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Soluble sugar availability of aerobically germinated barley, oat and rice coleoptiles in anoxia
Journal of Plant Physiology 167 (2010) 1571–1576
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Soluble sugar availability of aerobically germinated barley, oat and rice coleoptiles in anoxia Hisashi Kato-Noguchi ∗ , Yukihiro Yasuda, Ryosuke Sasaki Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki, Kagawa 761-0795, Japan
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Article history: Received 18 March 2010 Received in revised form 25 May 2010 Accepted 2 June 2010 Keywords: ␣-Amylase Anoxia tolerance Barley Ethanolic fermentation Oat Soluble sugar Rice
a b s t r a c t Physiological and metabolic responses to anoxia were compared for aerobically germinated seedlings of barley (Hordeum vulgare), oat (Avena sativa) and rice (Oryza sativa). Coleoptile growth of barley, oat and rice seedlings was suppressed by a 24 h-anoxic stress, but the growth of the rice coleoptiles was much greater than that of the barley and oat coleoptiles. ATP concentration in the anoxic rice coleoptiles was greater than that in the anoxic barley and oat coleoptiles. Concentrations of ethanol and activity of alcohol dehydrogenase (ADH) in the anoxic rice coleoptiles were also greater than those of the anoxic barley and oat coleoptiles, suggesting that ethanolic fermentation may be more active in the rice coleoptiles than in the barley and oat coleoptiles, where glycolysis and ethanolic fermentation are the main source of ATP production. Soluble sugar concentration in the anoxic rice coleoptiles was greater than that of the anoxic barley and oat coleoptiles. However, ␣-amylase, which catabolizes reserve starch to soluble sugars, was active in anoxic barley, oat and rice endosperms, and soluble sugar concentration in the anoxic barley, oat and rice endosperms was not significantly different. Therefore, anoxia stress may inhibit soluble sugar transport from the endosperms to the coleoptiles in barley and oat more than in rice. Since the availability of soluble sugar is essential for operation of glycolysis and fermentation in plant cells, ability for sugar transport from the endosperms to the coleoptiles may be one means to distinguish the coleoptile growth of these plant species in anoxia and anoxia tolerance of these plants. © 2010 Elsevier GmbH. All rights reserved.
Introduction Among cereal species, rice is one of the few plants that can germinate and grow its coleoptiles in complete anoxia (Alpi and Beevers, 1983; Cobb and Kennedy, 1987; Lasanthi-Kudahettige et al., 2007). Rice coleoptiles display a strong ethanolic fermentation system under conditions in which glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of ATP production (Setter et al., 1997; Boamfa et al., 2003; Loreti et al., 2003; Magneschi and Perata, 2009). Rice seeds also possess an ability to induce a complete set of starch-breakdown enzymes, such as ␣-amylase, -amylase, ␣-glucosidase and debranching enzymes, even in anoxia. The enzymes work for the degradation of reserve starch in the endosperm to soluble sugar, which is essential for the maintenance of glycolytic flux and ethanolic fermentation (Beck and Ziegler, 1989; Thomas, 1993; Guglielminetti et al., 2000; Mustroph et al., 2006).
Abbreviations: ADH, alcohol dehydrogenase. ∗ Corresponding author. Tel.: +81 87 891 3086; fax: +81 87 891 3086. E-mail address: [email protected] (H. Kato-Noguchi). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.06.017
Anoxia-intolerant cereal seeds such as barley, wheat and oat are not able to induce ␣-amylase and germinate in anoxia (Perata et al., 1997; Guglielminetti et al., 1995; Lasanthi-Kudahettige et al., 2007; Magneschi and Perata, 2009). These findings have been made using ungerminated seeds, and only rice germinated and grew in the conditions. After transfer to anoxia after germination in aerobic conditions, however, the coleoptiles of these anoxia-intolerant seedlings can grow and ethanol production has been observed in these seedlings (Raymond et al., 1985; Guglielminetti et al., 1995). These results indicate that these anoxia-intolerant cereals are also able to utilize soluble sugars for glycolysis and ethanolic fermentation, leading to ATP production under limited oxygen conditions. As noted by Goggin and Colmer (2007), however, the effect of anoxia on ␣-amylase activity in anoxia-intolerant cereals germinated in aerobic conditions has not been evaluated. Thus, the differences in ethanolic fermentation and reserve carbohydrate catabolism underlying coleoptile growth in anoxia between anoxia-tolerant and anoxia-intolerant cereal seedlings germinated in aerobic conditions is still not clear. In the present research, coleoptile elongation, ethanol and soluble sugar concentrations and alcohol dehydrogenase (ADH) and ␣-amylase activity in barley, oat and rice seedlings were determined under anoxic stress conditions after germination in aerobic conditions.
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Materials and methods
according to five repeated assays with pure enzyme in the extract.
