, Igor Mistrík, Katarína Valentoviˇcová, Veronika Zelinová Institute of Botany, Slovak Academy of Sciences, Dúbravská cesta 9, SK-84523 Bratislava, Slovak Republic
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Article history: Received 22 March 2012 Received in revised form 27 April 2012 Accepted 16 May 2012 Keywords: Cadmium Cell death Indole-3-acetic acid Root growth inhibition Root swelling
a b s t r a c t Short-term treatment (30 min) of barley roots with a low 10 M Cd concentration induced significant H2 O2 production in the elongation and differentiation zone of the root tip 3 h after treatment. This elevated H2 O2 production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60 M Cd concentration induced robust H2 O2 production in the elongation zone of the root tip already 1 h after short-term treatment. This robust H2 O2 generation caused extensive cell death 6 h after short-term treatment. Similarly to low Cd concentration, exogenously applied H2 O2 caused marked root growth inhibition, which at lower H2 O2 concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10 M Cd-induced root growth inhibition, H2 O2 production and root swelling, but was ineffective in the alleviation of 60 M Cd-induced root growth inhibition and H2 O2 production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death. © 2012 Elsevier GmbH. All rights reserved.
Introduction Oxygen as a terminal oxidant in respiration confers significant energy advantages on organisms, and was probably the most important change in Earth’s environment since life began. Due to its high reactivity, however, the production of reactive oxygen species (ROS) is an unavoidable by-product of aerobic metabolism. ROS is a collective term that describes the chemical species (including radicals and non-radicals) that are formed upon incomplete reduction of oxygen, such as superoxide anion, hydrogen peroxide, hydroxyl radical and nitric oxide (Halliwell, 2006). Contrary to the early hypothesis that ROS are only the toxic byproducts of aerobic metabolism, it has become clear that ROS play a crucial role in the regulation of different developmental and physiological processes (Foyer and Noctor, 2005). Hydrogen peroxide (H2 O2 ), in comparison to the other ROS, plays a key role as a signal
Abbreviations: IAA, indole-3-acetic acid; PCIB, p-chlorophenoxyisobutyric acid; ROS, reactive oxygen species; TIBA, triiodobenzoic acid. ∗ Corresponding author. E-mail address: [email protected] (L. Tamás). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.05.023
molecule due to its relative high stability and permeability through membranes. It acts as a signal molecule that induces gene expression (Desikan et al., 2000), germination (Barba-Espin et al., 2010), leaf abscission (Sakamoto et al., 2008), stomatal closure (Desikan et al., 2004) and gravitropic bending of roots (Joo et al., 2001). H2 O2 is also generated during abiotic and biotic stress conditions, where it is involved as a signal molecule in defense responses, including cell death (van Breusegem and Dat, 2006). In addition, a function for ROS in hormone responses has been reported in various developmental processes (Kwak et al., 2006). It is well known that the inhibition of root growth as a common response of roots to different stresses is associated with increased ROS production and alterations in phytohormone levels (Potters et al., 2009). Both Cd-induced alterations in the level of hormones and ROS have been observed in several plant species. It has previously been described that roots curved away from Cd, suggesting the inhibition of basipetal auxin flow by Cd (Hasenstein et al., 1988). In contrast to the auxin flow inhibition, ethylene production was highly stimulated by Cd treatment in carrot cell suspension cultures (Sanita di Toppi et al., 1998). Although Cd induces a perturbation in different cellular metabolism, an increasing number of publications suggest that several Cd-induced toxicity symptoms are attributable
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to oxidative damage arising from imbalance in the generation and removal of ROS (Sanita di Toppi and Gabbrielli, 1999; Benavides et al., 2005). Olmos et al. (2003) have reported that the early step in the oxidative burst induced by Cd in culture of tobacco cells is mediated through the activation of NADPH oxidase and superoxide dismutase, leading to superoxide and H2 O2 accumulation within some minutes. Similarly to cultured cells, the extracellular H2 O2 production was a very early response of alfalfa root segments to Cd or Hg exposure, which was detectable already just a few minutes after the treatment (Ortega-Villasante et al., 2007). Apart from Cd-induced root growth inhibition, in some cases, radial expansion of root cells has also been reported (Fusconi et al., 2007; Rascio et al., 2008). Pasternak et al. (2005) suggested that stress-induced phenotypes comprise a re-orientation rather than a cessation of growth. These stress-induced morphogenic responses, including inhibition of cell elongation, localized stimulation of cell division, alterations in cell differentiation and probably re-orientation of cell and organ growth are general acclimation strategies during various stresses (Potters et al., 2009). Using short-term treatments, the aim of this study was to analyze the interaction between H2 O2 and auxin signaling in root cell growth reorientation and cell death during Cd stress in barley root tips. Material 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, Czech Republic) moistened with distilled water in Petri dishes (5 ml per sheet of filter paper of 18 cm in diameter) 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. Trays were placed into a nearly vertical position to enable downward radical growth. In the case of root bending, assay trays were oriented with roots pointing upward. 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.
