Journal of Plant Research

, Volume 131, Issue 1, pp 157–163 | Cite as

Coumarin impairs redox homeostasis in wheat aleurone layers

  • Ahmed M. Saleh
  • Rashad Kebeish
Regular Paper


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.


Aleurone 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.

Incubation 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.

Statistical analysis

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).

Fig. 1

Effect of COU, GA3 and ABA on production of α-amylase by wheat aleurone layers. Layers were incubated for 3 days in 1000 μM COU or 5 μM GA3 in either absence (−ABA) or presence (+ABA) of 5 μM ABA. Each value is the mean of 3 independent replicates and vertical bars represent the standard error. Same letters on bars indicate no significant difference (P < 0.05) as analyzed by Duncan test

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.

Fig. 2

Effect of COU, GA3 and ABA on the activities of SOD (a), CAT (b) and APX (c) in wheat aleurone layers. Layers were incubated for 3 days in 1000 μM COU or 5 μM GA3 in either absence (−ABA) or presence (+ABA) of 5 μM ABA. Each value is the mean of 3 independent replicates and vertical bars represent the standard error. Same letters on bars indicate no significant difference (P < 0.05) as analyzed by Duncan test

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.

Fig. 3

Effect of COU, GA3 and ABA on accumulation of H2O2 (a) and MDA (b) in wheat aleurone layers. Layers were incubated for 3 days in 1000 μM COU or 5 μM GA3 in either absence (−ABA) or presence (+ABA) of 5 μM ABA. Each value is the mean of 3 independent replicates and vertical bars represent the standard error. Same letters on bars indicate no significant difference (P < 0.05) as analyzed by Duncan test

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).

Fig. 4

Effect of COU, GA3 and ABA on cell viability in wheat aleurone layers. Layers were incubated for 3 days in 1000 μM COU or 5 μM GA3 in either absence (−ABA) or presence (+ABA) of 5 μM ABA. Trypan blue staining was used to discriminate between dead (appeared blue) and live cells. Percentages of dead cells (a) and representative images of barley aleurone layers (b) are shown. Scale bars on images = 100 μm. Each value is the mean of 3 independent replicates and vertical bars represent the standard error. Same letters on bars indicate no significant difference (P < 0.05) as analyzed by Duncan test


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.


