Coumarin impairs redox homeostasis in wheat aleurone layers
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Many plant families produce coumarin (COU) and its derivatives as secondary metabolites via the phenylpropanoid biosynthetic pathway. This ubiquitous group of phytochemicals was shown to have diverse physiological effects on cellular, tissue, and organ levels. So far, research dealing with the hormonal like behavior of COU and its interaction with the activity and/or transport of phytohormones is very limited. In the current study, the impact of COU on redox homeostasis in aleurone layers of wheat grains was investigated. Aleurone layers were incubated in either 1000 μM COU or 5 μM gibberellic acid (GA3) alone or in combination with 5 μM abscisic acid (ABA). Results revealed that both COU and GA3 treatments induced the production of α-amylase but inhibited the activities of superoxide dismutase, catalase and ascorbate peroxidase. The downregulation of antioxidant enzymes that is provoked by COU and GA3 was accompanied by significant accumulation of both H2O2 and malondialdehyde. In contrast with the effect of ABA, both COU and GA3 treatments resulted in a significant reduction in cell viability as revealed by trypan blue staining. These results suggest that COU could disrupt the redox balance in aleurone layers through downregulation of the enzymatic antioxidant system. Therefore, the current study provides evidence for the gibberellin like activity of COU.
KeywordsAleurone cells Coumarin Gibberellic acid Abscisic acid Reactive oxygen species Antioxidant enzymes Lipid peroxidation
The aleurone layer of cereal grains is a key player in regulation of reserve weakening during germination because it produces several hydrolases such as amylase, protease and acid phosphatase. The synthesis and secretion of these enzymes from cells of aleurone layer is upregulated by gibberellic acid (GA3) and downregulated by abscisic acid (ABA) (Fath et al. 2001; Lovegrove and Hooley 2000). During the course of seed germination, aleurone cells undergo programmed cell death (PCD) due to the intensive production of reactive oxygen species (ROS), specially H2O2 (Fath et al. 2000; Wu et al. 2014). Beside its production by electron transport chain in the mitochondria, ROS is produced via the activity of NADPH dependent oxidases in aleurone plasma membrane and by the rapid conversion of triglycerides reserve to sucrose (Bethke et al. 2002; Ishibashi et al. 2015). There is evidence that GA3 induces PCD in aleurone through the elevated activity of NADPH dependent oxidases and downregulation of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and ascorbate peroxidase (APX) (Fath et al. 2001; Ishibashi et al. 2015). On the contrary, ABA maintains a redox balance by promoting the activity of antioxidant enzymes (Bethke et al. 1999, 2002). Moreover, ABA, but not GA3, downregulates PCD in aleurone cells via inhibiting the synthesis of some nucleases and cysteine proteases (Fath et al. 2000).
Coumarins represent a ubiquitous group of plant secondary metabolites that mainly arise from the general phenylpropanoid pathway (Brown and Zobel 1990). Despite some enzymes and genes involved in their biosynthesis being sufficiently documented, the exact biosynthetic pathway for coumarin production in plants is not fully characterized (Bourgaud et al. 2006). Coumarins are normally found in roots, stems, leaves, flowers, fruits and seeds of many plants (Matos et al. 2015), however their accumulation is induced by biotic and abiotic stresses suggesting a role for these phytochemicals in stress tolerance (Al-Wakeel et al. 2013; Cabello-Hurtado et al. 1998; Matern et al. 1999). The core structure of coumarins, coumarin (2H-chromen-2-one; COU), has several physiological implications on cellular, tissue and organ levels. In dose and species dependent manner, COU influences germination and seedling establishment (Abenavoli et al. 2006; Aliotta et al. 1993; Pergo et al. 2008). Morphology and ultrastructure of plant organs have also been reported to be affected in response to COU treatment (Abenavoli et al. 2004; Kupidlowska et al. 1994). In addition, COU could affect the rate of plant metabolic activities, induce qualitative and quantitative changes in the biochemical constituents and interfere with activities and/or transport of auxins (Abenavoli et al. 2001a, b; Lupini et al. 2014; Macias et al. 1999; Saleh et al. 2015; Saleh and Madany 2015). To the best of our knowledge, there is no evidence that COU is produced in aleurone layers, however, other forms of phenolic compounds, principally ferulic acids and its derivatives, have been detected in aleurone cells and other parts of cereal grains (Barron et al. 2007; Hernandez et al. 2011).
