Time requirement for hypoxic acclimation to anoxia tolerance in ricecoleoptiles

Hisashi Kato-NoguchiDepartment of Biochemistry and Food Science, Faculty of Agriculture, Kagawa University, Miki,Kagawa 761-0795, Japan (e-mail: hisashi@ag.kagawa-u.ac.jp; fax: +81-87-891-3086)

Received 18 April 2002; accepted in revised form 12 August 2002

Key words: Anoxia tolerance, ATP, Ethanolic fermentation, Hypoxic acclimation, Rice coleoptile

Abstract

Rice (Oriza sativa L.) seedlings were subjected to hypoxic pretreatment (H-PT; incubated in 5% O2 atmosphere)for various lengths of time followed by a 24-h anoxic stress. Anoxia tolerance of rice coleoptiles was improvedwith increasing duration of H-PT, but H-PT longer than 6 h gave no additional improvement. ATP and ethanolconcentrations in the coleoptiles were increased by H-PT, and the time and pattern of increase in ATP level andethanol production rate were similar to those of increase in the anoxia tolerance. These results suggest that theH-PT may increase anoxia tolerance due to maintenance of anaerobic glycolysis with induction of ethanolic fer-mentation to generate ATP, and hypoxic acclimation to anoxic stress in rice coleoptiles may occur within 6 h.

Abbreviations: H-PT – hypoxic pretreatment or hypoxic pretreated

Introduction

The ability of plant roots to survive anoxic stress canbe increased by hypoxic pretreatment (H-PT; Johnsonet al. (1989); Waters et al. (1991), Andrews et al.(1994), Germain et al. (1997), Ellis et al. (1999), Ka-to-Noguchi (2000)). Rice coleoptiles of the seedlingswere also acclimated by 24 h H-PT and the improvedanoxia tolerance was associated with greater concen-tration of ATP and sustained levels of glycolysis andethanolic fermentation (Kato-Noguchi 2002).

Most studies on this acclimation have used H-PTdurations of 16 h or more (Saglio et al. 1988; Johnsonet al. 1989; Germain et al. 1997; Ellis et al. 1999).These have been few studies to determine the mini-mal time requirement of H-PT for the acclimation toanoxic stress. In the present research, the time re-quirement for the acclimation in rice coleoptiles wasinvestigated in order to clarify the physiological traitsof rice seedlings under anoxic stress conditions.

Materials and methods

Seeds of rice (Oriza sativa L. cv. Nipponbare) wereallowed to germinate and grow in darkness at 25 °Cas described by Kato-Noguchi (2002). After 3 days,uniform seedlings were transferred, in groups of 20,to 9-cm Petri dishes each containing two sheets offilter paper (No 1; Toyo Ltd, Tokyo) moistened with10 ml distilled water.

For H-PT, the Perti dishes were placed into a gas-changing jar (5 l) at 25 °C as described by Kato-Noguchi (2000) and a stream of 5% O2 (balance N2)was passed continuously through the jar at a rate of200 ml min−1 for 0, 1, 2, 4, 6, 12, 18 or 24 h. AfterH-PT, a stream of N2 was passed continuouslythrough the jar at 200 ml min−1 for 24 h. The lengthof the rice coleoptiles was measured with a ruler justbefore and after the anoxic treatment to determinetheir elongation.

For determination of ATP and ethanol concentra-tion, rice coleoptiles were harvested and extracted asdescribed by Kato-Noguchi (2001). ATP was quanti-fied spectrophotometrically according to the methods

183Plant Growth Regulation 39: 183–185, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

described by Mohanty et al. (1993). Ethanol concen-tration was quantified using a gas chromatograph ac-cording to the method of Kato-Noguchi and Watada(1997).

Results and discussion

To determine the minimal time requirement for hy-poxic acclimation, rice seedlings were subjected toH-PT for various lengths of time followed by a 24-hanoxic stress (Figure 1). The elongation of the coleop-tiles of non-pretreated seedlings (0-h H-PT) was 2.9mm during the 24-h anoxia. This elongation was in-creased with increasing duration of H-PT and reacheda plateau value of around 8 mm with a 6-h H-PT. Thisresult indicates that the coleoptile elongation of riceseedlings in anoxia was improved with increasing du-ration of H-PT, but longer H-PT than 6 h gave nosignificant improvement.

A greater level of ATP was found to be a signifi-cant contributory factor to the plant survival underanoxic conditions (Raymond et al. 1985; Menegus etal. 1991; Germain et al. 1997). Thus, the effect ofH-PT on ATP level in rice coleoptiles was determinedafter a 24-h anoxic stress (Figure 2). ATP level wasincreased with increasing duration of H-PT, butlonger H-PT than 6 h gave no significant increase.Coleoptiles with 6-h H-PT contained 2.1-fold asmuch ATP as 0-h H-PT coleoptiles. This result indi-

cates that the availability of ATP in rice coleoptilesassociated with survival under the anoxic stress con-dition was increased with increasing duration of H-PTup to 6 h.

When oxygen becomes limiting, glycolysis re-places the Krebs cycle as the main source of energyto generate ATP and ethanolic fermentation is re-quired for the continuation of active glycolysis owingto pyruvate consumption (Kennedy et al. 1992;Ricard et al. 1994; Drew 1997). H-PT was consideredto increase anoxia tolerance in several plant roots be-cause H-PT immediately induced ethanolic fermenta-tion in them after exposure to anoxia (Germain et al.1997; Ellis et al. 1999). For the estimation of activityof ethanolic fermentation in rice coleoptiles immedi-ately after exposure to anoxia, the production rate ofethanol, the end product of the pathway, was deter-mined during the first h. The ethanol production ratewas low in 0-h H-PT coleoptiles, but increased withincreasing duration of H-PT up to 6 h exposure (Fig-ure 3). The rate of production in 6-h H-PT coleop-tiles was 3.1 �mol g−1 fresh weight, which was 3.9-fold greater than that of 0-h H-PT. This resultindicates that the activity of ethanol fermentation im-mediately after exposing anoxia in rice coleoptileswas increased with increasing duration of H-PT up to6 h.

Anoxia tolerance of rice coleoptiles was increasedwith increasing duration of H-PT until 6 h and thenleveled off (Figure 1). ATP concentration and ethanol

Figure 1. Effect of duration of H-PT on rice coleoptile elongation.Rice seedlings were treated with various durations of H-PT andexposed to a 24-h anoxic stress. The length of the coleoptiles wasthen measured. Means ± SE from three independent experimentswith 20 plants for each determination are shown.

Figure 2. Effect of duration of H-PT on ATP level in rice coleop-tiles. Rice seedlings were treated with various durations of H-PTand exposed to a 24-h anoxic stress. ATP concentration in rice co-leoptiles was then determined. Means ± SE from three independentexperiments with five assays for each determination are shown.

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production were also increased by H-PT (Figures 2and 3), and the time and pattern of increase in ATPlevel and ethanol production rate were similar tothose of the increase in anoxia tolerance. These re-sults suggest that hypoxic acclimation to anoxic stressin rice coleoptiles may occur within 6 h.

References

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Figure 3. Effect of duration of H-PT on the ethanol production ratefor the first h after the onset of anoxia. Rice seedlings were treatedwith various durations of H-PT and exposed to anoxia. The rate ofethanol production was determined 1 h after the onset of anoxia.Means ± SE from three independent experiments with five assaysfor each determination are shown.

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