Plant material and anoxic treatment Extraction and assay of ˛-amylase Seeds of barley (Hordeum vulgare L. cv. Ichibanboshi), oat (Avena sativa L. cv. Victory) and rice (Oryza sativa L. cv. Nipponbare) were surface sterilized in an aqueous solution of 25 mmol/L sodium hypochlorite for 15 min, rinsed four times in distilled water, and germinated on two sheets of moist filter paper (No 1; Toyo Ltd., Tokyo) for 4, 3 and 5 days, respectively, in darkness at 25 ◦ C. Uniform seedlings (coleoptile length about 2 mm) were then transferred, in groups of 10, to 9-cm Petri dishes each containing two sheets of filter paper moistened with 10 mL of distilled water, and subjected to an anoxic treatment. The Petri dishes were placed into 5-L jars at 25 ◦ C. A stream of N2 (99.9%) was passed continuously through the jar at a rate of 200 mL min−1 for 24 h. No oxygen was detected in the jars by an O2 analyzer (XO-326; Cosmos Electric Co., Osaka, Japan). Control seedlings were supplied with air flowing at 200 mL min−1 . Measurements of coleoptile length The length of coleoptiles of barley, oat and rice seedlings was measured with a ruler at the beginning and the end of a 24 h-anoxic treatment, and elongation of the coleoptiles during the treatment was determined. The experiments were repeated three times with 50 plants each. Significant differences were examined by Tukey’s multiple test. Extraction and determination of ATP and ethanol Barley, oat and rice seedlings were sampled at the beginning and the end of a 24 h-anoxic treatment and killed with liquid N2 in anoxia, and their coleoptiles were separated from the seedlings in liquid N2 and stored at −80 ◦ C until extraction. The frozen coleoptiles (10 coleoptiles for one determination) were powdered in a mortar containing liquid N2 using a pestle, and homogenized with five volumes of ice-cold 0.4 mol/L HClO4. The homogenate was centrifuged at 30,000 × g for 15 min at 4 ◦ C and the supernatant was neutralized with 3 M K2 CO3 . The precipitated potassium perchlorate was removed by centrifugation (30,000 × g, 5 min) and the resulting supernatant was used to analyze ATP and ethanol. ATP and ethanol were then quantified spectrophotometrically according to the methods described by Bergmeyer (1985). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ATP and ethanol added to the extraction medium containing coleoptile powder before homogenization was 83 ± 7% and 71 ± 11% (means ± SE) as calculated from five replications. Extraction and assay of ADH Coleoptiles of barley, oat and rice were powdered as described above. The powder was homogenized in an ice-cold solution containing 100 mmol/L Hepes-KOH (pH 7.5), 1 mmol/L EDTA, 5 mmol/L MgCl2 , 5 mmol/L DTT, 10 mmol/L NaHSO3 as described by Guglielminetti et al. (1995). The homogenate was then centrifuged at 30,000 × g for 20 min and the supernatant used for ADH analysis. ADH activity was determined spectrophotometrically by monitoring the oxidation of NADH at 340 nm for 15 min at 35 ◦ C in a 1 mL reaction mixture as described by Kato-Noguchi (2000). The experiments were repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ADH activity through the quantification process was 89 ± 6% (means ± SE)
Barley, oat and rice seedlings were killed with liquid N2 in anoxia and their coleoptiles and endosperms were separated from the seedlings in liquid N2 and powdered as described above. The frozen powder was then homogenized with 5 mL of ice-cold solution of 100 mM HEPES-KOH (pH 7.5) containing 1 mmol/L EDTA, 5 mmol/L MgCl2 , 5 mmol/L DTT, 10 mmol/L NaHSO3 and 50 mmol/L bovine serum albumin. The homogenate was centrifuged at 30,000 × g for 30 min, and the supernatant was heated with 3 mmol/L CaCl2 at 75 ◦ C for 15 min to inactivate -amylase and ␣-glucosidase (Sun and Henson, 1991; Guglielminetti et al., 1995). ␣-Amylase was assayed by measuring the rate of generation of reducing sugars from soluble starch as described by Kato-Noguchi and Macías (2006). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ␣-amylase activity through the quantification process was 92 ± 8% (means ± SE) according to five repeated assays with pure enzyme in the extract. Extraction and determination of soluble sugars Coleoptiles and endosperms of barley, oat and rice seedlings were powdered as described above. The powder was then extracted in 80% (v/v) ethanol, and soluble sugar concentration was determined as hexose units using anthrone (Yemm and Willis, 1954; Huang et al., 2003). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of soluble sugar (glucose) added to the extraction medium through the quantification process was 78 ± 9% (means ± SE) according to five replications. Results Coleoptile growth After germination in an aerobic condition, barley, oat and rice were subjected to 24-h of anoxic stress and the effects of the stress on the coleoptile elongation was determined (Fig. 1). The elongation of anoxia barley, oat and rice coleoptiles, respectively, was 29%, 25% and 71% that of control barley, oat and rice coleoptiles. Thus, rice coleoptiles were more tolerant than barley and oat coleoptiles in anoxia. ATP concentration Anoxic stress decreased ATP concentrations in all coleoptiles (Fig. 2). The ATP concentration in anoxic coleoptiles of barley, oat and rice, respectively, was 21%, 18% and 57% that in control barley, oat and rice coleoptiles. Thus, the anoxic rice coleoptiles maintained 2.7- and 3.2-fold higher level of ATP than the anoxic barley and oat coleoptiles, respectively. ADH activity and ethanol production Anoxic stress increased ADH activity in all coleoptiles (Fig. 3). The ADH activity in anoxic barley, oat and rice coleoptiles, respectively, became 9.3-, 8.5- and 26-fold greater than that in control barley, oat and rice coleoptiles. Ethanol concentrations in initial and control coleoptiles of barley, oat and rice were low and anoxic stress increased the concentrations in all coleoptiles (Fig. 4). However, the increase in the
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Fig. 1. Effects of anoxia on coleoptile elongation of barley, oat and rice seedlings. Barley, oat and rice seedlings were exposed to anoxic stress for 24 h. Control seedlings were grown in air for 24 h. Means ± SE from three independent experiments with 50 plants for each determination are shown. Different letters show significant difference (P < 0.05) for each panel according to Tukey’s multiple test.