Root length measurement For the determination of root length increases, the position of root tips following the treatments were marked on the filter paper. After 3, 6 or 24 h, roots were excised at the position of marks and the length increase was measured after recording with a stereomicroscope (STMPRO BELPhotonics, Italy) using a BEL micro image analyzer. Cd content determination Root segments (3 mm long) were oven-dried immediately after short-term treatments at 60 ◦ C for 2 d, and after digestion and ˇ and Poláˇceková, 2000) were analyzed using extraction (Korenovská an atomic absorption spectrometer (Perkin Elmer 4100, Norwalk, CT, USA). Hydrogen peroxide assay H2 O2 production was monitored fluorimetrically using the Amplex UltraRed Hydrogen Peroxide Assay Kit (Molecular Probes) according to manufacturer’s recommendations, with minor modifications. Segments from barley root tips (10 segments per reaction) were incubated in 200 l of 50 mM sodium phosphate buffer, pH 7.4 containing 25 M Amplex UltraRed reagent (from 10 mM DMSO stock solution) and 0.02 U of horse radish peroxidase for 20 min at 30 ◦ C. The fluorescence signal was recorded (170 l of reaction mixture without root segments) with the microplate reader (Synergy HT BIO-TEK, USA) using excitation at 530 (filter 530/25) nm and fluorescence detection at 590 (filter 590/20) nm. Histochemical localization of H2 O2 Intact roots were immersed in the solution of 10 mM sodium phosphate buffer pH 5.8 containing 0.1% 3,3 -diaminobenzidine – DAB and, after vacuum-infiltration for 5 min, were incubated for 1 h in the light. Brown staining characteristic of the DAB–H2 O2 interaction was photographed with a stereomicroscope (STMPRO BELPhotonics, Italy). Detection of cell death Intact roots were immersed in a solution of 0.25% Evans Blue for 15 min at room temperature. After washing with distilled water for 3 min × 5 min, roots were photographed with a stereomicroscope.
Short-term treatments Statistical analyses Roots of seedlings were immersed into distilled water (control) or into the appropriate test solution (see also figures and figure legends) for 30 min. For Cd exposure 10, 20, 30, 40, 50, 60 or 100 M concentrations were used. Hydrogen peroxide (H2 O2 ) was used at 0.5, 1, 5 or 10 mM concentrations. Indole-3-acetic acid (IAA) was applied at 10 M, and IAA efflux inhibitor 2,3,5triiodobenzoic acid (TIBA) at 20 M concentration. IAA signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) was used at 10, 20, or 30 M concentrations. After washing in distilled water for 5 min the seedlings were incubated as described above. After 1, 3 or 6 h of these short-term treatments, the individual barley root segments (3 mm in length) were obtained by the gradual cutting of each root from the tip to the base. Under control conditions, the first segment represents the meristem and the elongation zone, while the second segment the beginning of differentiation zone. Where indicated, Cd (stock at 1 mM CdCl2 ) or H2 O2 (stock at 10 mM) were dissolved in water; IAA (stock at 1 mM) were dissolved in ethanol, and TIBA or PCIB (stock at 10 mM) were dissolved in acetone and ethanol, respectively.
The experiments were carried out in five independent series with three replicates. The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Significant root growth inhibition was observed after relatively short-term (30 min) exposure of barley roots to low 10 M Cd concentration. The root increase of Cd-treated seedlings measured 3 or 6 h after the short-term treatment represented approximately 30% of controls (roots treated with distilled water for 30 min) (Fig. 1a). No root growth was observed during the first 6 h after short-term treatment with 30 or 60 M Cd. After that, the root growth of 30 M Cd short-term treated seedlings was renewed, while root growth was completely inhibited even 24 h after short-term treatment with the 60 M Cd concentration (Fig. 1a). Apart from the Cdinduced root growth inhibition, visible root swelling was detected
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Fig. 1. Root length increments (a) 3, 6 and 24 h after short-term treatments with 0, 10, 30 or 60 M concentration of Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of radial root swelling 3 and 6 h (b) after short-term treatment with 0 or 10 M Cd. After short-term treatment roots were arranged into the row that root tips were positioned on the line marked on the bottom of the tray. After subsequent incubation root part lying on this line was marked with black stain which is visible on the picture. The root part below this mark represents new root growth. Black arrows show swollen root part. MZ – meristematic zone, EZ – elongation zone.
within 6 h after short-term treatment with 10 M Cd immediately behind the root tip from 0 to 3 mm, representing the meristem and elongation zone during the short-term treatment (Fig. 1b). Similarly to the root growth restoration, a longer recovery time was required after short-term treatment for swelling appearing (8–9 h) in roots exposed to 30 M Cd (data not shown) in comparison with the 10 M Cd-treated roots. However, it was not detectable in roots treated with the 60 M Cd concentration. Analysis of H2 O2 production revealed that its level markedly increased after 1 h of short-term treatment with the concentration higher than 10 M Cd immediately behind the root apex (from 0 to 3 mm representing meristem and elongation zone) in a concentration-dependent manner (Fig. 2a). In contrast, in the next segment from 3 to 6 mm behind the root apex (representing the differentiation zone), Cd induced H2 O2 production to a smaller extent than in the first segment, and with minor differences between different Cd concentrations (Fig. 2b). The considerable 60 M Cd-induced H2 O2 production was also detectable with DAB staining and was localized immediately behind the root tip 1 h after short-term treatment (Fig. 2c). This robust 60 M Cd-induced H2 O2 production in the root apex detected 1 h after short-term treatment decreased over time, but it remained elevated in relation to control. H2 O2 production after short-term treatment with 10 M Cd was much lower, but increased in a time-dependent manner (Fig. 3a). In contrast to the first root segment, in the second segment from 3 to 6 mm behind the root apex, marked Cd-induced H2 O2 production
Fig. 2. H2 O2 production in root segments from 0 to 3 mm (a) and from 3 to 6 mm (b) behind the root apex 1 h after short-term treatment with different concentration of Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of H2 O2 production (c) 1 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show root part stained by DAB.
was detected at the 10 M Cd concentration 3 h after short-term treatment (Fig. 3b). In this segment, elevated H2 O2 production was also observed after short-term treatment with the 60 M Cd concentration, but to a much lower extent than after 10 M Cd treatment. In the case of 10 M Cd treatment, the elevated H2 O2 production was mainly associated with the radial expansion of roots. The swollen part was the first segment representing the meristem and elongation zone during the short-term treatment, but the second 3 mm segment was from 3 to 6 mm behind the root tip 6 h after short-term treatment due to the root growth (Fig. 3c, see also Fig. 1B). In contrast to 10 M Cd-induced H2 O2 production in the differentiation zone of root tip that probably invoked root swelling, 60 M Cd-induced robust H2 O2 production in the
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Fig. 4. Localization of cell death 6 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show a marked uptake of Evans blue into cells signalizing cell death.