Previous work of ours and others has investigated some growth regulating activities for COU, under both normal and stressful conditions (Al-Wakeel et al. 2013; Saleh et al. 2015; Saleh and Madany 2015; Tartoura et al. 2004). In this regard, our recent study, Saleh and Abu El-Soud (2015), points to some gibberellin like activities for COU. We have reported that COU could stimulate amylase synthesis and secretion from endospermic (de-embryonated) wheat half-grains that peaks at 1000 µM. Moreover, COU at concentrations less than 350 µM has promoted the elongation of second leaf sheath of wheat seedling. However, the impact of COU on redox homeostasis in aleurone layers has not been investigated. Therefore, the current work was to reinforce the gibberellin-like activity of COU by considering the antioxidant capacity and redox status in aleurone cells. To achieve this aim, the effect of both COU and GA3, alone or in combination with ABA, on activity of antioxidant enzymes, accumulation of H2O2 and lipid peroxidation was assessed. Aleurone layers were used as a model system not only for being a homogenous population of non-dividing cells, but also because they are free from bioactive gibberellins and/or COU, therefore the observed effect could be ascribed to exogenous COU.
Materials and methods
Preparation of aleurone layers
Aleurone layers of wheat (Triticum aestivum L.) grains were prepared according to the method outlined by Fath et al. (2001). Briefly, wheat grains were de-embryonated by removal of the embryo containing part to give 3 mm de-embryonated halves. The grain halves were surface sterilized with 5.0% commercial bleach for 20 min. Bleach solution was then replaced with sterilized distilled water. The grain halves were swirled well and the sterilized water was renewed five times. The de-embryonated half-grains were imbibed in sterile water at 25 °C for 3 days. Thereafter, aleurone layers were isolated by gentle removal of the starchy endosperm under aseptic conditions.
Fifty aleurone layers were incubated at 25 °C for 3 days in 10 mL of incubation medium (20 mM CaCl2 in 20 mM sodium acetate buffer, pH 5.0) alone, to provide a control, or supplemented with COU, and/or ABA stock solutions. Five treatments were tested comprising 1000 μM COU, 5 μM GA3, 5 μM ABA, 1000 μM COU + 5 μM ABA and 5 μM GA3 + 5 μM ABA. The concentration of COU was selected based on a dose response curve for amylase production (Saleh and Abu El-Soud 2015). GA3, ABA and COU were purchased from Sigma-Aldrich (St. Louis, MO).
Determination of cell viability
Staining of dead cells was performed using the method described by Joo et al. (2005). Five aleurone layers from each treatment were transferred to a small plastic tube and immersed with 0.4% trypan blue solution. The tubes were incubated in a boiling water bath for 1 min, then left for 3 h in the staining solution. Thereafter, the aleurone layers were de-stained with chloral hydrate solution. Layers were examined with a light microscope provided with a digital camera (Optika, Italy) using a 20× objective lens. Three different aleurone layers were observed for each treatment and five randomly selected fields from each aleurone layer were considered. The percent of dead cells was calculated by counting the blue colored cells relative to the total number of cells.
Determination of H2O2 and MDA content
The protocol outlined by Sergiev et al. (1997) was used for colorimetric determination of H2O2. Liquid nitrogen frozen aleurone layers were homogenized in 1% (w/v) trichloroacetic acid (TCA) and then the homogenate was centrifuged for 20 min at 12,000g and 4 °C. One-half mL of the supernatant was mixed with 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI (prepared in 10 mM potassium phosphate buffer, pH 7.0). After 60 min incubation at 25 °C, the absorbance of the mixture was measured at 390 nm. The concentration of H2O2 in each sample was calculated using a standard curve of H2O2 and expressed as nmol g FW−1.
Malondialdehyde (MDA) content was assayed as an indication of lipid peroxidation. The extraction and determination of MDA was performed as described by Heath and Packer (1968) with some modification. Twenty aleurone layers were grinded under liquid nitrogen and then homogenized in 2 mL phosphate buffer (pH 7.8). After centrifugation for 20 min at 4000g and 4 °C, 1 mL of each extract was mixed with 2 mL of 0.6% thiobarbituric acid (TBA) and incubated for 15 min in boiling water bath. After centrifugation for 10 min at 4000g, absorbance of the clear supernatant was read at 600, 532 and 450 nm. MDA content was calculated using the formula provided by Zheng et al. (2012) and expressed as nmol g FW−1.
