A MECHANISM FOR PROMOTING THE GERMINATION OF ZINNIA ELEGANS

Plant Cell Physiol. 42(3): 286–291 (2001) JSPP © 2001

A Mechanism for Promoting the Germination of Zinnia elegans Seeds by Hydrogen Peroxide Ken’ichi Ogawa1 and Masaki Iwabuchi Research Institute for Biological Sciences Okayama (RIBS), 7549-1 Yoshikawa, Kayou-cho, Okayama 716-1241, Japan ;

constantly exposed to the toxicity of H2O2. Ascorbate peroxidase scavenging H2O2 is localized at the site of H2O2 generation in the plant cell (Asada 1999), and is likely to be microcompartmentalized by organizing scavenging complex of enzymes such as superoxide dismutase and monodehydroascorbate reductase (Ogawa et al. 1995, Asada et al. 1996, Asada 1999). During the biosynthesis of lignin, H2O2 is produced at the site of lignification (Ogawa et al. 1996, Ogawa et al. 1997). Thereby, H2O2 generated is scavenged or utilized at its generation site in plants. If H2O2 is generated at an unexpected site or reaches a level that exceeds the scavenging capacity, it causes oxidative damage to plants. H2O2 impairs the photosynthetic activity of isolated chloroplasts when applied exogenously even at 1 M which is a level having little effect on the activity when generated in chloroplasts (Asada and Takahashi 1987). Nevertheless, exogenously applied H2O2 ameliorates seed germination in many plants (Fontaine et al. 1994, Chien and Lin 1994). This has been explained by the fact that the scavenging activity for H2O2 is high enough, resulting in the production of O2 for mitochondrial respiration. However, an early step of seed germination (dormancy breakage) dispenses with mitochondrial O2 respiration and requires the activation of the oxidative pentose phosphate pathway. Thioredoxin reduction by NADPH produced via the oxidative pentose phosphate pathway allows the mobilization of storage proteins of cereals, leading to germination (Fontaine et al. 1994). H2O2 treatment elevates the GSH level in barley seeds, in which the GSH/ GSSG ratio increases. Since GSH is reduced by NADPH in a reaction that is catalyzed by glutathione reductase, Fontaine et al. (1994) proposed that H2O2 would activate the oxidative pentose pathway, resulting in promotion of seed germination. Another explanation is that H2O2 is helpful in cracking the hard seeds, allowing them to interact with water (Chien and Lin 1994), although the concentration of H2O2 used is high enough to limit the seed germination and seedling growth of the seeds with no hard seed coat. Here we propose a mechanism for the promotion of seed germination by exogenously applied H2O2 in Zinnia elegans. We report the sites of H2O2 generation in the seedling during germination and the antioxidant effects on germination, and discuss the physiological significance of H2O2 in the germination process.

H2O2 promotes seed germination of cereal plants such as barley, wheat and rice, and several mechanisms have been proposed for its action [Naredo et al. (1998) Seed Sci. Technol. 26: 675–689]. We investigated the role of H2O2 in the germination of Zinnia elegans seeds. H2O2 promoted seed germination in a dose-dependent manner as did respiratory inhibitors, indicating that H2O2 itself possibly promotes seed germination rather than O2. Seed germination was promoted by removal of pericarp from seeds or by removal of ethanol-soluble compounds from the seeds with pericarp. The ethanol-soluble compounds suppressed the germination of seeds having no pericarp, and this effect was reversed by H2O2. These findings indicate that oxidation of the germination inhibitor(s) present in the pericarp by H2O2 promotes seed germination. Antioxidants which are derivatives of well-known germination inhibitors suppressed seed germination in a dose-dependent manner, suggesting that, to initiate seed germination, a germination inhibitor(s) should be decomposed by an oxidant such as H2 O2 . Key words: Development — Dormancy breakage — Germination — Hydrogen peroxide — Redox — Zinnia elegans. Abbreviations: GR, Glutathione reductase; MCLA, 2-methyl6-( p-methoxyphenyl)-3,7-dihydroimidazo[1,2-=]pyrazin-3-one; ROS, reactive oxygen species; SHAM, salicylhydroxamic acid.