(Fig. 6). IAA signaling inhibitor PCIB effectively inhibited both 15 M Cd-induced root growth inhibition and H2 O2 production in a concentration-dependent manner (Fig. 7) Cd-induced radial root swelling was completely eliminated in the presence of PCIB during the short-term Cd treatment of roots (Fig. 7c). By contrast, in seedlings treated with the high 60 M Cd, PCIB was ineffective in the alleviation of Cd-induced root growth inhibition and H2 O2 production (data not shown). The analysis of Cd uptake revealed that the Cd content of the root tip was not affected by the presence of PCIB (Table 1).
Fig. 3. H2 O2 production in root segments from 0 to 3 mm (a) and from 3 to 6 mm (b) behind the root apex 1, 3 and 6 h after short-term treatment with 0, 10 or 60 M Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of H2 O2 production (c) 6 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show root part stained by DAB.
beginning of the elongation zone of root tip already 1 h after shortterm treatment, causing extensive cell death clearly detectable 6 h after short-term treatment (Fig. 4). Short-term treatment of barley roots with H2 O2 strongly inhibited root growth (Fig. 5a). This H2 O2 -induced root growth inhibition was accompanied at lower H2 O2 concentrations by root swelling, which at higher H2 O2 concentration was not so obvious, but was accompanied with strong inhibition of root hairs development (Fig. 5b). Similarly to exogenously applied Cd or H2 O2 at lower concentrations, the IAA or IAA efflux inhibitor TIBA, apart from the marked root growth inhibition, caused radial cell expansion, which was accompanied by an elevated production of H2 O2
Discussion The results presented here show that a transient 30 min shortterm Cd-treatment induces significant root growth inhibition in barley seedlings. At low the 10 M Cd concentration, this Cdinduced root growth inhibition was accompanied by the radial expansion of root cells, while the higher concentration of Cd caused the cessation of root growth and cell death. The radial expansion of root cortex cells causing root swelling is a characteristic morphological symptom of several stress conditions, such as Fe or Cu deficiency (Romera et al., 2003), excess of Cu (Panou-Filotheou and Bosabalidis, 2004), salinity (Burssens et al., 2000), UV light (Ktitorova et al., 2006), mechanical impedance (Wilson et al., 1977) and hormones such as auxin and ethylene (Pulgarín et al., 1996). Therefore, it probably belongs to the general stress-induced morphogenic responses (Potters et al., 2009) as a consequence of growth reorientation from the anisodiametric elongation to isodiametric enlargement of cells in the elongation zone of root tips.
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Table 1 Cd content (mg/kg) of barely root segments measured immediately after short-term treatrment with 10 M Cd and 30 M PCIB for 30 min with or without washing of root segments in 5 mM CaCl2 for 10 min after short-term tratments. Root segments Distance from root apex
0–3 mm 3–6 mm
10 M Cd/30 min
10 M Cd + 30 M PCIB/30 min
10 M Cd/30 min 5 mM CaCl2 /10 min
10 M Cd + 30 M PCIB/30 min 5 mM CaCl2 /10 min
127.9 ± 9.6 248.1 ± 15.7
122.7 ± 11 248.5 ± 21.7
75.5 ± 4.7 135.4 ± 5
74.4 ± 3.7 134 ± 5.5
Root growth inhibition as a consequence of enhanced ROS production is a very early response of plants during various stress conditions, including Cd (Ortega-Villasante et al., 2007). Garnier et al. (2006) have reported that tobacco cells exposed to Cd responded with a transient accumulation of H2 O2 , which peaked at 30 min after the Cd treatment. This high transient H2 O2 accumulation was followed by enhanced O2 − production and lipid peroxidation, which similar to our results, led to the cell death. H2 O2 as a signal molecule is involved in numerous processes, including the induction of high-affinity K+ transport activity (Shin and Schachtman, 2004), which is involved in the IAA-induced swelling of protoplasts (Keller and van Volkenburgh, 1996). We showed that Cd at a non-lethal doses induced elevated generation of H2 O2 , which probably initiated the radial expansion of cells in the elongation zone of barley root tips. This elevated H2 O2 production was detected 1 h after Cd treatment and localized in the differentiation zone of root tips. The H2 O2 production increased with time, and 6 h after Cd treatment was associated mainly with the swollen part of root and probably was required for cell wall reorganization during this rapid isodiametric growth of cells. On the other hand, at a higher Cd concentration, the robust production of H2 O2 was localized in the elongation zone of root tip with a maximum peak 1 h after the Cd treatment and was associated with cell, and at very high Cd concentration, also with root death.
Fig. 5. Root length increments (a) and localization of radial root swelling (b) 6 h after short-term treatment with 0, 0.5, 1.0, 5.0 or 10 mM H2 O2 . Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrow shows swollen root part. Black mark – see Fig. 1.
This high Cd concentration generated H2 O2 in the elongation zone and inhibited both elongation and radial expansion of cells. This was also confirmed by the exogenously applied H2 O2 , where in addition to the inhibition of cell elongation at a lower (up to 1 mM) H2 O2 concentration, the radial expansion of cells occurred similarly to the low Cd concentration. In contrast, at a higher Cd or H2 O2 concentration, the root swelling was markedly inhibited,
Fig. 6. Root length increments 6 h (a), H2 O2 production in root segments from 3 to 6 mm behind the root apex 3 h (b) and localization of radial root swelling with elevated H2 O2 production 6 h (c) after short-term treatment with dw (control), 10 M IAA or 20 M TIBA. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrow shows swollen root part stained by DAB.
