JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2453-2463; doi:10.1093/jxb/eri238
RESEARCH PAPER |
Manipulation of ethanol production in anoxic rice coleoptiles by exogenous glucose determines rates of ion fluxes and provides estimates of energy requirements for cell maintenance during anoxia
1School of Plant Biology, Faculty of Natural and Agricultural Sciences, The University of Western Australia, 35 Stirling Highway, Crawley 6009 WA, Australia
2Department of Developmental Biology and Neuroscience, Graduate School of Life Sciences, Tohoku University, 980-8578 Sendai, Japan
To whom correspondence should be addressed. Fax: +61 8 6488 1108. E-mail: tdcolmer{at}cyllene.uwa.edu.au
Received 22 February 2005; Accepted 29 May 2005
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Abstract |
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Ethanol production by anoxic, excised, 7–10 mm tips of rice coleoptiles was manipulated using a range of exogenous glucose concentrations. Such a dose–response curve enabled good estimates at which level of ethanol production (and hence by inference ATP production), injury commenced and also allowed assessments of energy requirements for maintenance in anoxia. Rates of net uptake or loss of K+ and P by these excised coleoptile tips were related to rates of ethanol production (r2 of 0.59 and 0.68, respectively). At 72 h anoxia, ATP levels in excised tips were similar at 0, 2.5, and 50 mol m–3 exogenous glucose, despite large differences in the inferred rates of ATP production. At 96 h anoxia, tips without exogenous glucose had low ATP concentrations; these may be the cause or the consequence of cell injury. In tips without glucose, injury was indicated by losses of K+ and Cl– between 72–96 h anoxia, and during the first hour after re-aeration, while later than 1 h after re-aeration, rates of net uptake were substantially lower than for re-aerated tips previously in anoxia with exogenous glucose. Between 96 h and 124 h anoxia, ion losses from tips without exogenous glucose increased while recovery of net uptake after re-aeration was very sluggish and incomplete. The energy requirement for maintenance of health and survival of anoxic coleoptile tips, expressed on a fresh weight basis, was lower than for three other anoxia-tolerant plant tissues/cells, studied previously. However, the energy requirement on a protein basis was assessed at 1.4 µmol ATP mg–1 protein h–1 and this value is 2.6–5.4-fold higher than for the other plant tissues/cells. Yet, this requirement was still only 58–88% of the published values for aerated tissues. The reason for this relatively high ATP requirement per unit protein in anoxic rice coleoptiles remains to be elucidated.
Key words: Anoxia, ATP, cell maintenance, energy requirement, ethanol production, ion transport, re-aeration, rice coleoptile, sugar
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Introduction |
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Coleoptiles of rice (Oryza sativa L.) are very anoxia-tolerant (Menegus et al., 1991
; Pearce and Jackson, 1991). For example, shoots of intact rice seedlings survived 19 d of anoxia, initiated on the second day after germination (Couee et al., 1992). During anoxia, growth of the coleoptile continues, whereas growth of leaves and roots ceases (Atwell et al., 1982; Alpi and Beevers, 1983). The elongation of coleoptiles is of adaptive significance when seeds are sown in flooded fields, since they reach a source of O2, be it in the air or produced from photosynthesis, so that the coleoptile will function as a snorkel (Kordan, 1974).
Energy produced during anoxia in rice is nearly entirely via glycolysis linked to ethanol production (chemical analysis in Menegus et al., 1991, and 1H NMR spectroscopy in Menegus et al., 1988), as it is in many other species, at least after the first 3–4 h of anoxia (ap Rees et al., 1987; Ricard et al., 1994). In coleoptiles of an anoxia-tolerant rice genotype, the ratio of hexose flowing to ethanol under anoxia to hexose catabolized during oxidative phosphorylation was 2.0–2.7 (Gibbs et al., 2000), so that ATP production was 4.4–9.0-times lower in anoxia than in air (using the assumptions by Gibbs and Greenway, 2003). During such an energy crisis, the energy apportionment in rice coleoptiles favours elongation and fresh weight increments rather than protein synthesis (Alpi and Beevers, 1983). Energy-dependent solute transport may also have priority during an energy crisis. Firstly, sugar is required as the substrate for glycolysis. Secondly, osmotic solutes are required for cell expansion (Menegus et al., 1984; Huang et al., 2003a). Thirdly, the acid load, imposed by vacuoles, requires continued solute transport at the tonoplast to avoid acidosis of the cytosol (Greenway and Gibbs, 2003).
A key objective of the present work was to obtain a dose–response curve to exogeneous glucose for anoxic rice coleoptiles. Our previous papers on rice coleoptiles have evaluated responses to anoxia only in terms of the absence, or presence, of exogenous glucose at 20 mol m–3; assessing energy requirements for maintenance and reductions in membrane permeability to K+ (Colmer et al., 2001), as well as recovery of ion uptake after return to air (Colmer et al., 2001; Huang et al., 2003b). Further elucidation of the response of rice coleoptiles to anoxia would be greatly facilitated by measuring the response at a range of rates of ethanolic fermentation, and hence ATP production. Firstly, the dose–response curves can be used to measure glucose net uptake by anoxic tissues of a vascular plant, in the present experiments lasting 120 h. So far, glucose uptake in tissues of vascular plants under anoxia has only been measured for 1–2 h (reviewed by Greenway and Gibbs, 2003). Secondly, manipulation of the rate of ethanol formation is crucial to evaluate the consequences of reducing the energy production in tissues already under an energy crisis; so far this has only been done with yeast (Verduyn et al., 1990). Similar manipulation should be possible with vascular plants under anoxia, as indicated by the kinetics of glucose uptake over 15 min in anoxic maize root tips (Xia and Saglio, 1988). Thus, it should be possible to assess the hierarchy of energy allocation to different metabolic processes during an energy crisis and the minimum energy requirement for cellular maintenance (i.e. the ATP production below which injury occurs). Such a study requires tissues with a high tolerance to anoxia and, preferably, as homogeneous as possible; in the present experiments excised 7–11 mm tips of rice coleoptiles provided a suitable model system because these tips do not include leaf tissues (cf. Huang et al., 2003b).