ethanol concentration in the rice coleoptiles was 3.3- and 3.5-fold greater than that in the barley and oat coleoptiles, respectively. ˛-Amylase activity Activity of ␣-amylase was not detected in coleoptiles of barley, oat and rice under control (aerobic) and anoxic conditions (data not shown), whereas the activity was found in endosperms of barley, oat and rice under both conditions (Fig. 5). The activity increased in control conditions during 24 h-incubation in all endosperms, but anoxic stress inhibited the activities. There were no significant differences in the activity of all anoxic endosperms. Sugar concentration in coleoptiles and endosperms Anoxic stress decreased soluble sugar concentrations in coleoptiles and endosperms of barley, oat and rice (Figs. 6 and 7). Anoxia rice coleoptiles contained 69% of the soluble sugar in control rice Fig. 3. Effects of anoxia on ADH activity in barley, oat and rice coleoptiles. Other details are as for Fig. 2.
Fig. 2. Effects of anoxia on ATP concentration in of barley, oat and rice coleoptiles. Barley, oat and rice seedlings were exposed to a 24 h-anoxic stress and these coleoptiles were sampled at the beginning (initial) and the end of the anoxic treatment. Control seedlings were grown in air for 24 h. Means ± SE from three independent experiments with four assays for each determination (n = 12) are shown. Different letters show significant difference (P < 0.05) for each panel according to Tukey’s multiple test.
Fig. 4. Effects of anoxia on ethanol concentration in barley, oat and rice coleoptiles. Other details are as for Fig. 2.
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coleoptiles, but anoxia barley and oat coleoptiles contained only 9% and 11% of the sugar in control barley and oat coleoptiles, respectively. The concentration in anoxic rice coleoptiles became 5.6and 6.1-fold greater than that of anoxic barley and oat coleoptiles, respectively (Fig. 6). However, soluble sugar concentrations in anoxia barley, oat and rice endosperms were not significantly different (Fig. 7).
Discussion
Fig. 5. Effects of anoxia on ␣-amylase activity in barley, oat and rice endosperms. At the beginning (initial) and the end of a 24 h-anoxic treatment, endosperms of barley, oat and rice were sampled. Other details are as for Fig. 2.
Fig. 6. Effects of anoxia on soluble sugar concentration in coleoptiles of barley, oat and rice. Other details are as for Fig. 2.
Fig. 7. Effects of anoxia on soluble sugar concentration in endosperms of barley, oat and rice. At the beginning (initial) and the end of a 24 h-anoxic treatment, endosperms of barley, oat and rice were sampled. Other details are as for Fig. 2.
Coleoptiles of barley, oat and rice seedlings germinated in an aerobic condition were capable of growing in anoxia, but the capability of the growth was greater in rice than in barley and oat coleoptiles (Fig. 1). Thus, the anoxia tolerance with respect to the coleoptile growth was greater in the rice coleoptiles than in the barley and oat coleoptiles. ATP concentration in anoxic rice coleoptiles was also greater than that in anoxic barley and oat coleoptiles (Fig. 2). ATP production by oxidative phosphoryzation in anoxia is negligible relative to that by anaerobic glycolysis (Drew, 1997; Vartapetian and Jackson, 1997; Magneschi and Perata, 2009). Thus, anaerobic glycolysis may be greater in the anoxic rice coleoptiles than in anoxic barley and oat coleoptiles. It has also been reported that anaerobic glycolysis in rice coleoptiles was 2.7-fold faster than the rate of glycolysis in the air (Setter et al., 1997; Gibbs and Greenway, 2003). The stress increased ADH activity and ethanol concentration in all coleoptiles (Figs. 3 and 4), which suggests that the anoxic stress probably activates ethanolic fermentation in the coleoptiles of all plants. However, ADH activity in anoxic rice coleoptiles was 2.2and 2.5-fold greater than that in anoxic barley and oat coleoptiles, respectively, and ethanol concentration in anoxic rice coleoptiles was 3.3- and 3.5-fold greater than that in anoxic barley and oat coleoptiles, respectively. These results suggest that the ethanolic fermentation may be more active in the anoxic rice coleoptiles than in the anoxic barley and oat coleoptiles. The ethanol concentration was probably underestimated because ethanol diffusion from tissues was rapid, and only 21% of the ethanol produced was found in rice coleoptiles during 12 h in anoxia (Alpi and Beevers, 1983). Therefore, the amount of ethanol produced in the barley, oat and rice coleoptiles was probably greater than the ethanol concentrations determined in these coleoptiles (Fig. 4). However, it is unlikely that the ethanol diffusion rates from coleoptile tissues are much different among these species because the diffusion rates of rice, oat and wheat coleoptiles were similar (Alpi and Beevers, 1983). Thus, the ethanolic fermentation in the anoxic rice coleoptiles is probably greater than that in the anoxic barley and oat coleoptiles. An active ethanolic fermentation pathway is essential for the continuation of anaerobic glycolysis owing to pyruvate consumption and recycling NAD+ when oxygen supply is limited and glycolysis replaces the Krebs cycle as the main source of ATP (Drew, 1997; Saglio et al., 1999; Tadege et al., 1999; Agarwal et al., 2007). In addition, the importance of ethanolic fermentation under anoxia has been demonstrated by studies on the alcohol dehydrogenase null mutants of several plant species (Johnson et al., 1994; Matsumura et al., 1995; Ellis et al., 1999; Saika et al., 2006). Therefore, activation of ethanolic fermentation was considered to be one of the strategies for plants to survive under anoxia (Drew, 1997; Tadege et al., 1999; Gibbs and Greenway, 2003). The present results (Figs. 1–4) suggest that anoxia tolerance of rice coleoptiles of the seedlings germinated in aerobic conditions was greater than that of barley and oat coleoptiles of the seedlings germinated in aerobic conditions. The soluble sugar concentration in anoxic rice coleoptiles was much greater than that in anoxic barley and oat coleoptiles (Fig. 6). Anoxic barley and oat coleoptiles contained only 17% and 16% of
