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Research Paper
Improvement of adventitious root formation in flax using hydrogen peroxide Toma´sˇ Taka´cˇ1, Bohusˇ Obert1, Jakub Rolcˇı´k2 and Jozef Sˇamaj1 Q1 1 Centre of the Region Hana´ for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacky´ University, Olomouc 783 71, Czech Republic 2 Centre of the Region Hana´ for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Faculty of Science, Palacky´ University, Olomouc 783 71, Czech Republic
Flax (Linum usitatissimum L.) is an important crop for the production of oil and fiber. In vitro manipulations of flax are used for genetic improvement and breeding while improvements in adventitious root formation are important for biotechnological programs focused on regeneration and vegetative propagation of genetically valuable plant material. Additionally, flax hypocotyl segments possess outstanding morphogenetic capacity, thus providing a useful model for the investigation of flax developmental processes. Here, we investigated the crosstalk between hydrogen peroxide and auxin with respect to reprogramming flax hypocotyl cells for root morphogenetic development. Exogenous auxin induced the robust formation of adventitious roots from flax hypocotyl segments while the addition of hydrogen peroxide further enhanced this process. The levels of endogenous auxin (indole-3-acetic acid; IAA) were positively correlated with increased root formation in response to exogenous auxin (1Naphthaleneacetic acid; NAA). Histochemical staining of the hypocotyl segments revealed that hydrogen peroxide and peroxidase, but not superoxide, were positively correlated with root formation. Measurements of antioxidant enzyme activities showed that endogenous levels of hydrogen peroxide were controlled by peroxidases during root formation from hypocotyl segments. In conclusion, hydrogen peroxide positively affected flax adventitious root formation by regulating the endogenous auxin levels. Consequently, this agent can be applied to increase flax regeneration capacity for biotechnological purposes such as improved plant rooting. Introduction Q2 Reactive oxygen species (ROS) are produced by aerobic metabolic processes in living organisms and their generation is elevated under unfavorable conditions. At a physiological level, they play important roles in signal transduction and multiple developmental processes [1–3]. ROS are implicated in the regulation of the cell cycle [4], cell elongation [5,6], root hair formation [7], lateral and adventitious root formation [8,9], root elongation [10], stomatal closure [11] gravitropism [12] and embryogenesis [13,14]. Corresponding author: Taka´cˇ, T. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2016.02.008 1871-6784/ß 2016 Published by Elsevier B.V.
Crosstalk between ROS signalling and auxin or abscisic acid is involved in plant developmental processes [15–17]. ROS that control developmental processes are generated by plasma membrane-localized NADPH oxidase [3,6]. The phytohormones auxin and abscisic acid are capable of promoting the production of ROS through this mechanism [12,18–21]. Auxin-induced mitotic activity of Arabidopsis protoplasts occurred in conjunction with accelerated H2O2 generation [22]. ROS stimulated cell division only in the presence of auxin; in the absence of auxin, they exerted a rather damaging effect on protoplast culture [8]. Auxin controls gravitropic curvature www.elsevier.com/locate/nbt
Please cite this article in press as: Taka´cˇ, T. et al., Improvement of adventitious root formation in flax using hydrogen peroxide, New Biotechnol. (2016), http://dx.doi.org/10.1016/ j.nbt.2016.02.008
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through the generation of ROS [12] and this is dependent on phosphatidylinositol 3-kinase [23]. In addition, a hydrogen peroxide-inducing chemical compound called alloxan stimulates auxin-dependent lateral root formation [8]. The intracellular level of ROS under unfavorable conditions is controlled via enzymatic and non-enzymatic compounds working in co-operation [1,24,25]. Their role in stress response is well documented; however, little is known about the regulation of ROS during developmental processes. Until now, only a limited number of studies have been devoted to the role of positive redox homeostasis and active antioxidant defense during developmental processes such as mitotic activity [22,26], androgenesis [27], somatic embryogenesis [28] and root growth [29]. Hydrogen peroxide decomposing catalase 2 plays an essential role in maintaining root meristem activity in the presence of oxidative stress [26]. The elevated activity of another enzyme involved in hydrogen peroxide decomposition, namely ascorbate peroxidase, corresponded with the mitotic activity accelerated by external auxin in Arabidopsis protoplasts [8]. Flax (Linum usitatissimum L.) is an important crop for the production of oil and fiber and is therefore an important target for biotechnological use [30–32]. In vitro manipulation is an essential tool of flax genetic improvement and breeding. It is generally accepted that flax hypocotyl segments possess outstanding morphogenetic capacity, thus providing a useful model for the investigation of flax developmental processes [13,33]. In this study, we used flax hypocotyl segments to study the processes connected to the reprogramming of cells for root morphogenetic development. We investigated the effect of exogenous hydrogen peroxide and provided new information on hydrogen peroxide-auxin crosstalk in this process. The manipulation of root morphogenesis by hydrogen peroxide and auxin may serve as a new and efficient tool for plant rooting in flax biotechnology.
Materials and methods Plant material Flax (Linum usitatissimum L.; cv. Super, AGRITEC, Research, Breeding & Services, Ltd., Czech Republic) seedlings were grown on vertically oriented Phytagel square plates containing ½ Murashige and Skoog (MS) medium (pH 5.7) (16-h light/8-h dark; 228C) for 4 days. Then, the hypocotyls were collected and segmented into 2 mm long segments and incubated in liquid ½ MS media containing 0.5 or 1 mg l1 NAA with or without the addition of 100 mM H2O2 for two days. One part of the segments was transferred to hormone- and H2O2-free solid ½ MS media, while the remainder was used for biochemical and histochemical assays.