Assay of α-amylase
At the end of the experiment, aleurone layers were homogenized in the incubation medium and the homogenate was centrifuged for 20 min at 12,000g and 4 °C. The supernatant was heated at 70 °C for denaturation of the heat-labile β-amylase. For determination of α-amylase activity, 500 µL of the supernatant was mixed with 500 µL of 0.5% soluble starch prepared in 50 mM acetate buffer (pH 5.5) containing 20 mM CaCl2. The mixture was left to react for 30 min at 40 °C before it was terminated by HgCl2. The produced reducing sugars were estimated by the Nelson’s method (Clark and Switzer 1977). Maltose was used to construct a standard curve for reducing end sugars. One unit of amylase defined as the production of 1 µg maltose per h. Soluble protein content was estimated according to the Folin-Lowry method adopted by Hartree (1972).
Assays of antioxidant enzymes
For extraction of antioxidant enzymes, 20 frozen aleurone layers were homogenized in 200 mM pre-chilled phosphate buffer (pH 7.8) supplemented with 0.1 mM EDTA, 1% PVP and 0.2 mM ascorbic acid (AsA). The activities of SOD, CAT and APX were assayed according to Garcı́a-Limones et al. (2002). One unit of SOD (U) was defined as the amount of enzyme required to inhibit the reduction rate of nitro blue tetrazolium chloride (NBT) by 50%. One unit (U) of CAT or APX was defined as the amount of enzyme required to decompose 1 µmol H2O2 or AsA min−1, respectively.
Experiments were carried out following a randomized complete block design. Data normality and the homogeneity of variances were checked using the Kolmogorov–Smirnov test and Levene’s test, respectively. All the data was subjected to one-way Analysis of Variance (ANOVA). Duncan’s Multiple Range Test (P ≤ 0.05) was carried out as the post-hoc test for mean separations. Where needed, data were transformed by log (x + 1) before statistical analysis. All statistical tests were performed using the computer program PASW statistics 18.0 (SPSS Inc., Chicago, IL, USA).
Results and discussion
COU and GA3 induce α-amylase production in wheat aleurone cells
The synthesis and secretion of hydrolases from aleurone cells of cereals is upregulated by GA3 and could be blocked by ABA (Fath et al. 2001; Lovegrove and Hooley 2000). Here the impact of COU, GA3 and/or ABA on the production of amylase from wheat aleurone was investigated. Results represented in Fig. 1 show that COU resembled GA3 in motivating the synthesis of α-amylase in aleurone cells. However, the incorporation of ABA along with COU or GA3 markedly reduced the synthesis of α-amylase. Moreover, negligible activity was detected in both control and ABA alone treatment. In accordance with the present results, we have previously investigated the ability of COU to stimulate the synthesis and secretion of amylase from de-embryonated wheat grains that peaks at 16 h with maximum induction by 1000 µM COU (Saleh and Abu El-Soud 2015). Similarly, a plethora of research has investigated and elucidated the mechanism of GA3-induced amylase production from cereal aleurone and the antagonistic effect of ABA (Eastmond and Jones 2005; Ishibashi et al. 2015; Jacobson and Corcoran 1977; Lovegrove and Hooley 2000; Wu et al. 2014).
Effect of COU, GA3 and ABA on the activity of antioxidant enzymes
Activity of antioxidant enzymes such as CAT, SOD and APX is a fundamental factor for maintaining redox homeostasis in aleurone cells by keeping the level of ROS under control (Bethke et al. 2002; Zhang et al. 2015). In the present study, the activities of CAT, SOD and APX were estimated to reveal the influence of COU and GA3 alone or in combination with ABA on the efficiency of the antioxidant pool and redox status in aleurone. It is obvious from data presented in Fig. 2 that aleurone layers treated with either COU or GA3 alone contained significant lower activities of antioxidant enzymes, as compared with the control. The inhibitory effect of GA3 on the activity of antioxidant enzymes was more pronounced than that of COU. By contrast, ABA alone treatment stimulated the activity of antioxidant enzymes, however the stimulatory effect of ABA was more evident on CAT activity than on SOD or APX. Moreover, results showed that integration of ABA has antagonized the effect of both COU and GA3. In this regard, previous studies revealed that GA3 treatment brought about an inhibition of SOD, CAT and APX activities in cereal aleurone, meanwhile ABA had a stimulatory effect (Beligni et al. 2002; Fath et al. 2001; Wu et al. 2014). However there is evidence that GA3 treatment caused enhancements in the activity of antioxidant enzymes when applied to mature seedlings (Saeidi-sar et al. 2007; Siddiqui et al. 2011). In the same context, imbibition of durum wheat grains in 1000 µM COU for 36 h resulted in a significant inhibition in APX and SOD activities (Abenavoli et al. 2006). While, Mahmood et al. (2013) recorded an enhancement in the activity of both SOD and CAT in rice seedlings exposed to 400 µM COU for 7 days. Thus, the impact of COU on the activity of antioxidant enzymes seems to be dependent on the dose applied and the developmental stage at the time of application.