Introduction H2O2 is a toxic molecule due to its highly oxidative reactivity and long life. In the presence of catalytic metal ions such as iron and copper, it produces hydroxyl radical (·OH) which strongly oxidizes cell components such as membrane lipids and enzymes. It is generated via the disproportionation of O2– or the univalent reduction of O2– by ascorbate, thiols, ferredoxin and manganous ions. It is also generated from the divalent reduction of O2 that is catalyzed by oxidases such as glycolate, glucose and sulfite oxidases. H2O2 is generated under stressful conditions such as UV-irradiation, exposure to intense light, chilling, drought, wounding and biotic intrusion, and it is generated in tissues requiring it as a substrate for biosyntheses such as lignification and suberization. Therefore, the plant is 1

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Promotion of seed germination by H2O2

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Materials and Methods Plant material and its germination condition The dispersal unit of Z. elegans is usually called “seed”, but it is the fruit which is classified as cypsela, in which the embryo is covered with seed coat and pericarp. In the present work, we refer to the cypsela as the seed unless specified, and, even when the pericarp was removed from cypsela, the seed coat still covered the embryos. Seeds of Z. elegans (cv. Scarlet Flame) were purchased from a local seed distributor (Sugiyama-Shubyo, Okayama, Japan). Seeds were imbibed in a plastic dish (15090 mm) which contained 15 ml of solution under the following conditions: 50 E m–2; 16-h light / 8-h dark cycle; 25C. Under such a condition, seeds were not submerged but imbibed on moistened paper. Therefore seeds germinated under aerobic conditions. The H2O2 solution was freshly diluted when required. In the germination test using seeds with pericarp removed, the pericarp was removed from the seeds by hand. Seeds washed with ethanol and preparation of ethanol extract from the seeds Seeds having pericarp (500 g) were soaked in 200 ml of 99.5% (v/v) ethanol in a conical beaker for 5 min, and then washed three times with 100 ml ethanol each (300 ml in total) through suction filtration using a Buchner funnel. Seeds (ethanol-washed seeds) were dried at room temperature for over 2 d before use. The filtrate (ethanol extract) was concentrated to 5 ml in an evaporator and then immediately used or stored at –20C under nitrogen gas until use. When the extract was not stored under nitrogen gas, the inhibitor activity was lost (data not shown). Detection of O2– in intact plants Seeds with pericarp removed or seedlings 17 h after imbibition were preincubated with 100 M 2-methyl-6-( p-methoxyphenyl)-3,7dihydroimidazo[1,2-]pyrazin-3-one (MCLA) for 10 min. Subsequently they were transferred into a dish moistened and analyzed using a two-dimensional photon counting system (ARGUS-50; Hamamatsu Photonics, Hamamatsu, Japan). The MCLA-dependent chemiluminescence from the seed/seedling was reduced in the presence of exogenously applied SOD and enhanced by H2O2 (data not shown), implying that it was an indicator of O2–.

Results and Discussion Promotion of germination of Z. elegans seeds by H2O2 and respiratory inhibitors The germination frequency of Z. elegans seeds was enhanced by H2O2 (Fig. 1). The concentration of H2O2 giving the maximal promotional effect varied depending on the time after imbibition of the seeds. The concentrations of H2O2 that gave half the maximal germination frequency (the mean frequency of the maximum and minimum) achieved 24 h and 48 h after imbibition were obtained between 2 and 5 mM, whereas it was between 5 and 20 mM H2O2 at 36 h and 48 h after imbibition. These suggest that H2O2 influences more than two steps in the process of germination and/or is continuously consumed at a certain rate to promote seed germination. Similar promotional effects have been reported in cereal plants such as wheat, rice and barley (Naredo et al. 1998). The