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Fig. 7. Effect of auxin signaling inhibitor PCIB on 10 M Cd-induced root growth inhibition, radial root swelling and H2 O2 production. Root length increments 6 h (a) and H2 O2 production in root segments from 3 to 6 mm behind the root apex 3 h (b), radial swelling 6 h (c) after short-term treatments. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrows show swollen root part. Black mark – see Fig. 1.
indicating that different processes may be activated in the root tip of barley depending on the level of H2 O2 . On the other hand, it has recently been reported that apoplastic ROS strongly inhibited IAA signaling while activating ethylene signaling and cell death (Blomster et al., 2011). This may explain the blockage of radial cell expansion at both high Cd and H2 O2 concentrations. In addition, with high H2 O2 content, breakdown of IAA may occur through the peroxidase catalyzed reaction (Kawano, 2003), which may limit further the IAA-mediated responses under a strong oxidative stress condition. It has been observed that PCIB effectively impairs the IAA signaling pathway in Arabidopsis, affecting IAA-regulated root morphology (Oono et al., 2003). To better understand the involvement of IAA in the Cd-induced H2 O2 accumulation and morphogenic responses, we also studied the effect of the IAA signaling inhibitor PCIB. We showed that PCIB alleviates or even blocks the short-term Cd stress-induced root growth inhibition and radial root expansion, which was accompanied by a reduction of Cd-generated H2 O2 . Similar to PCIB, exogenous application of salicylic acid also alleviated Cd toxicity, which was associated with the reduction of H2 O2 accumulation in the root apoplast (Zhang et al., 2011). It has recently
been shown that salicylic acid inhibited the IAA signaling pathway (Wang et al., 2007), which may decrease H2 O2 accumulation in cells in a manner similar to PCIB. In our experiments, PCIB co-treatment did not block exogenously applied IAA-induced responses (data not shown), likely due to the lag time required for PCIB action (Oono et al., 2003). Our results provide strong evidence that the short-term Cdtreatment induced root growth inhibition and radial root expansion is mediated through the IAA-induced H2 O2 generation in barley root tissues. In contrast, strong oxidative stress induced by high Cd concentration caused extensive H2 O2 accumulation independent of IAA in the elongation zone of root tip, resulting in the cessation of root growth or even in root death. The function of H2 O2 as a signal and/or substrate is well known in the IAA-mediated H2 O2 generation in root gravitropic response (Joo et al., 2001). We showed that H2 O2 as a signal molecule acts downstream of IAA and, in addition, it is probably directly utilized in the cell wall metabolism during mild Cd stress-induced morphogenic changes. Therefore, root growth inhibition and ROS production as a toxicity symptom of several stresses reported in numerous publications may be associated only with morphogenic responses without marked damages of the root tissues. Accordingly, the IAA signaling mutant is more tolerant to both oxidative and salinity stress, exhibiting higher rates of root elongation than wild type seedlings due to the reduced accumulation of ROS under stress conditions (Iglesias et al., 2010). The up-regulation of GH3 auxin amido synthase caused a decrease of active IAA levels in poplar root tissues and might have contributed to growth attenuation under Cd stress (Elobeid et al., 2012). Similar to roots, in rice shoots, the activation of auxin amido synthase results in a decrease of free IAA levels and enhanced drought tolerance (Zhang et al., 2009). In conclusion, these results suggest that IAA plays a crucial role in various morphogenic responses during mild stress conditions, including Cd excess. On the other hand, they probably did not play a direct role in the detoxification of Cd. The role of these morphogenic changes is probably associated with the evasion of roots from the source of Cd supply (Potters et al., 2007). Interestingly, these general morphogenic responses are absent in the metal hyperaccumulator plants, where instead of high Cd concentration, no significant morphogenic and structural modifications are observed in roots (Ederli et al., 2004). Our results showed that the induction of morphogenic responses by mild Cd stress are not associated with toxicity symptoms because, due to the inhibition of IAA signaling pathways, roots grow without inhibition in spite of the presence of relatively high concentrations of Cd in roots during and after the short-term Cd exposure. These observations raise questions about the definition of root growth inhibition during abiotic stresses as an early toxicity symptom. Our results indicate that root growth inhibition under mild stresses is not a result of stressinduced damages, but rather a general stress-induced morphogenic response just to avoid damage to root tissues under unfavorable conditions. Acknowledgments We wish to thank Margita Vaˇsková for excellent technical assistance. This work was supported by the Grant agency VEGA, project no. 2/0050/10. References Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ, Albacete A, Faize L, Faize M, et al. Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ 2010;33:981–94. Benavides MP, Gallego SM, Tomaro ML. Cadmium toxicity in plants. Brazil J Plant Physiol 2005;17:21–34.