Protein extraction for enzymatic analyses Flax hypocotyl segments were ground in the presence of liquid nitrogen and extraction buffer (100 mM sodium-phosphate buffer, pH 7.8, 2 mM EDTA, 2 mM ascorbate) using a mortar and pestle. The homogenate was centrifuged at 15 000 g for 20 min at 48C. The supernatant was collected and desalted on Amicon Ultra YM10 centrifugation columns (Merck Millipore, Germany). The protein content was determined according to Bradford using BSA as standard [34]. 2
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Enzyme activity measurements Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by monitoring the inhibition of the reduction of 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) at 560 nm [35]. One unit of SOD is defined as the extract volume required for 50% inhibition of MTT reduction. Catalase (EC 1.11.1.6) activity was measured by following the decrease in absorbance of H2O2 at 240 nm [36], and guaiacol peroxidase (GPX) activity was determined by following the absorbance decrease in guaiacol concentration at 470 nm [37]. The values presented are the means from at least three independent experiments.
Histochemical and fluorescent staining of flax hypocotyls for the visualization of viability, hydrogen peroxide, superoxide, and peroxidase activity For the evaluation of viability, the hypocotyl segments were incubated in 0.001% fluorescein diacetate (FDA) (w/v) for 1 h in the dark at room temperature. Then, the segments were washed in distilled water and processed as described below for ROS localization. ROS localization was monitored using 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Invitrogen, Carlsbad, CA) according to Fryer [40]. The segments were incubated in 25 mM H2DCFDA on a variable speed rocker at room temperature in the dark for 2 h. Next, the segments were incubated in distilled water in the dark for 1 h. The fluorescent signal was documented using a stereomicroscope (LEICA M165FC, Wetzlar, Germany) equipped with a GFP3 filter set (excitation 450–490 nm, emission 500–550 nm). For visualization of superoxide, the hypocotyl segments were incubated in 1% (w/v) nitroblue tetrazolium chloride in NBT solution in 10 mM potassium phosphate buffer (pH 7.8) under vacuum for 10 min [39]. To localize peroxidase activity, the hypocotyl segments were incubated in 0.2 M potassium buffer containing 0.33 M o-dianisidine and 3 mM H2O2 at 258C for 1 h.
Endogenous free indole-3-acetic acid measurement The quantification of endogenous IAA was performed as described previously [41]. Briefly, approximately 10 mg of ground plant material (in triplicate) was incubated for 10 minutes in 1 ml of cold phosphate buffer (50 mM; pH 7.0) containing 0.02% sodium diethyldithiocarbamate and supplied with [2H5]IAA internal standard. After centrifugation at 36 000 g, the samples were acidified with 1 M HCl to pH 2.7, subjected to C8-based solid-phase extraction and subsequently evaporated to dryness under vacuum. Final analyses were conducted by means of ultra-high performance liquid chromatography (Acquity UPLC system, Waters) coupled with mass spectrometry (micro API tandem quadrupole mass spectrometer, Waters).
Results Hydrogen peroxide stimulated the NAA-induced morphogenetic reprogramming towards adventitious root formation We incubated the flax hypocotyl segments in liquid ½ MS media containing two different auxin (NAA) concentrations for 2 days. Afterwards, they were transferred to solid MS hormone-free media. As expected, these growing conditions caused the intensive
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formation of adventitious roots, consistently from one of the cutting edges of the segments, after only 2 days. The higher dose of external NAA resulted in a higher number of segments with adventitious roots as well as a higher number of adventitious roots formed from one segment (Fig. 1c, e, f). To investigate how ROS affected this morphogenetic reprogramming, we supplemented the NAA-containing liquid media with H2O2. We found that external H2O2 had a positive effect on the monitored parameters (Fig. 1b–f) in the presence of 0.5 mg l1 NAA. Interestingly, this H2O2-enhanced adventitious root formation depended on the external NAA concentration because in the presence of 1 mg l1 NAA, the H2O2 inhibited the formation of adventitious roots from the flax hypocotyl segments (Fig. 1d–f). These results showed that H2O2 either enhanced or repressed the re-programming of the flax hypocotyl cells for adventitious root formation, and this depended on the exogenous auxin concentration.
Hydrogen peroxide caused a higher accumulation of endogenous IAA Next, we examined the levels of free IAA in the NAA- and H2O2treated flax hypocotyl segments (Fig. 2). The higher concentration of external NAA enhanced the accumulation of endogenous free IAA in the segments compared with those treated with the lower dose of NAA (Fig. 2). Supplementing NAA-containing media with H2O2 increased the endogenous IAA level in the case of the lower NAA concentration, while no substantial IAA increase was observed in the case of the higher external NAA dose. These results showed that stimulation of adventitious root formation in flax hypocotyl segments by NAA, H2O2 or both was positively correlated with higher levels of endogenous free IAA. In addition, these results revealed that H2O2 has the capacity to induce IAA accumulation in flax hypocotyls, which can be used for future biotechnological applications.