Fig. 1 Promotion of germination by H2O2 in Z. elegans seeds. Seeds were imbibed in a plastic Petri dish as described in Materials and Methods. Germination was defined as the time when the length of the elongating and extruding part exceeds that of the seed or when the cotyledons opened. Germinated seeds were counted at the time indicated in each graph. Values are the means SE (n = 45) from three experiments.

disproportionation of H2O2 resulting in an increased O2 level is considered to enhance the oxidative respiration, which can be the reason why the promotion of seed germination was observed. However, respiratory inhibitors also promoted the germination of Z. elegans seeds (Table 1), which surely inhibited the O2 uptake of seeds (Table 2), suggesting that such promotional effects are not mainly attributable to the increased O2 level and that the oxidative respiration is not a rate-limiting step for the seed germination, at least, through 48 h after imbibition. High concentrations of O2 generally enhance the germination of plant seeds, and such promotion has also been considered to be due to the enhancement of mitochondrial oxidative respiration. Since seeds were not submerged and the O2 level in the solution was approximately 256 M, the O2 level in seeds would hardly be affected by H2O2 treatment. The Km value for mitochondrial Cyt c oxidase which participates in the ATP production metabolism accompanied with O2 consumption is estimated to be approximately 140 nM O2, whereas that for alternative oxidase whose reaction is not accompanied with ATP

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Table 1 Effects of respiratory inhibitors on the germination of Z. elegans seeds Inhibitors

Germination frequency (%)

n

36.65.5 64.73.7 56.06.2 65.34.6

150 150 135 135

None 10–5 M NaN3 10–5 M KCN 10–4 M SHAM

Oxygen uptake in Z. elegans seeds

Concentration of NaN3 (M) 0 10–5 10–3

Oxygen uptake, (nmol min–1 seed–1)

+ – + – + –

Oxygen uptake (nmol min–1 seed–1) 0.0340.009 0.4710.005 0.0980.020 0.6040.014 0.9370.063 1.7310.039

Oxygen uptake was measured using oxygen electrode as described in Materials and Methods. Values are the means ± SE from three experiments. a + and – show the seeds with and without pericarp, respectively.

Table 5 Effects of H2O2 on the germination of seeds with pericarp removed

1.730.03 1.460.07 1.500.05

Table 3 Effects of pericarp removal and extraction with ethanol on the germination of Z. elegans seeds

None Removal of pericarp Extraction with ethanol 5 mM H2O2 20 mM H2O2

Pericarp a

0 min 0 min 10 min 10 min 4h 4h

H2O2 concentration (mM)


n

0 1 10

77.88.9 11.15.9 0.000.0

45 45 45

Oxygen uptake was measured using an oxygen electrode 4 h after imbibition of seeds with pericarp removed. Values are the means ± SE from three experiments.

Treatments

Oxygen uptake of the seeds with and without peri-

Time after imbibition

Seeds were imbibed in solutions containing the indicated inhibitors as described in Materials and Methods, and the germination frequency was determined 48 h after imbibition. Values are the means ± SE from four experiments.

Table 2

Table 4 carp


n

37.84.4 97.82.2 80.04.7 65.74.6 95.62.2

45 60 150 45 45

Pretreatments of the seeds (removal of pericarp and extraction with ethanol) were done as described in Materials and Methods. Seeds were imbibed as described in Materials and Methods. The germination frequency was determined 48 h after imbibition. Values are the means ± SE from three experiments.

production is approximately 1.7 M O2 (Millar et al. 1994). Application of H2O2 at micromolar levels has inhibitory effects on photosynthetic activity. These findings suggest that the promotion of germination of Z. elegans seeds by H2O2 is due to other reasons. Promotion of germination by removal of pericarp and extraction with ethanol The germination frequency was enhanced by removal of the pericarp from the seed (Table 3) with elevated O2 uptake (Table 4). The effect of H2O2 on germination of the pericarpless seeds was opposite to that on the intact seeds (Table 5). Removal of ethanol-soluble compounds also promoted the ger-


Seeds were imbibed in H2O2 solutions at indicated concentrations as described in Materials and Methods, and the germination frequency was determined 24 h after imbibition. Values are the means ± SE of a typical experiment from three experiments.