Effect of COU, GA3 and ABA on H2O2 and MDA accumulation
In plants, ROS has a manifold role in hormonal signaling, growth regulation and responses to different types of stresses (Ishibashi et al. 2012). H2O2 is the major ROS that is known to be accumulated in aleurone cells (Fath et al. 2000; Wu et al. 2014). In the present study, the impact of COU on accumulation of H2O2 in wheat aleurone layers was investigated in comparison with GA3, in either presence or absence of ABA (Fig. 3a). Both COU and GA3 induced a significant accumulation of H2O2 in the treated aleurone layers by 4× and 5× fold, respectively, relative to the control. However, the application of ABA along with either COU or GA3 reduced the amount of H2O2 to approximately half of its value relative to the COU and GA3 alone treatments. The lowest values of H2O2 content were detected in aleurone layers treated with ABA alone. Similarly, it was investigated that exogenous GA3 caused marked accumulation of H2O2 in de-embryonated grains, isolated aleurone layers and aleurone protoplasts of wheat and barley (Ishibashi et al. 2012; Wu et al. 2014). The authors also reported that GA-induced H2O2 accumulation is restrained by ABA.
Aleurone layer cells contain considerable amounts of triglycerides reserve that undergo gluconeogenesis to provide sugars for the growing embryo. This process is hormonally regulated and starts with the hydrolysis of triglycerides to fatty acids by lipase. The fatty acids are then oxidized by lipoxygenase. The produced lipid hydroperoxides are further metabolized with the associate production of a variety of byproducts and ROS (Bethke et al. 2002; Eastmond and Jones 2005). In the current study, we have assessed the accumulation of MDA in aleurone layers as an indicator of membrane damage and lipid peroxidation in cells. Data presented in Fig. 3b indicate a significant reduction in the content of MDA in ABA alone treated aleurone layers relative to the control. On the contrary, both COU and GA3 treatments have induced remarkable accumulations of MDA. Moreover, application of ABA antagonized both COU- and GA3-induced MDA accumulation (Fig. 3b). These results support the existing body of information that GA3, but not ABA, promotes the conversion of triglycerides to sugars in aleurone of barley and wheat (Barba-Espín et al. 2014; Doig et al. 1975; Eastmond and Jones 2005). In the same context, previous study of Pergo et al. (2008) indicated that 25 and 50 μM COU promoted the activity of lipoxygenase in root tissues of Bidens pilosa L. seedlings. They claimed that COU treatment did not cause accumulation of MDA, while significantly increasing the level of conjugated dienes. They hypothesized that MDA is further oxidized or metabolized. Moreover, Mahmood et al. (2013) reported that different concentrations of COU, 100–400 µM, significantly stimulate the accumulation of MDA in the root of rice seedlings.
Impact of COU, GA3 and ABA on aleurone cell viability
Trypan blue staining was employed to observe cell viability in aleurone layers after 3 days of incubation in buffered solution alone or with COU or GA3 and/or ABA. Trypan blue is excluded by intact membranes of viable cells. Unlike viable cells, dead cells take up trypan blue through their damaged membranes and are therefore stained blue (Tran et al. 2011). The present results revealed that GA3 and COU treated aleurone layers showed abundant cell death (Fig. 4). On the other hand, ABA treatment maintained about 99% viability as compared with the control aleurone layers that have only 90% viable cells. The incorporation of ABA in the incubation medium along with COU or GA3 markedly reduced the number of dead cells, as compared with COU or GA3 alone treatments, however the protective effect of ABA was more pronounced in COU treated aleurone layers. In this regard, several studies have indicated that aleurone layers treated with GA3 exhibited a progressive increase in the number of cells undergoing PCD until they die completely 48 h after treatment (Beligni et al. 2002; Fath et al. 2001; Wu et al. 2014). The authors reported also that ABA could prevent cell death during the incubation period. Moreover, it is well known that GA3 and ABA act antagonistically regarding the process of PCD (Bethke et al. 2002). COU is reported to exert a cytostatic effect on cultured carrot cells with obvious cell necrosis and medium browning in few days (Abenavoli et al. 2003).
Results presented here indicate the potentiality of COU to impair the redox homeostasis in cells of wheat aleurone. This conclusion is supported by the elevated level of H2O2, the major ROS that is known to be accumulated in aleurone cells undergoing PCD, and the decreased activities of the antioxidant enzymes CAT, SOD and APX. Therefore, current study provides further evidence for the gibberellin like activity of COU. However, further detailed investigations are required to understand the biochemical and molecular aspects underlying the proposed gibberellin like behavior of COU.
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