The localization and accumulation of H2O2 is positively correlated with adventitious root formation
FIGURE 1
Evaluation of adventitious root formation on flax hypocotyl segments preincubated for 2 days in NAA and H2O2 and subsequently grown on MS media for 5 days. Overviews of flax hypocotyl segments pre-incubated in liquid ½ MS media containing (a) 0.5 mg l1 NAA, (b) 0.5 mg l1 NAA and 100 mM H2O2, (c) 1 mg l1 NAA or (d) 1 mg l1 NAA and 100 mM H2O2. Bars represent 1 cm. (e) Graph showing quantification of the number of hypocotyl segments with adventitious roots per 50 segments. (f) Graph depicting the
Next, we aimed to localize ROS accumulation in the hypocotyl segments using histochemical methods and examined their accumulation in response to external NAA and H2O2. To test the viability of the segments after the NAA and H2O2 treatments, we stained the hypocotyl segments with FDA, a marker of cell viability. This observation showed that the treatments did not affect the viability of the cells in the segments. The distribution of the dye was equal on both cutting edges of the segments (Fig. 3a–d). The staining of the hypocotyl segments with NBT (specific for the superoxide radical) did not show substantial differences among treatments. NBT stained mainly the cutting edges of the hypocotyl segments. Similar staining patterns were detected in the case of H2DCFDA and o-dianisidine. We observed slightly stronger NBT staining in response to 1 mg l1 NAA compared with the lower concentration of this compound (Fig. 4a, c). The combined treatment of 1 mg l1 NAA and H2O2 abolished the NBT staining of the hypocotyl segments (Fig. 4d). The H2DCFDA fluorescence quantification of the adventitious root number per 50 segments. Different letters above the bars in (e) and (f ) indicate significant differences at p 0.05 based on Student’s t-test.
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Research Paper FIGURE 2
Levels of endogenous free IAA in flax hypocotyl segments incubated in liquid ½ MS media containing 0.5 mg l1 NAA, 0.5 mg l1 NAA and 100 mM H2O2, 1 mg l1 NAA or 1 mg l1 NAA and 100 mM H2O2 for 2 days. Different letters above the bars indicate significant differences at p 0.05 based on Student’s t-test.
FIGURE 4
FIGURE 3
The evaluation of cell viability using fluorescein diacetate (FDA) in flax hypocotyl segments treated with NAA and H2O2. (a)–(d) Flax hypocotyl segments incubated in liquid ½ MS media containing: (a) 0.5 mg l1 NAA, (b) 0.5 mg l1 NAA and 100 mM H2O2, (c) 1 mg l1 NAA or (d) 1 mg l1 NAA and 100 mM H2O2 for 2 days and subsequently stained with FDA.
signal increased in response to H2O2 combined with 0.5 mg l1 NAA compared with NAA alone, suggesting that the H2DCFDA was sensitive to the exogenous H2O2 treatment. In addition, the H2DCFDA signal was increased in response to the higher dose of exogenous NAA, suggesting increased accumulation of ROS. In the case of H2DCFDA, however, the fluorescence signal intensity differed between the edges of the hypocotyl segments with different capacities for adventitious root formation. This indicates that ROS production was positively correlated with adventitious root formation. Such a difference was not observed in the case of NBT staining, indicating that superoxide did not contribute to the adventitious root formation.
Enzymatic antioxidant defense is affected by exogenous auxin and H2O2 Next, we examined the activities of superoxide dismutase, guaiacol peroxidase and catalase to determine whether the antioxidant 4
Histochemical localization of ROS and peroxidases in flax hypocotyl segments treated with (a, e, i) 0.5 mg l1 NAA (b, f, j) 0.5 mg l1 NAA and 100 mM H2O2, (c, g, k) 1 mg l1 NAA or (d, h, l) 1 mg l1 NAA and 100 mM H2O2 for 2 days. (a)–(d) Histochemical staining of superoxide in flax hypocotyl segments using NBT; (e)–(h) fluorescent staining of ROS in hypocotyl segments using H2DCFDA; (i)–(l) histochemical staining of peroxidases in flax hypocotyl segments using o-dianisidine. Bar represents 2 mm.
enzymes in flax hypocotyls respond to external NAA and H2O2. We found that SOD activity in the hypocotyl segments did not change significantly after the addition of H2O2 to the incubation media (Fig. 5a). However, SOD activity significantly decreased in the response to the higher exogenous NAA concentration. A different pattern was observed in the case of the antioxidant enzymes decomposing H2O2. Guaiacol peroxidase (GPX) was activated by H2O2 at the lower exogenous NAA concentration but inhibited at the higher NAA concentration (Fig. 5b). In contrast, catalase showed increased activity at the higher NAA concentration, whereas it remained unaltered when H2O2 was added to the media containing 0.5 mg l1 NAA (Fig. 5c). Notably, the catalase activity was also increased in hypocotyls exposed to 1 mg l1 NAA together with H2O2. These results revealed that enzymatic antioxidant defense occurred in response to exogenous NAA and/or H2O2. Peroxidase was activated by external H2O2, while catalase activity was stimulated by higher external NAA levels. In addition, we also took advantage of the histochemical staining of peroxidase activity in the flax hypocotyl segments by using o-dianisidine as a
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substrate (Fig. 4 i–l). The peroxidase activity showed polar localization, which was similar to the H2DCFDA fluorescent staining pattern. This implies that peroxidase might be involved in the scavenging of H2O2 generated by endogenous auxin.