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Impact of the auxin signaling inhibitor p-chlorophenoxyisobutyric acid on short-term Cd-induced hydrogen peroxide production and growth response in barley root tip Ladislav Tamás ∗ , Beáta Boˇcová, Jana Huttová, L’ubica Liptáková, Igor Mistrík, Katarína Valentoviˇcová, Veronika Zelinová 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 22 March 2012 Received in revised form 27 April 2012 Accepted 16 May 2012 Keywords: Cadmium Cell death Indole-3-acetic acid Root growth inhibition Root swelling
a b s t r a c t Short-term treatment (30 min) of barley roots with a low 10 M Cd concentration induced significant H2 O2 production in the elongation and differentiation zone of the root tip 3 h after treatment. This elevated H2 O2 production was accompanied by root growth inhibition and probably invoked root swelling in the elongation zone of the root tip. By contrast, a high 60 M Cd concentration induced robust H2 O2 production in the elongation zone of the root tip already 1 h after short-term treatment. This robust H2 O2 generation caused extensive cell death 6 h after short-term treatment. Similarly to low Cd concentration, exogenously applied H2 O2 caused marked root growth inhibition, which at lower H2 O2 concentration was accompanied by root swelling. The auxin signaling inhibitor p-chlorophenoxyisobutyric acid effectively inhibited 10 M Cd-induced root growth inhibition, H2 O2 production and root swelling, but was ineffective in the alleviation of 60 M Cd-induced root growth inhibition and H2 O2 production. Our results demonstrated that Cd-induced mild oxidative stress caused root growth inhibition, likely trough the rapid reorientation of cell growth in which a crucial role was played by IAA signaling in the root tip. Strong oxidative stress induced by high Cd concentration caused extensive cell death in the elongation zone of the root tip, resulting in the cessation of root growth or even in root death. © 2012 Elsevier GmbH. All rights reserved.
Introduction Oxygen as a terminal oxidant in respiration confers significant energy advantages on organisms, and was probably the most important change in Earth’s environment since life began. Due to its high reactivity, however, the production of reactive oxygen species (ROS) is an unavoidable by-product of aerobic metabolism. ROS is a collective term that describes the chemical species (including radicals and non-radicals) that are formed upon incomplete reduction of oxygen, such as superoxide anion, hydrogen peroxide, hydroxyl radical and nitric oxide (Halliwell, 2006). Contrary to the early hypothesis that ROS are only the toxic byproducts of aerobic metabolism, it has become clear that ROS play a crucial role in the regulation of different developmental and physiological processes (Foyer and Noctor, 2005). Hydrogen peroxide (H2 O2 ), in comparison to the other ROS, plays a key role as a signal
Abbreviations: IAA, indole-3-acetic acid; PCIB, p-chlorophenoxyisobutyric acid; ROS, reactive oxygen species; TIBA, triiodobenzoic acid. ∗ Corresponding author. E-mail address: [email protected] (L. Tamás). 0176-1617/$ – see front matter © 2012 Elsevier GmbH. All rights reserved. http://dx.doi.org/10.1016/j.jplph.2012.05.023
molecule due to its relative high stability and permeability through membranes. It acts as a signal molecule that induces gene expression (Desikan et al., 2000), germination (Barba-Espin et al., 2010), leaf abscission (Sakamoto et al., 2008), stomatal closure (Desikan et al., 2004) and gravitropic bending of roots (Joo et al., 2001). H2 O2 is also generated during abiotic and biotic stress conditions, where it is involved as a signal molecule in defense responses, including cell death (van Breusegem and Dat, 2006). In addition, a function for ROS in hormone responses has been reported in various developmental processes (Kwak et al., 2006). It is well known that the inhibition of root growth as a common response of roots to different stresses is associated with increased ROS production and alterations in phytohormone levels (Potters et al., 2009). Both Cd-induced alterations in the level of hormones and ROS have been observed in several plant species. It has previously been described that roots curved away from Cd, suggesting the inhibition of basipetal auxin flow by Cd (Hasenstein et al., 1988). In contrast to the auxin flow inhibition, ethylene production was highly stimulated by Cd treatment in carrot cell suspension cultures (Sanita di Toppi et al., 1998). Although Cd induces a perturbation in different cellular metabolism, an increasing number of publications suggest that several Cd-induced toxicity symptoms are attributable
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to oxidative damage arising from imbalance in the generation and removal of ROS (Sanita di Toppi and Gabbrielli, 1999; Benavides et al., 2005). Olmos et al. (2003) have reported that the early step in the oxidative burst induced by Cd in culture of tobacco cells is mediated through the activation of NADPH oxidase and superoxide dismutase, leading to superoxide and H2 O2 accumulation within some minutes. Similarly to cultured cells, the extracellular H2 O2 production was a very early response of alfalfa root segments to Cd or Hg exposure, which was detectable already just a few minutes after the treatment (Ortega-Villasante et al., 2007). Apart from Cd-induced root growth inhibition, in some cases, radial expansion of root cells has also been reported (Fusconi et al., 2007; Rascio et al., 2008). Pasternak et al. (2005) suggested that stress-induced phenotypes comprise a re-orientation rather than a cessation of growth. These stress-induced morphogenic responses, including inhibition of cell elongation, localized stimulation of cell division, alterations in cell differentiation and probably re-orientation of cell and organ growth are general acclimation strategies during various stresses (Potters et al., 2009). Using short-term treatments, the aim of this study was to analyze the interaction between H2 O2 and auxin signaling in root cell growth reorientation and cell death during Cd stress in barley root tips. Material 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, Czech Republic) moistened with distilled water in Petri dishes (5 ml per sheet of filter paper of 18 cm in diameter) 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. Trays were placed into a nearly vertical position to enable downward radical growth. In the case of root bending, assay trays were oriented with roots pointing upward. 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.