  1. Abenavoli MR, De Santis C, Sidari M et al (2001a) Influence of coumarin on the net nitrate uptake in durum wheat. New Phytol 150:619–627CrossRefGoogle Scholar
  2. Abenavoli MR, Sorgonà A, Muscolo A (2001b) Morphophysiological changes in tissue culture of Petunia hybridain response to the allelochemical coumarin. Allelopath J 8:171–177Google Scholar
  3. Abenavoli MR, Sorgonà A, Sidari M et al (2003) Coumarin inhibits the growth of carrot (Daucus carota L. cv. Saint Valery) cells in suspension culture. J Plant Physiol 160:227–237CrossRefPubMedGoogle Scholar
  4. Abenavoli MR, Sorgonà A, Albano S, Cacco G (2004) Coumarin differentially affects the morphology of different root types of maize seedlings. J Chem Ecol 30:1871–1883CrossRefPubMedGoogle Scholar
  5. Abenavoli MR, Cacco G, Sorgona A et al (2006) The inhibitory effects of coumarin on the germination of durum wheat (Triticum turgidum ssp. durum, CV. Simeto) seeds. J Chem Ecol 32:489–506CrossRefPubMedGoogle Scholar
  6. Aliotta G, Cafiero G, Fiorentino A, Strumia S (1993) Inhibition of radish germination and root growth by coumarin and phenylpropanoids. J Chem Ecol 19:175–183CrossRefPubMedGoogle Scholar
  7. Al-Wakeel SM, Gabr MMA, Abu-El-Soud WM, Saleh AM (2013) Coumarin and salicylic acid activate resistance to Macrophomina phaseolina in Helianthus annuus. Acta Agron Hung 61:23–35CrossRefGoogle Scholar
  8. Barba-Espín G, Dedvisitsakul P, Hägglund P et al (2014) Gibberellic acid-induced aleurone layers responding to heat shock or tunicamycin provide insight into the N-glycoproteome, protein secretion, and endoplasmic reticulum stress. Plant Physiol 164:951–965CrossRefPubMedGoogle Scholar
  9. Barron C, Surget A, Rouau X (2007) Relative amounts of tissues in mature wheat (Triticum aestivum L.) grain and their carbohydrate and phenolic acid composition. J Cereal Sci 45:88–96CrossRefGoogle Scholar
  10. Beligni MV, Fath A, Bethke PC et al (2002) Nitric oxide acts as an antioxidant and delays programmed cell death in barley aleurone layers. Plant Physiol 129:1642–1650CrossRefPubMedPubMedCentralGoogle Scholar
  11. Bethke PC, Lonsdale J, Fath A, Jones R (1999) Hormonally regulated programmed cell death in barley aleurone cells. Plant Cell 11:1033–1046CrossRefPubMedPubMedCentralGoogle Scholar
  12. Bethke PC, Fath A, Spiegel YN et al (2002) Abscisic acid, gibberellin and cell viability in cereal aleurone. Euphytica 126:3–11CrossRefGoogle Scholar
  13. Bourgaud F, Hehn A, Larbat R et al (2006) Biosynthesis of coumarins in plants: a major pathway still to be unravelled for cytochrome P450 enzymes. Phytochem Rev 5:293–308CrossRefGoogle Scholar
  14. Brown SA, Zobel A (1990) Biosynthesis and distribution of coumarins in the plant. Proceedings of the conference “Coumarins: Research and Applications”. PaduaGoogle Scholar
  15. Cabello-Hurtado F, Durst F, Jorrin JV, Werck-Reichhart D (1998) Coumarins in Helianthus tuberosus: Characterization, induced accumulation and biosynthesis. Phytochemistry 49:1029–1036CrossRefGoogle Scholar
  16. Clark JM, Switzer RL (1977) Experimental Biochemistry, 2nd edn. W.H. Freeman & Company, San FranciscoGoogle Scholar
  17. Doig RI, Colborne AJ, Morris G, Laidman DL (1975) The induction of glyoxysomal enzyme activities in the aleurone cells of germinating wheat. J Exp Bot 26:387–398CrossRefGoogle Scholar
  18. Eastmond PJ, Jones RL (2005) Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar-dependent and gibberellin action involves SLENDER1 but not GAMYB. Plant J 44:483–493CrossRefPubMedGoogle Scholar
  19. Fath A, Bethke P, Lonsdale J et al (2000) Programmed cell death in cereal aleurone. Plant Mol Biol 44:255–266CrossRefPubMedGoogle Scholar
  20. Fath A, Bethke PC, Jones RL (2001) Enzymes that scavenge reactive oxygen species are down-regulated prior to gibberellic acid-induced programmed cell death in barley aleurone. Plant Physiol 126:156–166CrossRefPubMedPubMedCentralGoogle Scholar
  21. García-Limones C, Hervás A, Navas-Cortés JA et al (2002) Induction of an antioxidant enzyme system and other oxidative stress markers associated with compatible and incompatible interactions between chickpea (Cicer arietinum L.) and Fusarium oxysporum f. sp.ciceris. Physiol Mol Plant Pathol 61:325–337CrossRefGoogle Scholar
  22. Hartree EF (1972) Determination of protein: a modification of the Lowry method that gives a linear photometric response. Anal Biochem 48:422–427CrossRefPubMedGoogle Scholar
  23. Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189–198CrossRefPubMedGoogle Scholar
  24. Hernandez L, Afonso D, Rodriguez EM, Diaz C (2011) Phenolic compounds in wheat grain cultivars. Plant Foods Hum Nutr 66:408–415CrossRefPubMedGoogle Scholar
  25. Ishibashi Y, Tawaratsumida T, Kondo K et al (2012) Reactive oxygen species are involved in gibberellin/abscisic acid signaling in barley aleurone cells. Plant Physiol 158:1705–1714CrossRefPubMedPubMedCentralGoogle Scholar