In addition to the role of ROS in stress response and signalling, emphasis has also been placed on their role in developmental processes. For example, ROS stimulate cell division in alfalfa leaf protoplasts [4,22], mediate root hair elongation [6], control cell elongation in maize roots [5], regulate the elongation of the primary root of Arabidopsis and promote the formation of lateral roots in cucumber [8,9]. All of these processes are very important for the improvement of growth performance and crop yield. We report that H2O2 positively affects the developmental and biochemical events leading to adventitious root formation from flax hypocotyls. This contributes to the above-mentioned evidence that ROS are important modulators of plant developmental processes and they might be exploited in biotechnological crop improvement.
Positive effect of H2O2 on morphogenetic reprogramming depends on endogenous auxin level Important for biotechnological applications, the simultaneous exposure of flax hypocotyl segments to H2O2 and 0.5 mg l1 NAA enhanced the formation of adventitious roots compared with NAA alone. This indicates the activation of cell division in response to H2O2, which is consistent with previously published data [22]. At the higher exogenous NAA level, H2O2 inhibited the formation of the adventitious roots. It was also reported that ROS have deleterious effects on adventitious root formation when applied without auxin [8]. These data suggest that balanced homeostasis between H2O2 and auxin is necessary to achieve a positive effect of ROS on adventitious root formation. Interestingly, the formation of adventitious roots was positively correlated with endogenous IAA levels, regardless of exogenous H2O2 treatment. This shows that the external application of H2O2 could positively regulate auxin signalling or transport in flax hypocotyl segments. A previous genomic study showed transient early suppression of auxin signalling and subsequent recovery after 24 h by ozone-induced apoplastic ROS in Arabidopsis [42]. The ROS-induced attenuation of auxin signalling was recently explained by oxidative IAA catabolism by ROS produced in response to IAA accumulation [43]. In our system, the hypocotyl segments were simultaneously supplied with H2O2 and external NAA, which likely maintained the intracellular pool of active (non-oxidized) auxin. Under these conditions, H2O2 appears to positively affect adventitious root formation. FIGURE 5
Characterization of enzymatic antioxidant defense in flax hypocotyl segments treated with NAA and H2O2. Graphs depict activity of superoxide dismutase (SOD) (a), catalase (b) and guaiacol peroxidase (c) in flax hypocotyl segments incubated in liquid ½ MS media containing 0.5 mg l1 NAA, 0.5 mg l1 NAA and 100 mM H2O2, 1 mg l1 NAA or 1 mg l1 NAA and 100 mM H2O2 for 2 days. Different letters above the bars indicate significant differences at p 0.05 based on Student’s t-test.
Adventitious root formation likely depends on the polar auxin gradient Adventitious roots regularly formed only on one of the cutting edges of the hypocotyl segments. Considering the basipetal auxin flow in hypocotyls, we assume that adventitious roots formed in areas with endogenous auxin accumulation. Similar results were obtained for hypocotyl explants of Mesembryanthemum crystallinum, which formed adventitious roots only in their basal parts [44]. Interestingly, neither the elevated level of exogenous auxin
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Discussion
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nor the addition of H2O2 caused adventitious root formation at the apical end of the hypocotyl segments. This indicates that polar localization of endogenous auxin is required for hypocotyl reprogramming towards adventitious root formation. Such differential auxin accumulation at the two cutting edges of the hypocotyl segments was positively correlated with different localization of H2O2 in the segments. On the other hand, it was not related to the superoxide localization. Previously, it was reported that H2O2 and superoxide display distinct accumulation and roles in Arabidopsis root development [45]. Superoxide was predominantly located in the apoplast of the cell elongation zone, whereas H2O2 accumulated in the differentiation zone and in the cell wall of root hairs. Superoxide appeared to promote, while H2O2 supressed cell elongation and root hair formation [45]. Our results emphasize the role of H2O2, but not superoxide in adventitious root formation from flax hypocotyl segments, and thus provide new information on the versatility of ROS developmental functions in this biotechnologically important crop.
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to exogenous NAA and H2O2. Enhanced levels of H2O2 in response to NAA alone or combined with H2O2 clearly necessitated an induction of H2O2-scavenging enzymes in the flax hypocotyl segments. Different enzymes might be involved in H2O2 removal, depending on external treatment. In the flax hypocotyls, catalase activity was induced by 0.5 mg l1 of exogenous NAA while peroxidases responded to combined H2O2 and 0.5 mg l1 NAA treatment. Moreover, histochemical staining of peroxidase activity by o-dianisidine showed polar localization in the NAA- and H2O2-treated flax hypocotyl segments. Among other roles, peroxidases are involved in cell wall loosening through the generation of hydroxyl radicals from superoxide [10]. Our results support the role of peroxidases in H2O2 removal, mainly after exogenous H2O2 application, and the polar localization of peroxidase activity in hypocotyl segments. Moreover, the latter might be related to the presumably polar localization of endogenous auxin.
Uncited references Enzymatic antioxidant defense during adventitious root formation
[38,46].