Table 6 Inhibition of seed germination of Z. elegans by ethanol extract from the seeds and the reversal of the effects by H2O2 Treatment None a Ext b Ext b+5 mM H2O2


n

78.33.2 38.33.1 66.74.3

150 75 75

Ethanol extract from the seeds (Ext) was prepared as described in Materials and Methods. Seeds with pericarp removed were imbibed as described in Materials and Methods. The germination frequency was determined 48 h after imbibition. Values are the means ± SE of three experiments. a Seeds were imbibed in 0.5% ethanol (v/v) which is the corresponding concentration of ethanol for treatment with Ext. Ethanol used for the germination test was preconcentrated in a similar way to preparation of Ext. b The amount of Ext added to one dish was equivalent to that from the seed weight of 7.5 g.

mination of seeds with pericarp. Both promotional effects were similar to that of H2O2. The ethanol-extract had an inhibitory effect on seed germination, which was nullified by H2O2 (Table 6). These findings suggest the presence of an inhibitor(s) of seed germination in the pericarp that is(are) decomposed or


Fig. 2 Suppressive effects of antioxidants on the germination of Z. elegans seeds. Seeds were imbibed as described in Materials and Methods except that antioxidants were applied at concentrations indicated below each bar. The germination frequency was determined 48 h after imbibition. Values are the means ± SE (n = 150) from four experiments. Cont, No antioxidants applied; NDGA, nordihydro-guaiareitic acid applied; PDTC, pyrrolidine dithiocarbamate applied; and n-PG, npropyl gallate applied.

denatured via the oxidation by H2O2. The pericarp and seed coat often contain phenolic compounds and alkaloids which inhibit seed germination (Bhattacharyya et al. 1999, Tao and Buta 1986). Oxidation of this germination inhibitor(s) is likely to exclude a reaction that is mediated by peroxidases because strong inhibitors of peroxidases that have micromolar Ki value promoted seed germination (Table 1). It is likely that H2O2 oxidatively denatures germination inhibitor(s) such as ferulic and coumaric acids. Generation of reactive oxygen species and suppression of seed germination by antioxidants Phenolic compounds which act as an antioxidant suppressed seed germination in a dose-dependent manner (Fig. 2). The O2 level in the solutions was not affected by such phenolic compounds (data not shown), suggesting that their suppressive effects were not due to the O2 deficiency caused by their autooxidation and that the oxidative decomposition of the germination inhibitor(s) such as phenolic compounds by reactive oxygen species (ROS) probably occurs even in the absence of exogenous H2O2. These findings indicate that phenolic compounds in the pericarp can function as a germination inhibitor. Indeed, phenolic compounds in the pericarp and/or seed coat have been reported to function as a germination inhibitor (Qi et al. 1993), which indicates the possibility that H2O2 is produced by plants for germination. We measured the ROS levels in the


289

Fig. 3 Generation of O2– during germination of Z. elegans seeds. Imaging of MCLA-dependent chemiluminescence on the seed/seedling was carried out as in Materials and Methods. The colored areas indicate the chemiluminescent part that indicate site of O2– generation. The order in terms of decreasing chemiluminescent intensity is as follows: Green, pink, red-purple, blue-purple, gray and black. Bar = 5 mm. Radicle was still covered with seed coat 10 min after imbibition (left-hand side). Seventeen h after imbibition (right-hand side), the cotyledons were no longer covered with the seed coat; cotyledon was unopen; hypocotyl and root were elongating.