Root length measurement For the determination of root length increases, the position of root tips following the treatments were marked on the filter paper. After 3, 6 or 24 h, roots were excised at the position of marks and the length increase was measured after recording with a stereomicroscope (STMPRO BELPhotonics, Italy) using a BEL micro image analyzer. Cd content determination Root segments (3 mm long) were oven-dried immediately after short-term treatments at 60 ◦ C for 2 d, and after digestion and ˇ and Poláˇceková, 2000) were analyzed using extraction (Korenovská an atomic absorption spectrometer (Perkin Elmer 4100, Norwalk, CT, USA). Hydrogen peroxide assay H2 O2 production was monitored fluorimetrically using the Amplex UltraRed Hydrogen Peroxide Assay Kit (Molecular Probes) according to manufacturer’s recommendations, with minor modifications. Segments from barley root tips (10 segments per reaction) were incubated in 200 l of 50 mM sodium phosphate buffer, pH 7.4 containing 25 M Amplex UltraRed reagent (from 10 mM DMSO stock solution) and 0.02 U of horse radish peroxidase for 20 min at 30 ◦ C. The fluorescence signal was recorded (170 l of reaction mixture without root segments) with the microplate reader (Synergy HT BIO-TEK, USA) using excitation at 530 (filter 530/25) nm and fluorescence detection at 590 (filter 590/20) nm. Histochemical localization of H2 O2 Intact roots were immersed in the solution of 10 mM sodium phosphate buffer pH 5.8 containing 0.1% 3,3 -diaminobenzidine – DAB and, after vacuum-infiltration for 5 min, were incubated for 1 h in the light. Brown staining characteristic of the DAB–H2 O2 interaction was photographed with a stereomicroscope (STMPRO BELPhotonics, Italy). Detection of cell death Intact roots were immersed in a solution of 0.25% Evans Blue for 15 min at room temperature. After washing with distilled water for 3 min × 5 min, roots were photographed with a stereomicroscope.
Short-term treatments Statistical analyses Roots of seedlings were immersed into distilled water (control) or into the appropriate test solution (see also figures and figure legends) for 30 min. For Cd exposure 10, 20, 30, 40, 50, 60 or 100 M concentrations were used. Hydrogen peroxide (H2 O2 ) was used at 0.5, 1, 5 or 10 mM concentrations. Indole-3-acetic acid (IAA) was applied at 10 M, and IAA efflux inhibitor 2,3,5triiodobenzoic acid (TIBA) at 20 M concentration. IAA signaling inhibitor p-chlorophenoxyisobutyric acid (PCIB) was used at 10, 20, or 30 M concentrations. After washing in distilled water for 5 min the seedlings were incubated as described above. After 1, 3 or 6 h of these short-term treatments, the individual barley root segments (3 mm in length) were obtained by the gradual cutting of each root from the tip to the base. Under control conditions, the first segment represents the meristem and the elongation zone, while the second segment the beginning of differentiation zone. Where indicated, Cd (stock at 1 mM CdCl2 ) or H2 O2 (stock at 10 mM) were dissolved in water; IAA (stock at 1 mM) were dissolved in ethanol, and TIBA or PCIB (stock at 10 mM) were dissolved in acetone and ethanol, respectively.
The experiments were carried out in five independent series with three replicates. The data were analyzed by one-way analysis of variance (ANOVA test), and the means were separated using Tukey’s test. Results Significant root growth inhibition was observed after relatively short-term (30 min) exposure of barley roots to low 10 M Cd concentration. The root increase of Cd-treated seedlings measured 3 or 6 h after the short-term treatment represented approximately 30% of controls (roots treated with distilled water for 30 min) (Fig. 1a). No root growth was observed during the first 6 h after short-term treatment with 30 or 60 M Cd. After that, the root growth of 30 M Cd short-term treated seedlings was renewed, while root growth was completely inhibited even 24 h after short-term treatment with the 60 M Cd concentration (Fig. 1a). Apart from the Cdinduced root growth inhibition, visible root swelling was detected
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Fig. 1. Root length increments (a) 3, 6 and 24 h after short-term treatments with 0, 10, 30 or 60 M concentration of Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of radial root swelling 3 and 6 h (b) after short-term treatment with 0 or 10 M Cd. After short-term treatment roots were arranged into the row that root tips were positioned on the line marked on the bottom of the tray. After subsequent incubation root part lying on this line was marked with black stain which is visible on the picture. The root part below this mark represents new root growth. Black arrows show swollen root part. MZ – meristematic zone, EZ – elongation zone.
within 6 h after short-term treatment with 10 M Cd immediately behind the root tip from 0 to 3 mm, representing the meristem and elongation zone during the short-term treatment (Fig. 1b). Similarly to the root growth restoration, a longer recovery time was required after short-term treatment for swelling appearing (8–9 h) in roots exposed to 30 M Cd (data not shown) in comparison with the 10 M Cd-treated roots. However, it was not detectable in roots treated with the 60 M Cd concentration. Analysis of H2 O2 production revealed that its level markedly increased after 1 h of short-term treatment with the concentration higher than 10 M Cd immediately behind the root apex (from 0 to 3 mm representing meristem and elongation zone) in a concentration-dependent manner (Fig. 2a). In contrast, in the next segment from 3 to 6 mm behind the root apex (representing the differentiation zone), Cd induced H2 O2 production to a smaller extent than in the first segment, and with minor differences between different Cd concentrations (Fig. 2b). The considerable 60 M Cd-induced H2 O2 production was also detectable with DAB staining and was localized immediately behind the root tip 1 h after short-term treatment (Fig. 2c). This robust 60 M Cd-induced H2 O2 production in the root apex detected 1 h after short-term treatment decreased over time, but it remained elevated in relation to control. H2 O2 production after short-term treatment with 10 M Cd was much lower, but increased in a time-dependent manner (Fig. 3a). In contrast to the first root segment, in the second segment from 3 to 6 mm behind the root apex, marked Cd-induced H2 O2 production
Fig. 2. H2 O2 production in root segments from 0 to 3 mm (a) and from 3 to 6 mm (b) behind the root apex 1 h after short-term treatment with different concentration of Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of H2 O2 production (c) 1 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show root part stained by DAB.
was detected at the 10 M Cd concentration 3 h after short-term treatment (Fig. 3b). In this segment, elevated H2 O2 production was also observed after short-term treatment with the 60 M Cd concentration, but to a much lower extent than after 10 M Cd treatment. In the case of 10 M Cd treatment, the elevated H2 O2 production was mainly associated with the radial expansion of roots. The swollen part was the first segment representing the meristem and elongation zone during the short-term treatment, but the second 3 mm segment was from 3 to 6 mm behind the root tip 6 h after short-term treatment due to the root growth (Fig. 3c, see also Fig. 1B). In contrast to 10 M Cd-induced H2 O2 production in the differentiation zone of root tip that probably invoked root swelling, 60 M Cd-induced robust H2 O2 production in the
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Fig. 4. Localization of cell death 6 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show a marked uptake of Evans blue into cells signalizing cell death.