ROS are controlled by multiple enzymatic or non-enzymatic
Acknowledgements
Q3 antioxidant mechanisms [24,47]. Very little is known about the
control of ROS levels during developmental processes. Our results point to distinct changes in flax antioxidant enzymes in response
This work was supported by the grant No. LO1204 (Sustainable development of research in the Centre of the Region Hana´) from the National Program of Sustainability I, MEYS. Q5
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Improvement of adventitious root formation in flax using hydrogen peroxide Toma´sˇ Taka´cˇ1, Bohusˇ Obert1, Jakub Rolcˇı´k2 and Jozef Sˇamaj1 Q1 1 Centre of the Region Hana´ for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacky´ University, Olomouc 783 71, Czech Republic 2 Centre of the Region Hana´ for Biotechnological and Agricultural Research, Department of Chemical Biology and Genetics, Faculty of Science, Palacky´ University, Olomouc 783 71, Czech Republic
Flax (Linum usitatissimum L.) is an important crop for the production of oil and fiber. In vitro manipulations of flax are used for genetic improvement and breeding while improvements in adventitious root formation are important for biotechnological programs focused on regeneration and vegetative propagation of genetically valuable plant material. Additionally, flax hypocotyl segments possess outstanding morphogenetic capacity, thus providing a useful model for the investigation of flax developmental processes. Here, we investigated the crosstalk between hydrogen peroxide and auxin with respect to reprogramming flax hypocotyl cells for root morphogenetic development. Exogenous auxin induced the robust formation of adventitious roots from flax hypocotyl segments while the addition of hydrogen peroxide further enhanced this process. The levels of endogenous auxin (indole-3-acetic acid; IAA) were positively correlated with increased root formation in response to exogenous auxin (1Naphthaleneacetic acid; NAA). Histochemical staining of the hypocotyl segments revealed that hydrogen peroxide and peroxidase, but not superoxide, were positively correlated with root formation. Measurements of antioxidant enzyme activities showed that endogenous levels of hydrogen peroxide were controlled by peroxidases during root formation from hypocotyl segments. In conclusion, hydrogen peroxide positively affected flax adventitious root formation by regulating the endogenous auxin levels. Consequently, this agent can be applied to increase flax regeneration capacity for biotechnological purposes such as improved plant rooting. Introduction Q2 Reactive oxygen species (ROS) are produced by aerobic metabolic processes in living organisms and their generation is elevated under unfavorable conditions. At a physiological level, they play important roles in signal transduction and multiple developmental processes [1–3]. ROS are implicated in the regulation of the cell cycle [4], cell elongation [5,6], root hair formation [7], lateral and adventitious root formation [8,9], root elongation [10], stomatal closure [11] gravitropism [12] and embryogenesis [13,14]. Corresponding author: Taka´cˇ, T. ([email protected]) http://dx.doi.org/10.1016/j.nbt.2016.02.008 1871-6784/ß 2016 Published by Elsevier B.V.
Crosstalk between ROS signalling and auxin or abscisic acid is involved in plant developmental processes [15–17]. ROS that control developmental processes are generated by plasma membrane-localized NADPH oxidase [3,6]. The phytohormones auxin and abscisic acid are capable of promoting the production of ROS through this mechanism [12,18–21]. Auxin-induced mitotic activity of Arabidopsis protoplasts occurred in conjunction with accelerated H2O2 generation [22]. ROS stimulated cell division only in the presence of auxin; in the absence of auxin, they exerted a rather damaging effect on protoplast culture [8]. Auxin controls gravitropic curvature www.elsevier.com/locate/nbt
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through the generation of ROS [12] and this is dependent on phosphatidylinositol 3-kinase [23]. In addition, a hydrogen peroxide-inducing chemical compound called alloxan stimulates auxin-dependent lateral root formation [8]. The intracellular level of ROS under unfavorable conditions is controlled via enzymatic and non-enzymatic compounds working in co-operation [1,24,25]. Their role in stress response is well documented; however, little is known about the regulation of ROS during developmental processes. Until now, only a limited number of studies have been devoted to the role of positive redox homeostasis and active antioxidant defense during developmental processes such as mitotic activity [22,26], androgenesis [27], somatic embryogenesis [28] and root growth [29]. Hydrogen peroxide decomposing catalase 2 plays an essential role in maintaining root meristem activity in the presence of oxidative stress [26]. The elevated activity of another enzyme involved in hydrogen peroxide decomposition, namely ascorbate peroxidase, corresponded with the mitotic activity accelerated by external auxin in Arabidopsis protoplasts [8]. Flax (Linum usitatissimum L.) is an important crop for the production of oil and fiber and is therefore an important target for biotechnological use [30–32]. In vitro manipulation is an essential tool of flax genetic improvement and breeding. It is generally accepted that flax hypocotyl segments possess outstanding morphogenetic capacity, thus providing a useful model for the investigation of flax developmental processes [13,33]. In this study, we used flax hypocotyl segments to study the processes connected to the reprogramming of cells for root morphogenetic development. We investigated the effect of exogenous hydrogen peroxide and provided new information on hydrogen peroxide-auxin crosstalk in this process. The manipulation of root morphogenesis by hydrogen peroxide and auxin may serve as a new and efficient tool for plant rooting in flax biotechnology.
Materials and methods Plant material Flax (Linum usitatissimum L.; cv. Super, AGRITEC, Research, Breeding & Services, Ltd., Czech Republic) seedlings were grown on vertically oriented Phytagel square plates containing ½ Murashige and Skoog (MS) medium (pH 5.7) (16-h light/8-h dark; 228C) for 4 days. Then, the hypocotyls were collected and segmented into 2 mm long segments and incubated in liquid ½ MS media containing 0.5 or 1 mg l1 NAA with or without the addition of 100 mM H2O2 for two days. One part of the segments was transferred to hormone- and H2O2-free solid ½ MS media, while the remainder was used for biochemical and histochemical assays.