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soluble sugar of rice coleoptiles. The availability of soluble sugar is essential for operation of glycolysis and fermentation leading to ATP production in plant cells (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). However, the amount of soluble sugars in coleoptiles is very limited, and soluble sugars are mostly supplied to the coleoptiles from their endosperms where starch is the main reserve carbohydrate (Perata et al., 1997; Saglio et al., 1999; Loreti et al., 2003). ␣-Amylase plays a major role in the degradation of reserve carbohydrates to soluble sugars in anoxia (Sun and Henson, 1991; Guglielminetti et al., 1995). Thus, the function of ␣-amylase may be essential to anoxia tolerance. ␣-Amylase was active in all anoxic endosperms (Fig. 5), and soluble sugar concentrations in all anoxia endosperms were not significantly different (Fig. 7). It has also been reported that anoxia wheat seeds, which germinated in an aerobic condition, showed ␣-amylase activity (Goggin and Colmer, 2007). The ratio of soluble sugar concentration in control coleoptiles to the concentration in control endosperms was 12%, 10% and 13% for barley, oat and rice, respectively (Figs. 6 and 7). Therefore, the ratios were similar in all plant species, which suggests that an ability of sugar transport from the endosperms to the coleoptiles of barley, oat and rice may be dependent on sugar concentration in the endosperms. It was also reported that sugar transport among rice cultivars was dependent on sugar concentrations in the endosperms (Huang et al., 2003). On the other hand, the ratio of the concentration in anoxic coleoptiles to the concentration in anoxic endosperms was 2.9%, 2.8% and 16% for barley, oat and rice, respectively (Figs. 6 and 7). Although anoxia stress may inhibit the soluble sugar transport in barley, oat and rice, the inhibition may be much greater in anoxia-intolerant barley and oat than anoxia-tolerant rice. In addition, despite the fact that the consumption of soluble sugars in rice coleoptiles was probably greater than that in barley and oat coleoptiles because of faster rate of ethanol fermentation in rice coleoptiles (Fig. 4), the soluble sugar concentration in rice coleoptiles was greater than that in barley and oat coleoptiles (Fig. 6). These results suggest that the ability to transport sugar from the endosperms to the coleoptiles in rice may be greater than that in barley and oat in anoxia. Consequently, although soluble sugar concentrations among the anoxic endosperms were not significantly different, the sugar availability of anoxic rice coleoptiles became greater than that of anoxic barley and oat coleoptiles (Figs. 6 and 7). The ability for sugar transport from the endosperms to the coleoptiles is probably very important for the availability of soluble sugar in coleoptiles, as soluble sugar is essential to operate anoxic glycolysis and fermentation (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). This ability may be one way to distinguish the capability of coleoptile growth of these plant species in anoxia as well as an anoxia tolerance of these plants.
Conclusion The effects of anoxia on ␣-amylase activity and reserve carbohydrate catabolism underlying coleoptile growth between anoxia-tolerant and anoxia-intolerant cereals germinated in aerobic conditions have not been evaluated thus far Goggin and Colmer (2007). Coleoptile growth and ATP concentration in barley, oat and rice seedlings germinated in an aerobic condition were suppressed by a 24 h-anoxic stress, but the growth and concentration in the rice coleoptiles was much greater than that of the barley and oat coleoptiles (Figs. 1 and 2). According to measures of ethanol concentration and ADH activity (Figs. 3 and 4), ethanolic fermentation may be more active in the rice coleoptiles than in the barley and oat coleoptiles, where glycolysis and ethanolic fermentation are the main source of ATP production. The availability of soluble sugar is essen-
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tial for operation of the fermentation (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). Soluble sugars in the coleoptiles are very limited and mostly supplied from the endosperms (Perata et al., 1997; Saglio et al., 1999; Loreti et al., 2003). ␣-Amylase was active in anoxic barley, oat and rice endosperms, and soluble sugar concentrations in the anoxic barley, oat and rice endosperms was not significantly different (Figs. 5 and 7). However, the soluble sugar concentration in anoxic rice coleoptiles was much greater than that in anoxic barley and oat coleoptiles (Fig. 6). Therefore, anoxia stress may inhibit the soluble sugar transport to the coleoptiles from the endosperms to a much greater extent in anoxia-intolerant barley and oat than in anoxiatolerant rice. An ability to transport sugar from the endosperms to the coleoptiles may be one way to distinguish the coleoptile growth of these plant species in anoxia and anoxia tolerance of these plants.
