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Ethylene and ABA production in germinating seeds during allelopathy stress

Renata Bogatek, Krystyna Oracz and Agnieszka Gniazdowska

Warsaw Agricultural University, Nowoursynowska 159, 02-776 Warsaw, Poland, www.sggw.waw.pl Email agniazdowska@alpha.sggw.waw.pl

Abstract

Sunflower (Heliantus annuus L.) leaves extract inhibited germination of mustard (Sinapis alba) seeds. It was accompanied by increased membrane leakage and MDA concentration. These effects were correlated with the enhanced H2O2 production indicating induction of secondary oxidative stress. Allelopathy stress increased ABA level in seeds, whereas ethylene production was inhibited almost completely in prolonged allelopathic stress. Even the addition of exogenous ACC did not significantly enhanced ethylene production. Moreover ACC oxidase activity determined in allelopathy stressed seeds decreased with extended allelopathy treatment. Addition of sunflower extract to assay mixture did not affect ACO activity in control seeds, suggesting indirect effect of allelochemicals to ACO.

Media summary

Increased level of ABA accompanied by decreased ethylene emission and ACO activity was detected in non germinating mustard seeds during allelopathy stress.

Key Words

ABA, ethylene, oxidative stress, membrane deterioration, H2O2 concentration

Introduction

Sunflower allelochemicals are known to inhibit or delay germination of many seeds, but the knowledge of the mechanism of their action is still fragmentary. It was reported that allelochemicals may induce secondary, oxidative stress, which is manifested as increased production of reactive oxygen species (ROS) and activation of cellular antioxidant system (Bais et al. 2003; Romero-Romero et al. 2005). Ethylene and abscisic acid (ABA) are so-called stress hormones, which production is enhanced in response to many stresses (Chinnusamy et al. 2004), they are also involved in regulation of seed germination (Finkelstein et al. 2002). Ethylene stimulates germination of many seeds (Kępczyński and Kępczyńska 1997). Its action appears to be limited to an early step of germination (Nascimento 2003), before visible germination, since a peak of ethylene production is correlated with radicle protrusion (Kępczyński and Kępczyńska 1997). Its interaction with ABA is suggested due to the fact that ethylene releases the inhibitory effect of ABA during seed germination, therefore it was proposed that ethylene might act as negative regulator of ABA during germination (Beaudoin et al. 2002; Ghassemian et al. 2002). In germinating seeds ethylene is produced almost exclusively from 1-aminocyclopropane-1-carboxylic acid (ACC) that is synthesized de novo. Endogenous ACC stimulates the germination and reduces the inhibitory effect on germination caused by some stresses. This reactions regard to ACC oxidase activity, catalyzing the last step of ethylene biosynthesis pathway.

The aim of our study was to estimate if sunflower allelochemicals may provoke changes in the level of stress hormones: ABA and ethylene in germinating mustard seeds. We correlated also oxidative stress, a result of allelopathic stress, with alteration in ABA concentration and ethylene emission.

Material and methods

Sunflower leaf 10 % (w/v) water extract was prepared as described by Bogatek et al. (2005). Mustard (Sinapis alba) seeds were germinated in the darkness at 20 C (Bogatek et al. 2005).

Plant hormone determination

ABA concentration in seeds was assayed by the method of enzyme-linked immunoabsorbent assay (ELISA) as described by Weiler (1982). Results are expressed as ng/g FW.

Ethylene emission by seeds was measured using gas chromatograph. Results are expressed as nl/g FW/h.

ACC oxidase (ACO) activity

ACO was extracted as described by Petruzzelli et al. (2000) with some modifications. ACO activity was determined by gas chromatography according to Malerba et al. (1995) with some modifications. Results are expressed as nl/g FW/h.

Hydrogen peroxide (H2O2) concentration

H2O2 contents of seeds were determined according to the method described by O’Kane et al. (1996). Results are expressed as μmol/g FW.

Electrolyte leakage

20 seeds were placed in 15 ml distilled water at room temperature (20 C) in darkness. The electric conductivity in the medium was measured with a conductivity meter after 2 hours incubation. Results are expressed as % of total leakage from seeds boiled for 20 min in water.

Malondialdehyde (MDA) measurements

Lipid peroxidation was estimated by measuring spectrophotometrically MDA contents of seeds. MDA determination was carried out as described by Bailly et al. (1996). Results are expressed as pmol /g DW.

All presented data correspond to the means of the values SD from at least 3 independent repetitions.

Results and discussion

Almost all (95%) control mustard seeds germinated within 3 days and after 8 days formed typically etiolated seedlings. Water extract from sunflower leaves (10 % w/v) inhibited germination of mustard seeds. After 8 days of exposure to allelopathics compounds only 5 % of seeds were germinated but they failed to develop into normal seedlings (Bogatek et al. 2005).

Table 1. Electrolyte leakage, MDA and H2O2 concentration in control seeds after 18 hours of imbibition in water or in mustard seeds germinated in the presence of sunflower leaf allelochemicals.