seed/seedling using a two-dimensional photon counting system as described in Materials and Methods (Fig. 3). The MCLA-dependent chemiluminescence, which is specific for O2– (Tampo et al. 1998), was detected as colored areas on the images of the seeds/seedlings. The colors indicate the chemiluminescence intensities determined by photon counting and their order in terms of decreasing intensity is as follows: green, pink, red-purple, blue-purple, gray and black. In the seed analyzed immediately after imbibition, intensive chemiluminescence signals were detected on the cotyledon which was still covered with the seed coat and on the extruding part that will elongate and proliferate (left-hand side in Fig. 3). The signals were also detected on the root of the seedling 17 h after imbibition but were rarely found on the cotyledons which were no longer covered with seed coat (right-hand side in Fig. 3). These results were consistent with those obtained using luminol which can produce a chemiluminescence signal in its reaction with H2O2 (data not shown). The parts where an intensive chemiluminescence signal was observed could be regarded as the proliferating and/or elongating parts, leading to a conclusion that H2O2 produced at the proliferating and elongating parts contributes to the decomposition of a germination inhibitor(s).

290


Imbibition of dry seeds is associated with a rapid increase in oxygen uptake and mitochondrial respiration supporting ATP synthesis (Bewley and Black 1985). It is estimated that up to 2% of mitochondrial O2 consumption in seeds is involved in the generation of H2O2 (Cakmak et al. 1993). H2O2 is generated in soybean embryonic axes (Puntarulo et al. 1988) and in vascular tissues of the spinach hypocotyl (Ogawa et al. 1996) and was also observed in root tissues (Fig. 3). The H2O2 generated would be lost in the presence of a germination inhibitor(s) because the pericarp trapped H2O2 (Table 5). KCN, which inhibits catalase, peroxidase and Cyt c oxidase and provokes production of high levels of H2O2, promoted seed germination of Z. elegans, suggesting that this germination inhibitor(s) may block other processes involved in germination aside from mitochondrial respiration. Activation of the oxidative pentose phosphate pathway generally leads to seed germination, and has been proposed to be promoted by H2O2 (Fontaine et al. 1994). The oxidative pentose phosphate pathway provides NADPH which is used for the reduction of redox-regulating proteins such as thioredoxin. Such proteins regulate mobilization of storage proteins and the modulation of activities of enzymes and transcriptional factors by their reduction of disulfide bond in the target molecules (Kobrehel et al. 1992). This is why activation of the pentose phosphate pathway leads to germination. Antioxidant germination inhibitors such as phenolic compounds may block activation of the pathway. A bacterial system for adaptation to oxidative stresses involves enzymes scavenging ROS such as Mn-superoxide dismutase and catalase that are encoded in SoxRS and OxyR regulons (Prieto-Álamo et al. 2000). O2– regulates the SoxRS regulon including genes encoding respiratory enzymes such as glucose-6-phosphate dehydrogenase, which provides NADPH, while H2O2 regulates the OxyR regulon including genes encoding thioredoxin, glutaredoxin, glutathione reductase (GR) and C-glutamylcystein synthetase. ROS generated during germination might function in a similar way although such homologous transcriptional factors have not been found in plant genomes. H2O2 increases the GSH level in barley seeds due to its induction of the GR activity (Fontaine et al. 1994) and, possibly, of the C-glutamylcystein synthetase activity. Catalase and ascorbate peroxidase activities increase and dehydroascorbate reductase activity decreases with germination of wheat seeds (Cakmak et al. 1993), suggesting that there is low scavenging activity of H2O2 in seeds at the initial stage of germination. These findings suggest that the temporal oxidized state of the seed embryo that is induced by H2O2 might initiate germination and that antioxidant germination inhibitor(s) might prevent the induction of the oxidized state in seeds. Concluding remarks Here we proposed a mechanism for the promotion of seed germination by exogenously applied H2O2 and also the possibility that H2O2 endogenously generated actually functions as a


promoter of seed germination by oxidizing germination inhibitors.

Acknowledgments The present work was supported by “Research for the Future” Program of The Japan Society for the Promotion of Science (JSPSRFTF 00L01605).

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(Received July 31, 2000; Accepted December 12, 2000)


A MECHANISM FOR PROMOTING THE GERMINATION OF ZINNIA ELEGANS

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