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Journal of Plant Physiology journal homepage: www.elsevier.de/jplph
Soluble sugar availability of aerobically germinated barley, oat and rice coleoptiles in anoxia Hisashi Kato-Noguchi ∗ , Yukihiro Yasuda, Ryosuke Sasaki Department of Applied Biological Science, Faculty of Agriculture, Kagawa University, Ikenobe 2393, Miki, Kagawa 761-0795, Japan
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Article history: Received 18 March 2010 Received in revised form 25 May 2010 Accepted 2 June 2010 Keywords: ␣-Amylase Anoxia tolerance Barley Ethanolic fermentation Oat Soluble sugar Rice
a b s t r a c t Physiological and metabolic responses to anoxia were compared for aerobically germinated seedlings of barley (Hordeum vulgare), oat (Avena sativa) and rice (Oryza sativa). Coleoptile growth of barley, oat and rice seedlings was suppressed by a 24 h-anoxic stress, but the growth of the rice coleoptiles was much greater than that of the barley and oat coleoptiles. ATP concentration in the anoxic rice coleoptiles was greater than that in the anoxic barley and oat coleoptiles. Concentrations of ethanol and activity of alcohol dehydrogenase (ADH) in the anoxic rice coleoptiles were also greater than those of the anoxic barley and oat coleoptiles, suggesting that ethanolic fermentation may be more active in the rice coleoptiles than in the barley and oat coleoptiles, where glycolysis and ethanolic fermentation are the main source of ATP production. Soluble sugar concentration in the anoxic rice coleoptiles was greater than that of the anoxic barley and oat coleoptiles. However, ␣-amylase, which catabolizes reserve starch to soluble sugars, was active in anoxic barley, oat and rice endosperms, and soluble sugar concentration in the anoxic barley, oat and rice endosperms was not significantly different. Therefore, anoxia stress may inhibit soluble sugar transport from the endosperms to the coleoptiles in barley and oat more than in rice. Since the availability of soluble sugar is essential for operation of glycolysis and fermentation in plant cells, ability for sugar transport from the endosperms to the coleoptiles may be one means to distinguish the coleoptile growth of these plant species in anoxia and anoxia tolerance of these plants. © 2010 Elsevier GmbH. All rights reserved.
Introduction Among cereal species, rice is one of the few plants that can germinate and grow its coleoptiles in complete anoxia (Alpi and Beevers, 1983; Cobb and Kennedy, 1987; Lasanthi-Kudahettige et al., 2007). Rice coleoptiles display a strong ethanolic fermentation system under conditions in which glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of ATP production (Setter et al., 1997; Boamfa et al., 2003; Loreti et al., 2003; Magneschi and Perata, 2009). Rice seeds also possess an ability to induce a complete set of starch-breakdown enzymes, such as ␣-amylase, -amylase, ␣-glucosidase and debranching enzymes, even in anoxia. The enzymes work for the degradation of reserve starch in the endosperm to soluble sugar, which is essential for the maintenance of glycolytic flux and ethanolic fermentation (Beck and Ziegler, 1989; Thomas, 1993; Guglielminetti et al., 2000; Mustroph et al., 2006).
Abbreviations: ADH, alcohol dehydrogenase. ∗ Corresponding author. Tel.: +81 87 891 3086; fax: +81 87 891 3086. E-mail address: [email protected] (H. Kato-Noguchi). 0176-1617/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jplph.2010.06.017
Anoxia-intolerant cereal seeds such as barley, wheat and oat are not able to induce ␣-amylase and germinate in anoxia (Perata et al., 1997; Guglielminetti et al., 1995; Lasanthi-Kudahettige et al., 2007; Magneschi and Perata, 2009). These findings have been made using ungerminated seeds, and only rice germinated and grew in the conditions. After transfer to anoxia after germination in aerobic conditions, however, the coleoptiles of these anoxia-intolerant seedlings can grow and ethanol production has been observed in these seedlings (Raymond et al., 1985; Guglielminetti et al., 1995). These results indicate that these anoxia-intolerant cereals are also able to utilize soluble sugars for glycolysis and ethanolic fermentation, leading to ATP production under limited oxygen conditions. As noted by Goggin and Colmer (2007), however, the effect of anoxia on ␣-amylase activity in anoxia-intolerant cereals germinated in aerobic conditions has not been evaluated. Thus, the differences in ethanolic fermentation and reserve carbohydrate catabolism underlying coleoptile growth in anoxia between anoxia-tolerant and anoxia-intolerant cereal seedlings germinated in aerobic conditions is still not clear. In the present research, coleoptile elongation, ethanol and soluble sugar concentrations and alcohol dehydrogenase (ADH) and ␣-amylase activity in barley, oat and rice seedlings were determined under anoxic stress conditions after germination in aerobic conditions.
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Materials and methods
according to five repeated assays with pure enzyme in the extract.