Protein extraction for enzymatic analyses Flax hypocotyl segments were ground in the presence of liquid nitrogen and extraction buffer (100 mM sodium-phosphate buffer, pH 7.8, 2 mM EDTA, 2 mM ascorbate) using a mortar and pestle. The homogenate was centrifuged at 15 000 g for 20 min at 48C. The supernatant was collected and desalted on Amicon Ultra YM10 centrifugation columns (Merck Millipore, Germany). The protein content was determined according to Bradford using BSA as standard [34]. 2
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Enzyme activity measurements Superoxide dismutase (SOD, EC 1.15.1.1) activity was determined by monitoring the inhibition of the reduction of 3-(4,5-dimethyl2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) at 560 nm [35]. One unit of SOD is defined as the extract volume required for 50% inhibition of MTT reduction. Catalase (EC 1.11.1.6) activity was measured by following the decrease in absorbance of H2O2 at 240 nm [36], and guaiacol peroxidase (GPX) activity was determined by following the absorbance decrease in guaiacol concentration at 470 nm [37]. The values presented are the means from at least three independent experiments.
Histochemical and fluorescent staining of flax hypocotyls for the visualization of viability, hydrogen peroxide, superoxide, and peroxidase activity For the evaluation of viability, the hypocotyl segments were incubated in 0.001% fluorescein diacetate (FDA) (w/v) for 1 h in the dark at room temperature. Then, the segments were washed in distilled water and processed as described below for ROS localization. ROS localization was monitored using 20 ,70 -dichlorodihydrofluorescein diacetate (H2DCFDA, Molecular Probes, Invitrogen, Carlsbad, CA) according to Fryer [40]. The segments were incubated in 25 mM H2DCFDA on a variable speed rocker at room temperature in the dark for 2 h. Next, the segments were incubated in distilled water in the dark for 1 h. The fluorescent signal was documented using a stereomicroscope (LEICA M165FC, Wetzlar, Germany) equipped with a GFP3 filter set (excitation 450–490 nm, emission 500–550 nm). For visualization of superoxide, the hypocotyl segments were incubated in 1% (w/v) nitroblue tetrazolium chloride in NBT solution in 10 mM potassium phosphate buffer (pH 7.8) under vacuum for 10 min [39]. To localize peroxidase activity, the hypocotyl segments were incubated in 0.2 M potassium buffer containing 0.33 M o-dianisidine and 3 mM H2O2 at 258C for 1 h.
Endogenous free indole-3-acetic acid measurement The quantification of endogenous IAA was performed as described previously [41]. Briefly, approximately 10 mg of ground plant material (in triplicate) was incubated for 10 minutes in 1 ml of cold phosphate buffer (50 mM; pH 7.0) containing 0.02% sodium diethyldithiocarbamate and supplied with [2H5]IAA internal standard. After centrifugation at 36 000 g, the samples were acidified with 1 M HCl to pH 2.7, subjected to C8-based solid-phase extraction and subsequently evaporated to dryness under vacuum. Final analyses were conducted by means of ultra-high performance liquid chromatography (Acquity UPLC system, Waters) coupled with mass spectrometry (micro API tandem quadrupole mass spectrometer, Waters).
Results Hydrogen peroxide stimulated the NAA-induced morphogenetic reprogramming towards adventitious root formation We incubated the flax hypocotyl segments in liquid ½ MS media containing two different auxin (NAA) concentrations for 2 days. Afterwards, they were transferred to solid MS hormone-free media. As expected, these growing conditions caused the intensive
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formation of adventitious roots, consistently from one of the cutting edges of the segments, after only 2 days. The higher dose of external NAA resulted in a higher number of segments with adventitious roots as well as a higher number of adventitious roots formed from one segment (Fig. 1c, e, f). To investigate how ROS affected this morphogenetic reprogramming, we supplemented the NAA-containing liquid media with H2O2. We found that external H2O2 had a positive effect on the monitored parameters (Fig. 1b–f) in the presence of 0.5 mg l1 NAA. Interestingly, this H2O2-enhanced adventitious root formation depended on the external NAA concentration because in the presence of 1 mg l1 NAA, the H2O2 inhibited the formation of adventitious roots from the flax hypocotyl segments (Fig. 1d–f). These results showed that H2O2 either enhanced or repressed the re-programming of the flax hypocotyl cells for adventitious root formation, and this depended on the exogenous auxin concentration.
Hydrogen peroxide caused a higher accumulation of endogenous IAA Next, we examined the levels of free IAA in the NAA- and H2O2treated flax hypocotyl segments (Fig. 2). The higher concentration of external NAA enhanced the accumulation of endogenous free IAA in the segments compared with those treated with the lower dose of NAA (Fig. 2). Supplementing NAA-containing media with H2O2 increased the endogenous IAA level in the case of the lower NAA concentration, while no substantial IAA increase was observed in the case of the higher external NAA dose. These results showed that stimulation of adventitious root formation in flax hypocotyl segments by NAA, H2O2 or both was positively correlated with higher levels of endogenous free IAA. In addition, these results revealed that H2O2 has the capacity to induce IAA accumulation in flax hypocotyls, which can be used for future biotechnological applications.