(Fig. 6). IAA signaling inhibitor PCIB effectively inhibited both 15 M Cd-induced root growth inhibition and H2 O2 production in a concentration-dependent manner (Fig. 7) Cd-induced radial root swelling was completely eliminated in the presence of PCIB during the short-term Cd treatment of roots (Fig. 7c). By contrast, in seedlings treated with the high 60 M Cd, PCIB was ineffective in the alleviation of Cd-induced root growth inhibition and H2 O2 production (data not shown). The analysis of Cd uptake revealed that the Cd content of the root tip was not affected by the presence of PCIB (Table 1).
Fig. 3. H2 O2 production in root segments from 0 to 3 mm (a) and from 3 to 6 mm (b) behind the root apex 1, 3 and 6 h after short-term treatment with 0, 10 or 60 M Cd. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Localization of H2 O2 production (c) 6 h after short-term treatment with 0, 10 or 60 M Cd. Black arrows show root part stained by DAB.
beginning of the elongation zone of root tip already 1 h after shortterm treatment, causing extensive cell death clearly detectable 6 h after short-term treatment (Fig. 4). Short-term treatment of barley roots with H2 O2 strongly inhibited root growth (Fig. 5a). This H2 O2 -induced root growth inhibition was accompanied at lower H2 O2 concentrations by root swelling, which at higher H2 O2 concentration was not so obvious, but was accompanied with strong inhibition of root hairs development (Fig. 5b). Similarly to exogenously applied Cd or H2 O2 at lower concentrations, the IAA or IAA efflux inhibitor TIBA, apart from the marked root growth inhibition, caused radial cell expansion, which was accompanied by an elevated production of H2 O2
Discussion The results presented here show that a transient 30 min shortterm Cd-treatment induces significant root growth inhibition in barley seedlings. At low the 10 M Cd concentration, this Cdinduced root growth inhibition was accompanied by the radial expansion of root cells, while the higher concentration of Cd caused the cessation of root growth and cell death. The radial expansion of root cortex cells causing root swelling is a characteristic morphological symptom of several stress conditions, such as Fe or Cu deficiency (Romera et al., 2003), excess of Cu (Panou-Filotheou and Bosabalidis, 2004), salinity (Burssens et al., 2000), UV light (Ktitorova et al., 2006), mechanical impedance (Wilson et al., 1977) and hormones such as auxin and ethylene (Pulgarín et al., 1996). Therefore, it probably belongs to the general stress-induced morphogenic responses (Potters et al., 2009) as a consequence of growth reorientation from the anisodiametric elongation to isodiametric enlargement of cells in the elongation zone of root tips.
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Table 1 Cd content (mg/kg) of barely root segments measured immediately after short-term treatrment with 10 M Cd and 30 M PCIB for 30 min with or without washing of root segments in 5 mM CaCl2 for 10 min after short-term tratments. Root segments Distance from root apex
0–3 mm 3–6 mm
10 M Cd/30 min
10 M Cd + 30 M PCIB/30 min
10 M Cd/30 min 5 mM CaCl2 /10 min
10 M Cd + 30 M PCIB/30 min 5 mM CaCl2 /10 min
127.9 ± 9.6 248.1 ± 15.7
122.7 ± 11 248.5 ± 21.7
75.5 ± 4.7 135.4 ± 5
74.4 ± 3.7 134 ± 5.5
Root growth inhibition as a consequence of enhanced ROS production is a very early response of plants during various stress conditions, including Cd (Ortega-Villasante et al., 2007). Garnier et al. (2006) have reported that tobacco cells exposed to Cd responded with a transient accumulation of H2 O2 , which peaked at 30 min after the Cd treatment. This high transient H2 O2 accumulation was followed by enhanced O2 − production and lipid peroxidation, which similar to our results, led to the cell death. H2 O2 as a signal molecule is involved in numerous processes, including the induction of high-affinity K+ transport activity (Shin and Schachtman, 2004), which is involved in the IAA-induced swelling of protoplasts (Keller and van Volkenburgh, 1996). We showed that Cd at a non-lethal doses induced elevated generation of H2 O2 , which probably initiated the radial expansion of cells in the elongation zone of barley root tips. This elevated H2 O2 production was detected 1 h after Cd treatment and localized in the differentiation zone of root tips. The H2 O2 production increased with time, and 6 h after Cd treatment was associated mainly with the swollen part of root and probably was required for cell wall reorganization during this rapid isodiametric growth of cells. On the other hand, at a higher Cd concentration, the robust production of H2 O2 was localized in the elongation zone of root tip with a maximum peak 1 h after the Cd treatment and was associated with cell, and at very high Cd concentration, also with root death.
Fig. 5. Root length increments (a) and localization of radial root swelling (b) 6 h after short-term treatment with 0, 0.5, 1.0, 5.0 or 10 mM H2 O2 . Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrow shows swollen root part. Black mark – see Fig. 1.