Plant material and anoxic treatment Extraction and assay of ˛-amylase Seeds of barley (Hordeum vulgare L. cv. Ichibanboshi), oat (Avena sativa L. cv. Victory) and rice (Oryza sativa L. cv. Nipponbare) were surface sterilized in an aqueous solution of 25 mmol/L sodium hypochlorite for 15 min, rinsed four times in distilled water, and germinated on two sheets of moist filter paper (No 1; Toyo Ltd., Tokyo) for 4, 3 and 5 days, respectively, in darkness at 25 ◦ C. Uniform seedlings (coleoptile length about 2 mm) were then transferred, in groups of 10, to 9-cm Petri dishes each containing two sheets of filter paper moistened with 10 mL of distilled water, and subjected to an anoxic treatment. The Petri dishes were placed into 5-L jars at 25 ◦ C. A stream of N2 (99.9%) was passed continuously through the jar at a rate of 200 mL min−1 for 24 h. No oxygen was detected in the jars by an O2 analyzer (XO-326; Cosmos Electric Co., Osaka, Japan). Control seedlings were supplied with air flowing at 200 mL min−1 . Measurements of coleoptile length The length of coleoptiles of barley, oat and rice seedlings was measured with a ruler at the beginning and the end of a 24 h-anoxic treatment, and elongation of the coleoptiles during the treatment was determined. The experiments were repeated three times with 50 plants each. Significant differences were examined by Tukey’s multiple test. Extraction and determination of ATP and ethanol Barley, oat and rice seedlings were sampled at the beginning and the end of a 24 h-anoxic treatment and killed with liquid N2 in anoxia, and their coleoptiles were separated from the seedlings in liquid N2 and stored at −80 ◦ C until extraction. The frozen coleoptiles (10 coleoptiles for one determination) were powdered in a mortar containing liquid N2 using a pestle, and homogenized with five volumes of ice-cold 0.4 mol/L HClO4. The homogenate was centrifuged at 30,000 × g for 15 min at 4 ◦ C and the supernatant was neutralized with 3 M K2 CO3 . The precipitated potassium perchlorate was removed by centrifugation (30,000 × g, 5 min) and the resulting supernatant was used to analyze ATP and ethanol. ATP and ethanol were then quantified spectrophotometrically according to the methods described by Bergmeyer (1985). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ATP and ethanol added to the extraction medium containing coleoptile powder before homogenization was 83 ± 7% and 71 ± 11% (means ± SE) as calculated from five replications. Extraction and assay of ADH Coleoptiles of barley, oat and rice were powdered as described above. The powder was homogenized in an ice-cold solution containing 100 mmol/L Hepes-KOH (pH 7.5), 1 mmol/L EDTA, 5 mmol/L MgCl2 , 5 mmol/L DTT, 10 mmol/L NaHSO3 as described by Guglielminetti et al. (1995). The homogenate was then centrifuged at 30,000 × g for 20 min and the supernatant used for ADH analysis. ADH activity was determined spectrophotometrically by monitoring the oxidation of NADH at 340 nm for 15 min at 35 ◦ C in a 1 mL reaction mixture as described by Kato-Noguchi (2000). The experiments were repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ADH activity through the quantification process was 89 ± 6% (means ± SE)
Barley, oat and rice seedlings were killed with liquid N2 in anoxia and their coleoptiles and endosperms were separated from the seedlings in liquid N2 and powdered as described above. The frozen powder was then homogenized with 5 mL of ice-cold solution of 100 mM HEPES-KOH (pH 7.5) containing 1 mmol/L EDTA, 5 mmol/L MgCl2 , 5 mmol/L DTT, 10 mmol/L NaHSO3 and 50 mmol/L bovine serum albumin. The homogenate was centrifuged at 30,000 × g for 30 min, and the supernatant was heated with 3 mmol/L CaCl2 at 75 ◦ C for 15 min to inactivate -amylase and ␣-glucosidase (Sun and Henson, 1991; Guglielminetti et al., 1995). ␣-Amylase was assayed by measuring the rate of generation of reducing sugars from soluble starch as described by Kato-Noguchi and Macías (2006). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of ␣-amylase activity through the quantification process was 92 ± 8% (means ± SE) according to five repeated assays with pure enzyme in the extract. Extraction and determination of soluble sugars Coleoptiles and endosperms of barley, oat and rice seedlings were powdered as described above. The powder was then extracted in 80% (v/v) ethanol, and soluble sugar concentration was determined as hexose units using anthrone (Yemm and Willis, 1954; Huang et al., 2003). The experiment was repeated three times with four assays for each determination. Significant differences were examined by Tukey’s multiple test. The overall recovery of soluble sugar (glucose) added to the extraction medium through the quantification process was 78 ± 9% (means ± SE) according to five replications. Results Coleoptile growth After germination in an aerobic condition, barley, oat and rice were subjected to 24-h of anoxic stress and the effects of the stress on the coleoptile elongation was determined (Fig. 1). The elongation of anoxia barley, oat and rice coleoptiles, respectively, was 29%, 25% and 71% that of control barley, oat and rice coleoptiles. Thus, rice coleoptiles were more tolerant than barley and oat coleoptiles in anoxia. ATP concentration Anoxic stress decreased ATP concentrations in all coleoptiles (Fig. 2). The ATP concentration in anoxic coleoptiles of barley, oat and rice, respectively, was 21%, 18% and 57% that in control barley, oat and rice coleoptiles. Thus, the anoxic rice coleoptiles maintained 2.7- and 3.2-fold higher level of ATP than the anoxic barley and oat coleoptiles, respectively. ADH activity and ethanol production Anoxic stress increased ADH activity in all coleoptiles (Fig. 3). The ADH activity in anoxic barley, oat and rice coleoptiles, respectively, became 9.3-, 8.5- and 26-fold greater than that in control barley, oat and rice coleoptiles. Ethanol concentrations in initial and control coleoptiles of barley, oat and rice were low and anoxic stress increased the concentrations in all coleoptiles (Fig. 4). However, the increase in the
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Fig. 1. Effects of anoxia on coleoptile elongation of barley, oat and rice seedlings. Barley, oat and rice seedlings were exposed to anoxic stress for 24 h. Control seedlings were grown in air for 24 h. Means ± SE from three independent experiments with 50 plants for each determination are shown. Different letters show significant difference (P < 0.05) for each panel according to Tukey’s multiple test.