The localization and accumulation of H2O2 is positively correlated with adventitious root formation
FIGURE 1
Evaluation of adventitious root formation on flax hypocotyl segments preincubated for 2 days in NAA and H2O2 and subsequently grown on MS media for 5 days. Overviews of flax hypocotyl segments pre-incubated in liquid ½ MS media containing (a) 0.5 mg l1 NAA, (b) 0.5 mg l1 NAA and 100 mM H2O2, (c) 1 mg l1 NAA or (d) 1 mg l1 NAA and 100 mM H2O2. Bars represent 1 cm. (e) Graph showing quantification of the number of hypocotyl segments with adventitious roots per 50 segments. (f) Graph depicting the
Next, we aimed to localize ROS accumulation in the hypocotyl segments using histochemical methods and examined their accumulation in response to external NAA and H2O2. To test the viability of the segments after the NAA and H2O2 treatments, we stained the hypocotyl segments with FDA, a marker of cell viability. This observation showed that the treatments did not affect the viability of the cells in the segments. The distribution of the dye was equal on both cutting edges of the segments (Fig. 3a–d). The staining of the hypocotyl segments with NBT (specific for the superoxide radical) did not show substantial differences among treatments. NBT stained mainly the cutting edges of the hypocotyl segments. Similar staining patterns were detected in the case of H2DCFDA and o-dianisidine. We observed slightly stronger NBT staining in response to 1 mg l1 NAA compared with the lower concentration of this compound (Fig. 4a, c). The combined treatment of 1 mg l1 NAA and H2O2 abolished the NBT staining of the hypocotyl segments (Fig. 4d). The H2DCFDA fluorescence quantification of the adventitious root number per 50 segments. Different letters above the bars in (e) and (f ) indicate significant differences at p 0.05 based on Student’s t-test.
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Levels of endogenous free IAA in flax hypocotyl segments incubated in liquid ½ MS media containing 0.5 mg l1 NAA, 0.5 mg l1 NAA and 100 mM H2O2, 1 mg l1 NAA or 1 mg l1 NAA and 100 mM H2O2 for 2 days. Different letters above the bars indicate significant differences at p 0.05 based on Student’s t-test.
FIGURE 4
FIGURE 3
The evaluation of cell viability using fluorescein diacetate (FDA) in flax hypocotyl segments treated with NAA and H2O2. (a)–(d) Flax hypocotyl segments incubated in liquid ½ MS media containing: (a) 0.5 mg l1 NAA, (b) 0.5 mg l1 NAA and 100 mM H2O2, (c) 1 mg l1 NAA or (d) 1 mg l1 NAA and 100 mM H2O2 for 2 days and subsequently stained with FDA.
signal increased in response to H2O2 combined with 0.5 mg l1 NAA compared with NAA alone, suggesting that the H2DCFDA was sensitive to the exogenous H2O2 treatment. In addition, the H2DCFDA signal was increased in response to the higher dose of exogenous NAA, suggesting increased accumulation of ROS. In the case of H2DCFDA, however, the fluorescence signal intensity differed between the edges of the hypocotyl segments with different capacities for adventitious root formation. This indicates that ROS production was positively correlated with adventitious root formation. Such a difference was not observed in the case of NBT staining, indicating that superoxide did not contribute to the adventitious root formation.
Enzymatic antioxidant defense is affected by exogenous auxin and H2O2 Next, we examined the activities of superoxide dismutase, guaiacol peroxidase and catalase to determine whether the antioxidant 4
Histochemical localization of ROS and peroxidases in flax hypocotyl segments treated with (a, e, i) 0.5 mg l1 NAA (b, f, j) 0.5 mg l1 NAA and 100 mM H2O2, (c, g, k) 1 mg l1 NAA or (d, h, l) 1 mg l1 NAA and 100 mM H2O2 for 2 days. (a)–(d) Histochemical staining of superoxide in flax hypocotyl segments using NBT; (e)–(h) fluorescent staining of ROS in hypocotyl segments using H2DCFDA; (i)–(l) histochemical staining of peroxidases in flax hypocotyl segments using o-dianisidine. Bar represents 2 mm.
enzymes in flax hypocotyls respond to external NAA and H2O2. We found that SOD activity in the hypocotyl segments did not change significantly after the addition of H2O2 to the incubation media (Fig. 5a). However, SOD activity significantly decreased in the response to the higher exogenous NAA concentration. A different pattern was observed in the case of the antioxidant enzymes decomposing H2O2. Guaiacol peroxidase (GPX) was activated by H2O2 at the lower exogenous NAA concentration but inhibited at the higher NAA concentration (Fig. 5b). In contrast, catalase showed increased activity at the higher NAA concentration, whereas it remained unaltered when H2O2 was added to the media containing 0.5 mg l1 NAA (Fig. 5c). Notably, the catalase activity was also increased in hypocotyls exposed to 1 mg l1 NAA together with H2O2. These results revealed that enzymatic antioxidant defense occurred in response to exogenous NAA and/or H2O2. Peroxidase was activated by external H2O2, while catalase activity was stimulated by higher external NAA levels. In addition, we also took advantage of the histochemical staining of peroxidase activity in the flax hypocotyl segments by using o-dianisidine as a
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substrate (Fig. 4 i–l). The peroxidase activity showed polar localization, which was similar to the H2DCFDA fluorescent staining pattern. This implies that peroxidase might be involved in the scavenging of H2O2 generated by endogenous auxin.