   

Electrolyte leakage
(% total)

MDA concentration
(nmol/gDW)

H2O2 concentration
(μmol/gFW)

Control

0.75 day

9.0 2.00

7.0 0.51

0.5 0.03

Allelopathy

0.75 day

18.5 2.21

6.0 0.52

1.9 0.25

 

2 days

22.2 3.10

8.3 0.81

2.4 0.42

 

4 days

30.0 2.90

16.5 1.00

2.7 0.50

Ion leakage from control mustard seeds after 18 hours of germination process was low (4 %) (Table 1). Sunflower allelochemicals increased electrolyte leakage from seeds. It represented 20 % of total electrolytes just after 18 hours of allelopathy stress and increased continuously during the experiment, reaching 32 % after 4 days. That observations are in agreement with data obtained on cucumber cotyledons treated with dehydrozaluzanin C, causing a rapid plasma membrane leakage (Galindo et al. 1999). Increased electrolyte leakage during seed germination reflects a loss in membrane integrity. This may indicate an inability to maintain coherent membranes, resulting finally in losses in germinability. The increase in membrane permeability in allelopathy stress corresponded well with increased lipid peroxidation determined as the increase in MDA concentration (Table 1). After 4 days of allelopathy stress it was more than double as compared to the control, while the concentration of MDA in control seeds and seedlings was low (for seedlings data not shown) during the whole period of culture. These data support the idea that loss of seed viability is associated with enhanced lipid peroxidation. The similar increased lipid peroxidation in the presence of aqueous leachate of Callicarpa acuminata and Sicyos deppei was detected in tomato roots (Cruz-Ortega et al. 2002; Romero-Romero et al. 2005). Damage of the membrane system of the plant examined at ultrastructural level as well as lipid peroxidation was also observed in cucumber, sorghum, rice and rape seedlings treated by secalonic acid F (Zeng et al. 2001).

It is well known that many environmental stresses, disrupt the cellular homeostasis, enhancing production of reactive oxygen species (ROS), but a correlation between allelochemicals and increase in ROS production has been elucidated only recently (Bais et al. 2003). In seed physiology ROS are usually considered as toxic molecules, the accumulation of which leads to cell injury and disturbances in seed development or germination processes (Bailly 2004). Hydrogen peroxide concentration in control mustard seed was low (0.5 μmol/g FW) (Table 1). After 18 hours of allelopathy treatment it was more than three times higher than in the control and increased progressively (Table 1). Thus, we can conclude that a consequence of allelopathic stress in germinating mustard seeds was the excessive generation of ROS (H2O2) which brings about lipids peroxidation, leading to membrane damage (Scandalios 1993; Foyer et al. 1994). Although activation of the cellular detoxication and antioxidant system is detected rapidly after allelopathy treatment it does not function sufficiently to avoid some cellular damage (Bogatek et al. 2002).

Figure 1. ABA concentration in control mustard seeds germinated in water (□) or in the presence of water extracts of sunflower leaf allelochemicals (■).

Synthesis of two stress hormones was detected in seeds germinating in the presence of sunflower allelochemicals. ABA concentration was more than doubled 18 hours after plant sown under allelopathic conditions compared to the control, and decreased slightly during the prolonged experiment (Figure 1). Similar increase of ABA level in roots of mustard and wheat seedlings in allelopathic stress was also reported by Bernat et al. (2004). The high level of ABA is characteristic for dormant seeds (e.g. dormant apple embryos Bogatek et al. (2003)), therefore we may suspect that in allelopathic stress (prolonged for a few days only) seeds became “artificially dormant”, even though they do not loose viability they do not germinate (Bogatek et al. 2005).

A

B

Figure 2. Ethylene emission (A) and ACO activity determined in the presence of exogenous 0.5μM ACC (B) in control mustard seeds germinated in water (□) or in the presence of sunflower leaf allelochemicals (■).

Ethylene emission in control material was typical for germinating seeds and seedlings. Positive correlation between ethylene concentration, ACO activity and seedling growth parameters was observed (data not shown). Inhibition of ethylene production by seeds subjected to many different stresses (high temperature, osmotic or salinity) was observed (Zapata et al. 2004; Morgan and Drew 1997). The

decreased ethylene emission was detected also in mustard seeds exposed to sunflower allelochemicals (Figure 2A). Exogenous ACC did not significantly increase ethylene production in allelopathy treated seeds, while in control seeds ACC drastically enhanced ethylene production (data not shown). In short period of allelopathy treatment (6 hours) the transient increase in ethylene emission was detected, reflecting probably so-called stress ethylene emission. Prolonged exposition to allelochemicals resulted in inhibition of ethylene biosynthesis, which may be the result of increased membrane deterioration (Table 1).

Ethylene production is restricted by ACC availability and activity of ACC oxidase (ACO). ACO activity correlated well with ethylene emission data. In control seeds its activity increased as the experiment prolonged (data not shown), while in allelopathy treated seeds it was lower than in the control and decreased as the allelopathic stress was extended. ACO activity in allelopathy treated seeds 2 days after sowing was 3 times lower as compared to control (Figure 2B). Additional determinations of ACO activity in seeds germinating on water with allelopathic extract added directly into measurement solution did not effect ethylene emission (data not shown), indicating indirect effect of sunflower allelochemicals on ACO. On the other hand, Prakash et al. (2003) reported that germinating pigeonpea seeds secreted factor(s) having antiethylene-like effects on rice and A. thaliana. Therefore we can not exclude the possibility that sunflower allelopathic extract may include compounds that inhibit ethylene biosynthesis.

Sunflower allelochemical seems to disturb hormonal balance between ABA and ethylene, which is regarded as the factor controlling germination and growth processes.

Conclusions

Inhibition of seed germination by sunflower allelochemicals is correlated with induction of oxidative stress and enhanced membrane lipid peroxidation. These effects result in alteration in plant hormone (ABA and ethylene) content in mustard seeds. Increased level of ABA accompanied by decreased ethylene emission and ACO activity may explain inhibition of mustard seed germination by sunflower allelochemicals.

Acknowledgments

The authors wish to express their thanks to Professor E.W. Weiler from Ruhr University, Bochum, Germany for kindly supplying antibodies and tracer used for ABA assay and to Mrs. M. Dzięcioł for her skilful assistance.

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