  26. Ishibashi Y, Kasa S, Sakamoto M et al (2015) A role for reactive oxygen species produced by NADPH oxidases in the embryo and aleurone cells in barley seed germination. Plos One 10:1–17Google Scholar
  27. Jacobson A, Corcoran MR (1977) Tannins as gibberellin antagonists in the synthesis of α-amylase and acid phosphatase by barley seeds. Plant Physiol 59:129–133CrossRefPubMedPubMedCentralGoogle Scholar
  28. Joo JH, Wang S, Chen JG et al (2005) Different signaling and cell death roles of heterotrimeric G protein a and b subunits in the arabidopsis oxidative stress response to ozone. Plant Cell 17:957–970CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kupidlowska E, Kowalec M, Sulkowski G, Zobel AM (1994) The effect of coumarins on root elongation and ultrastructure of meristematic cell protoplast. Ann Bot 73:525–530CrossRefGoogle Scholar
  30. Lovegrove A, Hooley R (2000) Gibberellin and abscisic acid signalling in aleurone. Trends Plant Sci 5:102–110CrossRefPubMedGoogle Scholar
  31. Lupini A, Araniti F, Sunseri F, Abenavoli MMR (2014) Coumarin interacts with auxin polar transport to modify root system architecture in Arabidopsis thaliana. Plant Growth Regul 72:1–9CrossRefGoogle Scholar
  32. Macias ML, Rojas IS, Mata R, Lotina-Hennsen B (1999) Effect of selected coumarins on spinach chloroplast photosynthesis. J Agric Food Chem 47:2137–2140CrossRefPubMedGoogle Scholar
  33. Mahmood K, Khan MB, Song YY et al (2013) Differential morphological, cytological and biochemical responses of two rice cultivars to coumarin. Allelopath J 31:281–296.Google Scholar
  34. Matern U, Lüer P, Kreusch D (1999) Biosynthesis of coumarins. In: Sankawa U (ed) Comprehensive natural products chemistry, vol 1, polyketides and other secondary metabolites including fatty acids and their derivatives. Pergamon, Oxford, pp 623–637Google Scholar
  35. Matos MJ, Santana L, Uriarte E et al (2015) Coumarins—an important class of phytochemicals. In: Rao AV, Rao LG (eds) Phytochemcals—isolation, characterisation and role in human health. ISBN 978-953-51-2170-1, pp 113–140Google Scholar
  36. Pergo EM, Abrahim D, Soares da Silva PC et al (2008) Bidens pilosa L. exhibits high sensitivity to coumarin in comparison with three other weed species. J Chem Ecol 34:499–507CrossRefPubMedGoogle Scholar
  37. Saeidi-sar S, Khavari-nejad RA, Fahimi H et al (2007) Interactive effects of gibberellin A3 and ascorbic acid on lipid peroxidation and antioxidant enzyme activities in glycine max seedlings under nickel stress 1. Russ J Plant Physiol 54:74–79CrossRefGoogle Scholar
  38. Saleh AM, Abu El-Soud W (2015) Evidence for “gibberellin-like” activity of coumarin. S Afr J Bot 100:51–57.CrossRefGoogle Scholar
  39. Saleh AM, Madany MMY (2015) Coumarin pretreatment alleviates salinity stress in wheat seedlings. Plant Physiol Biochem 88:27–35CrossRefPubMedGoogle Scholar
  40. Saleh AM, Madany MMY, González L (2015) The effect of coumarin application on early growth and some physiological parameters in faba bean (Vicia faba L.). J Plant Growth Regul 34:233–241CrossRefGoogle Scholar
  41. Sergiev I, Alexieva V, Karanov E (1997) Effect of spermine, atrazine and combination between them on some endogenous protective systems and stress markers in plants. C R Acad Bulg Sci 51:121–124Google Scholar
  42. Siddiqui MH, Al-Whaibi MH, Basalah MO (2011) Interactive effect of calcium and gibberellin on nickel tolerance in relation to antioxidant systems in Triticum aestivum L. Protoplasma 248:503–511CrossRefPubMedGoogle Scholar
  43. Tartoura K, da Rocha A, Youssef S (2004) Synergistic interaction between coumarin 1, 2-benzopyrone and indole-3-butyric acid in stimulating adventitious root formation in Vigna radiata (L.) Wilczek cuttings: I. Endogenous free and conjugated IAA and basic isoperoxidases. Plant Growth Regul 42:253–262CrossRefGoogle Scholar
  44. Tran SL, Puhar A, Ngo-Camus M, Ramarao N (2011) Trypan blue dye enters viable cells incubated with the pore-forming toxin HlyII of Bacillus cereus. Plos One 6:2–6Google Scholar
  45. Wu M, Li J, Wang F et al (2014) Cobalt alleviates GA-Induced programmed cell death in wheat aleurone layers via the regulation of H2O2 production and heme oxygenase-1 expression. Int J Mol Sci 15:21155–21178CrossRefPubMedPubMedCentralGoogle Scholar
  46. Zhang YX, Hu K Di, Lv K et al (2015) The hydrogen sulfide donor nahs delays programmed cell death in barley aleurone layers by acting as an antioxidant. Oxid Med Cell Longev 2015:1–11. doi: 10.1155/2015/714756
  47. Zheng YH, Li X, Li YG et al (2012) Contrasting responses of salinity-stressed salt-tolerant and intolerant winter wheat (Triticum aestivum L.) cultivars to ozone pollution. Plant Physiol Biochem 52:169–178CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan KK 2017

Authors and Affiliations

  1. 1.Biology Department, Faculty of Science YanbuTaibah UniversityYanbu El-BahrSaudi Arabia
  2. 2.Department of Botany and Microbiology, Faculty of ScienceCairo UniversityGizaEgypt
  3. 3.Botany Department, Faculty of ScienceZagazig UniversityZagazigEgypt

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