In addition to the role of ROS in stress response and signalling, emphasis has also been placed on their role in developmental processes. For example, ROS stimulate cell division in alfalfa leaf protoplasts [4,22], mediate root hair elongation [6], control cell elongation in maize roots [5], regulate the elongation of the primary root of Arabidopsis and promote the formation of lateral roots in cucumber [8,9]. All of these processes are very important for the improvement of growth performance and crop yield. We report that H2O2 positively affects the developmental and biochemical events leading to adventitious root formation from flax hypocotyls. This contributes to the above-mentioned evidence that ROS are important modulators of plant developmental processes and they might be exploited in biotechnological crop improvement.
Positive effect of H2O2 on morphogenetic reprogramming depends on endogenous auxin level Important for biotechnological applications, the simultaneous exposure of flax hypocotyl segments to H2O2 and 0.5 mg l1 NAA enhanced the formation of adventitious roots compared with NAA alone. This indicates the activation of cell division in response to H2O2, which is consistent with previously published data [22]. At the higher exogenous NAA level, H2O2 inhibited the formation of the adventitious roots. It was also reported that ROS have deleterious effects on adventitious root formation when applied without auxin [8]. These data suggest that balanced homeostasis between H2O2 and auxin is necessary to achieve a positive effect of ROS on adventitious root formation. Interestingly, the formation of adventitious roots was positively correlated with endogenous IAA levels, regardless of exogenous H2O2 treatment. This shows that the external application of H2O2 could positively regulate auxin signalling or transport in flax hypocotyl segments. A previous genomic study showed transient early suppression of auxin signalling and subsequent recovery after 24 h by ozone-induced apoplastic ROS in Arabidopsis [42]. The ROS-induced attenuation of auxin signalling was recently explained by oxidative IAA catabolism by ROS produced in response to IAA accumulation [43]. In our system, the hypocotyl segments were simultaneously supplied with H2O2 and external NAA, which likely maintained the intracellular pool of active (non-oxidized) auxin. Under these conditions, H2O2 appears to positively affect adventitious root formation. FIGURE 5
Characterization of enzymatic antioxidant defense in flax hypocotyl segments treated with NAA and H2O2. Graphs depict activity of superoxide dismutase (SOD) (a), catalase (b) and guaiacol peroxidase (c) in flax hypocotyl segments incubated in liquid ½ MS media containing 0.5 mg l1 NAA, 0.5 mg l1 NAA and 100 mM H2O2, 1 mg l1 NAA or 1 mg l1 NAA and 100 mM H2O2 for 2 days. Different letters above the bars indicate significant differences at p 0.05 based on Student’s t-test.
Adventitious root formation likely depends on the polar auxin gradient Adventitious roots regularly formed only on one of the cutting edges of the hypocotyl segments. Considering the basipetal auxin flow in hypocotyls, we assume that adventitious roots formed in areas with endogenous auxin accumulation. Similar results were obtained for hypocotyl explants of Mesembryanthemum crystallinum, which formed adventitious roots only in their basal parts [44]. Interestingly, neither the elevated level of exogenous auxin
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Discussion
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nor the addition of H2O2 caused adventitious root formation at the apical end of the hypocotyl segments. This indicates that polar localization of endogenous auxin is required for hypocotyl reprogramming towards adventitious root formation. Such differential auxin accumulation at the two cutting edges of the hypocotyl segments was positively correlated with different localization of H2O2 in the segments. On the other hand, it was not related to the superoxide localization. Previously, it was reported that H2O2 and superoxide display distinct accumulation and roles in Arabidopsis root development [45]. Superoxide was predominantly located in the apoplast of the cell elongation zone, whereas H2O2 accumulated in the differentiation zone and in the cell wall of root hairs. Superoxide appeared to promote, while H2O2 supressed cell elongation and root hair formation [45]. Our results emphasize the role of H2O2, but not superoxide in adventitious root formation from flax hypocotyl segments, and thus provide new information on the versatility of ROS developmental functions in this biotechnologically important crop.
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to exogenous NAA and H2O2. Enhanced levels of H2O2 in response to NAA alone or combined with H2O2 clearly necessitated an induction of H2O2-scavenging enzymes in the flax hypocotyl segments. Different enzymes might be involved in H2O2 removal, depending on external treatment. In the flax hypocotyls, catalase activity was induced by 0.5 mg l1 of exogenous NAA while peroxidases responded to combined H2O2 and 0.5 mg l1 NAA treatment. Moreover, histochemical staining of peroxidase activity by o-dianisidine showed polar localization in the NAA- and H2O2-treated flax hypocotyl segments. Among other roles, peroxidases are involved in cell wall loosening through the generation of hydroxyl radicals from superoxide [10]. Our results support the role of peroxidases in H2O2 removal, mainly after exogenous H2O2 application, and the polar localization of peroxidase activity in hypocotyl segments. Moreover, the latter might be related to the presumably polar localization of endogenous auxin.
Uncited references Enzymatic antioxidant defense during adventitious root formation
[38,46].
ROS are controlled by multiple enzymatic or non-enzymatic
Acknowledgements
Q3 antioxidant mechanisms [24,47]. Very little is known about the
control of ROS levels during developmental processes. Our results point to distinct changes in flax antioxidant enzymes in response
This work was supported by the grant No. LO1204 (Sustainable development of research in the Centre of the Region Hana´) from the National Program of Sustainability I, MEYS. Q5
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Research Paper
New Biotechnology Volume 00, Number 00 February 2016