This high Cd concentration generated H2 O2 in the elongation zone and inhibited both elongation and radial expansion of cells. This was also confirmed by the exogenously applied H2 O2 , where in addition to the inhibition of cell elongation at a lower (up to 1 mM) H2 O2 concentration, the radial expansion of cells occurred similarly to the low Cd concentration. In contrast, at a higher Cd or H2 O2 concentration, the root swelling was markedly inhibited,
Fig. 6. Root length increments 6 h (a), H2 O2 production in root segments from 3 to 6 mm behind the root apex 3 h (b) and localization of radial root swelling with elevated H2 O2 production 6 h (c) after short-term treatment with dw (control), 10 M IAA or 20 M TIBA. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrow shows swollen root part stained by DAB.
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Fig. 7. Effect of auxin signaling inhibitor PCIB on 10 M Cd-induced root growth inhibition, radial root swelling and H2 O2 production. Root length increments 6 h (a) and H2 O2 production in root segments from 3 to 6 mm behind the root apex 3 h (b), radial swelling 6 h (c) after short-term treatments. Different letters indicate statistical significance according to Tukey’s test (P < 0.05). Black arrows show swollen root part. Black mark – see Fig. 1.
indicating that different processes may be activated in the root tip of barley depending on the level of H2 O2 . On the other hand, it has recently been reported that apoplastic ROS strongly inhibited IAA signaling while activating ethylene signaling and cell death (Blomster et al., 2011). This may explain the blockage of radial cell expansion at both high Cd and H2 O2 concentrations. In addition, with high H2 O2 content, breakdown of IAA may occur through the peroxidase catalyzed reaction (Kawano, 2003), which may limit further the IAA-mediated responses under a strong oxidative stress condition. It has been observed that PCIB effectively impairs the IAA signaling pathway in Arabidopsis, affecting IAA-regulated root morphology (Oono et al., 2003). To better understand the involvement of IAA in the Cd-induced H2 O2 accumulation and morphogenic responses, we also studied the effect of the IAA signaling inhibitor PCIB. We showed that PCIB alleviates or even blocks the short-term Cd stress-induced root growth inhibition and radial root expansion, which was accompanied by a reduction of Cd-generated H2 O2 . Similar to PCIB, exogenous application of salicylic acid also alleviated Cd toxicity, which was associated with the reduction of H2 O2 accumulation in the root apoplast (Zhang et al., 2011). It has recently
been shown that salicylic acid inhibited the IAA signaling pathway (Wang et al., 2007), which may decrease H2 O2 accumulation in cells in a manner similar to PCIB. In our experiments, PCIB co-treatment did not block exogenously applied IAA-induced responses (data not shown), likely due to the lag time required for PCIB action (Oono et al., 2003). Our results provide strong evidence that the short-term Cdtreatment induced root growth inhibition and radial root expansion is mediated through the IAA-induced H2 O2 generation in barley root tissues. In contrast, strong oxidative stress induced by high Cd concentration caused extensive H2 O2 accumulation independent of IAA in the elongation zone of root tip, resulting in the cessation of root growth or even in root death. The function of H2 O2 as a signal and/or substrate is well known in the IAA-mediated H2 O2 generation in root gravitropic response (Joo et al., 2001). We showed that H2 O2 as a signal molecule acts downstream of IAA and, in addition, it is probably directly utilized in the cell wall metabolism during mild Cd stress-induced morphogenic changes. Therefore, root growth inhibition and ROS production as a toxicity symptom of several stresses reported in numerous publications may be associated only with morphogenic responses without marked damages of the root tissues. Accordingly, the IAA signaling mutant is more tolerant to both oxidative and salinity stress, exhibiting higher rates of root elongation than wild type seedlings due to the reduced accumulation of ROS under stress conditions (Iglesias et al., 2010). The up-regulation of GH3 auxin amido synthase caused a decrease of active IAA levels in poplar root tissues and might have contributed to growth attenuation under Cd stress (Elobeid et al., 2012). Similar to roots, in rice shoots, the activation of auxin amido synthase results in a decrease of free IAA levels and enhanced drought tolerance (Zhang et al., 2009). In conclusion, these results suggest that IAA plays a crucial role in various morphogenic responses during mild stress conditions, including Cd excess. On the other hand, they probably did not play a direct role in the detoxification of Cd. The role of these morphogenic changes is probably associated with the evasion of roots from the source of Cd supply (Potters et al., 2007). Interestingly, these general morphogenic responses are absent in the metal hyperaccumulator plants, where instead of high Cd concentration, no significant morphogenic and structural modifications are observed in roots (Ederli et al., 2004). Our results showed that the induction of morphogenic responses by mild Cd stress are not associated with toxicity symptoms because, due to the inhibition of IAA signaling pathways, roots grow without inhibition in spite of the presence of relatively high concentrations of Cd in roots during and after the short-term Cd exposure. These observations raise questions about the definition of root growth inhibition during abiotic stresses as an early toxicity symptom. Our results indicate that root growth inhibition under mild stresses is not a result of stressinduced damages, but rather a general stress-induced morphogenic response just to avoid damage to root tissues under unfavorable conditions. Acknowledgments We wish to thank Margita Vaˇsková for excellent technical assistance. This work was supported by the Grant agency VEGA, project no. 2/0050/10. References Barba-Espin G, Diaz-Vivancos P, Clemente-Moreno MJ, Albacete A, Faize L, Faize M, et al. Interaction between hydrogen peroxide and plant hormones during germination and the early growth of pea seedlings. Plant Cell Environ 2010;33:981–94. Benavides MP, Gallego SM, Tomaro ML. Cadmium toxicity in plants. Brazil J Plant Physiol 2005;17:21–34.
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