ethanol concentration in the rice coleoptiles was 3.3- and 3.5-fold greater than that in the barley and oat coleoptiles, respectively. ˛-Amylase activity Activity of ␣-amylase was not detected in coleoptiles of barley, oat and rice under control (aerobic) and anoxic conditions (data not shown), whereas the activity was found in endosperms of barley, oat and rice under both conditions (Fig. 5). The activity increased in control conditions during 24 h-incubation in all endosperms, but anoxic stress inhibited the activities. There were no significant differences in the activity of all anoxic endosperms. Sugar concentration in coleoptiles and endosperms Anoxic stress decreased soluble sugar concentrations in coleoptiles and endosperms of barley, oat and rice (Figs. 6 and 7). Anoxia rice coleoptiles contained 69% of the soluble sugar in control rice Fig. 3. Effects of anoxia on ADH activity in barley, oat and rice coleoptiles. Other details are as for Fig. 2.
Fig. 2. Effects of anoxia on ATP concentration in of barley, oat and rice coleoptiles. Barley, oat and rice seedlings were exposed to a 24 h-anoxic stress and these coleoptiles were sampled at the beginning (initial) and the end of the anoxic treatment. Control seedlings were grown in air for 24 h. Means ± SE from three independent experiments with four assays for each determination (n = 12) are shown. Different letters show significant difference (P < 0.05) for each panel according to Tukey’s multiple test.
Fig. 4. Effects of anoxia on ethanol concentration in barley, oat and rice coleoptiles. Other details are as for Fig. 2.
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coleoptiles, but anoxia barley and oat coleoptiles contained only 9% and 11% of the sugar in control barley and oat coleoptiles, respectively. The concentration in anoxic rice coleoptiles became 5.6and 6.1-fold greater than that of anoxic barley and oat coleoptiles, respectively (Fig. 6). However, soluble sugar concentrations in anoxia barley, oat and rice endosperms were not significantly different (Fig. 7).
Discussion
Fig. 5. Effects of anoxia on ␣-amylase activity in barley, oat and rice endosperms. At the beginning (initial) and the end of a 24 h-anoxic treatment, endosperms of barley, oat and rice were sampled. Other details are as for Fig. 2.
Fig. 6. Effects of anoxia on soluble sugar concentration in coleoptiles of barley, oat and rice. Other details are as for Fig. 2.
Fig. 7. Effects of anoxia on soluble sugar concentration in endosperms of barley, oat and rice. At the beginning (initial) and the end of a 24 h-anoxic treatment, endosperms of barley, oat and rice were sampled. Other details are as for Fig. 2.
Coleoptiles of barley, oat and rice seedlings germinated in an aerobic condition were capable of growing in anoxia, but the capability of the growth was greater in rice than in barley and oat coleoptiles (Fig. 1). Thus, the anoxia tolerance with respect to the coleoptile growth was greater in the rice coleoptiles than in the barley and oat coleoptiles. ATP concentration in anoxic rice coleoptiles was also greater than that in anoxic barley and oat coleoptiles (Fig. 2). ATP production by oxidative phosphoryzation in anoxia is negligible relative to that by anaerobic glycolysis (Drew, 1997; Vartapetian and Jackson, 1997; Magneschi and Perata, 2009). Thus, anaerobic glycolysis may be greater in the anoxic rice coleoptiles than in anoxic barley and oat coleoptiles. It has also been reported that anaerobic glycolysis in rice coleoptiles was 2.7-fold faster than the rate of glycolysis in the air (Setter et al., 1997; Gibbs and Greenway, 2003). The stress increased ADH activity and ethanol concentration in all coleoptiles (Figs. 3 and 4), which suggests that the anoxic stress probably activates ethanolic fermentation in the coleoptiles of all plants. However, ADH activity in anoxic rice coleoptiles was 2.2and 2.5-fold greater than that in anoxic barley and oat coleoptiles, respectively, and ethanol concentration in anoxic rice coleoptiles was 3.3- and 3.5-fold greater than that in anoxic barley and oat coleoptiles, respectively. These results suggest that the ethanolic fermentation may be more active in the anoxic rice coleoptiles than in the anoxic barley and oat coleoptiles. The ethanol concentration was probably underestimated because ethanol diffusion from tissues was rapid, and only 21% of the ethanol produced was found in rice coleoptiles during 12 h in anoxia (Alpi and Beevers, 1983). Therefore, the amount of ethanol produced in the barley, oat and rice coleoptiles was probably greater than the ethanol concentrations determined in these coleoptiles (Fig. 4). However, it is unlikely that the ethanol diffusion rates from coleoptile tissues are much different among these species because the diffusion rates of rice, oat and wheat coleoptiles were similar (Alpi and Beevers, 1983). Thus, the ethanolic fermentation in the anoxic rice coleoptiles is probably greater than that in the anoxic barley and oat coleoptiles. An active ethanolic fermentation pathway is essential for the continuation of anaerobic glycolysis owing to pyruvate consumption and recycling NAD+ when oxygen supply is limited and glycolysis replaces the Krebs cycle as the main source of ATP (Drew, 1997; Saglio et al., 1999; Tadege et al., 1999; Agarwal et al., 2007). In addition, the importance of ethanolic fermentation under anoxia has been demonstrated by studies on the alcohol dehydrogenase null mutants of several plant species (Johnson et al., 1994; Matsumura et al., 1995; Ellis et al., 1999; Saika et al., 2006). Therefore, activation of ethanolic fermentation was considered to be one of the strategies for plants to survive under anoxia (Drew, 1997; Tadege et al., 1999; Gibbs and Greenway, 2003). The present results (Figs. 1–4) suggest that anoxia tolerance of rice coleoptiles of the seedlings germinated in aerobic conditions was greater than that of barley and oat coleoptiles of the seedlings germinated in aerobic conditions. The soluble sugar concentration in anoxic rice coleoptiles was much greater than that in anoxic barley and oat coleoptiles (Fig. 6). Anoxic barley and oat coleoptiles contained only 17% and 16% of
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soluble sugar of rice coleoptiles. The availability of soluble sugar is essential for operation of glycolysis and fermentation leading to ATP production in plant cells (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). However, the amount of soluble sugars in coleoptiles is very limited, and soluble sugars are mostly supplied to the coleoptiles from their endosperms where starch is the main reserve carbohydrate (Perata et al., 1997; Saglio et al., 1999; Loreti et al., 2003). ␣-Amylase plays a major role in the degradation of reserve carbohydrates to soluble sugars in anoxia (Sun and Henson, 1991; Guglielminetti et al., 1995). Thus, the function of ␣-amylase may be essential to anoxia tolerance. ␣-Amylase was active in all anoxic endosperms (Fig. 5), and soluble sugar concentrations in all anoxia endosperms were not significantly different (Fig. 7). It has also been reported that anoxia wheat seeds, which germinated in an aerobic condition, showed ␣-amylase activity (Goggin and Colmer, 2007). The ratio of soluble sugar concentration in control coleoptiles to the concentration in control endosperms was 12%, 10% and 13% for barley, oat and rice, respectively (Figs. 6 and 7). Therefore, the ratios were similar in all plant species, which suggests that an ability of sugar transport from the endosperms to the coleoptiles of barley, oat and rice may be dependent on sugar concentration in the endosperms. It was also reported that sugar transport among rice cultivars was dependent on sugar concentrations in the endosperms (Huang et al., 2003). On the other hand, the ratio of the concentration in anoxic coleoptiles to the concentration in anoxic endosperms was 2.9%, 2.8% and 16% for barley, oat and rice, respectively (Figs. 6 and 7). Although anoxia stress may inhibit the soluble sugar transport in barley, oat and rice, the inhibition may be much greater in anoxia-intolerant barley and oat than anoxia-tolerant rice. In addition, despite the fact that the consumption of soluble sugars in rice coleoptiles was probably greater than that in barley and oat coleoptiles because of faster rate of ethanol fermentation in rice coleoptiles (Fig. 4), the soluble sugar concentration in rice coleoptiles was greater than that in barley and oat coleoptiles (Fig. 6). These results suggest that the ability to transport sugar from the endosperms to the coleoptiles in rice may be greater than that in barley and oat in anoxia. Consequently, although soluble sugar concentrations among the anoxic endosperms were not significantly different, the sugar availability of anoxic rice coleoptiles became greater than that of anoxic barley and oat coleoptiles (Figs. 6 and 7). The ability for sugar transport from the endosperms to the coleoptiles is probably very important for the availability of soluble sugar in coleoptiles, as soluble sugar is essential to operate anoxic glycolysis and fermentation (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). This ability may be one way to distinguish the capability of coleoptile growth of these plant species in anoxia as well as an anoxia tolerance of these plants.
Conclusion The effects of anoxia on ␣-amylase activity and reserve carbohydrate catabolism underlying coleoptile growth between anoxia-tolerant and anoxia-intolerant cereals germinated in aerobic conditions have not been evaluated thus far Goggin and Colmer (2007). Coleoptile growth and ATP concentration in barley, oat and rice seedlings germinated in an aerobic condition were suppressed by a 24 h-anoxic stress, but the growth and concentration in the rice coleoptiles was much greater than that of the barley and oat coleoptiles (Figs. 1 and 2). According to measures of ethanol concentration and ADH activity (Figs. 3 and 4), ethanolic fermentation may be more active in the rice coleoptiles than in the barley and oat coleoptiles, where glycolysis and ethanolic fermentation are the main source of ATP production. The availability of soluble sugar is essen-
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tial for operation of the fermentation (Perata et al., 1997; Saglio et al., 1999; Gibbs and Greenway, 2003; Mustroph et al., 2006). Soluble sugars in the coleoptiles are very limited and mostly supplied from the endosperms (Perata et al., 1997; Saglio et al., 1999; Loreti et al., 2003). ␣-Amylase was active in anoxic barley, oat and rice endosperms, and soluble sugar concentrations in the anoxic barley, oat and rice endosperms was not significantly different (Figs. 5 and 7). However, the soluble sugar concentration in anoxic rice coleoptiles was much greater than that in anoxic barley and oat coleoptiles (Fig. 6). Therefore, anoxia stress may inhibit the soluble sugar transport to the coleoptiles from the endosperms to a much greater extent in anoxia-intolerant barley and oat than in anoxiatolerant rice. An ability to transport sugar from the endosperms to the coleoptiles may be one way to distinguish the coleoptile growth of these plant species in anoxia and anoxia tolerance of these plants.
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