The dose–response curve chosen was for exogenous glucose between 0 and 50 mol m–3. The primary achievement of the present study was the determination of the consequences of a range of ATP production rates, with emphasis on gaining a more precise estimate of energy requirements for maintenance in anoxia than in earlier studies, and on rates of net K+, Cl–, and P uptake, or loss, during anoxia as related to energy production. After re-aeration, detailed time-courses of ion uptake or loss were also obtained, but only at 0 and 20 mol m–3 exogenous glucose. The assessed energy requirements for maintenance during anoxia, of these coleoptile tips, were compared with energy requirements of other anoxia-tolerant tissues, as reviewed by Greenway and Gibbs (2003).
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Plant culture
Dehulled seeds of rice (Oryza sativa L. cv. Amaroo) were surface-sterilized in dilute sodium hypochlorite for 10 min and then washed thoroughly in deionized water. The seeds were then transferred to a PVC vessel containing 4.0 l of aerated solution with final composition in mol m–3: Ca2+, 0.68;
0.5; MES, 0.5; the pH had been adjusted to 6.5 using Ca(OH)2. The vessels were sterilized and the solutions were autoclaved (before adding MES); all procedures were performed in a laminar-flow hood to minimize contamination. Approximately 200 seeds were placed in each vessel and they were kept in the dark at 30 °C. The seedlings were then exposed to a 16 h hypoxic pretreatment (0.028 mol O2 m–3) before starting the experiments.
Healing and anoxic treatments
All treatments were at 30 °C. Coleoptile tips of 7–10 mm were excised from hypoxically pretreated seedlings, these tips did not contain leaf tissues. Tips were the top half of the coleoptile of each seedling; the bottom one-third contains leaf tissues inside the coleoptile and was therefore avoided. After excision, there was always 10 g m–3 carbenicillin in the incubation solutions. Excised coleoptile tips were placed in ‘Thunberg tubes’ and then incubated for 5 h in the hypoxic solution, containing 20 mol m–3 exogenous glucose, prior to imposing anoxia in these tubes. This procedure allowed healing of the damage due to cutting (called ‘ageing’ in Colmer et al., 2001), while avoiding any confounding between anoxia and injury, which might have occurred if the tissues had been transferred to other vessels at the start of anoxia. Thunberg tubes were made as described by Zhang et al. (1992).
Anoxia was imposed by replacement of the hypoxic solution in the Thunberg tubes by 10 ml of anoxic nutrient solution followed by continuous flushing with high-purity N2. The nutrient solution was at pH 6.5 and contained in mol m–3: MES, 0.50; K+, 0.25; Ca2+, 0.50; Mg2+, 0.10; 
0.10; 
0.20; 0.44; 
0.10; Fe-EDTA, 0.0125, and in mmol m–3: Cl–, 250; 
6.25; Mn2+, 0.50; Zn2+, 0.50; Cu2+, 0.125; 
0.125; and Ni2+, 0.25. Exogenous glucose concentrations were: 0, 1.0, 2.5, 5.0, 20, and 50 mol m–3. All gases were flushed through water columns to avoid the loss of solution from the tubes, which would have confounded calculations of ion uptake or loss from the medium. Analyses of solutions from tubes without tissues confirmed that this flushing prevented changes in ion concentration.
Coleoptile tips were exposed to anoxia for up to 120 h. (There was no long-term aerated treatment, since under such conditions even intact coleoptiles senesce; the accompanying physiological changes have best been shown by Ishizawa et al. [1999]; the energy charge in aerobic coleoptiles was lower than in anoxic coleoptiles at 3 d and onwards after germination.) The O2 concentration in the gas space of the Thunberg tubes during anoxic treatments was below the detection limit of 0.01%, as analysed by gas chromatography (KOR-75, GL Science, Tokyo, Japan). The solutions were refreshed with anoxic solution and collected every 24 h. Ethanol was trapped in vials containing ice-cold water. All samples were stored at –20 °C. The recovery of ethanol from this system, after 24 h of flushing with N2 gas, was 91% and the data were corrected accordingly. For counting bacteria in the incubation solutions, serial dilutions were plated on Petri dishes containing tryptic soy agar and then placed in an anaerobic PVC vessel at 30 °C. Bacterial numbers in incubation solutions after 120 h of anoxia at 20 mol m–3 exogenous glucose were less than 1.7±0.3 x106 ml–1. The total calculated fresh weight of bacteria in culture solution was less than 0.013% of the fresh weight of coleoptile tips.
Coleoptile tips sampled for fresh weight, sugar, and protein measurements were washed for 3x3 min with anoxic culture solution without glucose and then stored at –20 °C. However, tips designated for ATP analysis were not washed, but were collected on a sieve and then killed by plunging the sieve into liquid N2 within 5 s after the Thunberg tube had been opened. This procedure caused only a few per cent increase in ATP, as verified by a time sequence over 20 s (data not shown). Each treatment had three replicates.
Re-aeration
Coleoptile tips which had been in anoxia at 0 or 20 mol m–3 exogenous glucose were re-aerated for 48 h following 72, 96, or 120 h of anoxia. During re-aeration, all nutrient solutions contained 20 mol m–3 exogenous glucose and were sampled and renewed after 1, 2, 4, 6, 8, 24, 36, and 48 h. The tips remained in the same Thunberg tubes with 10 ml of solution for the first 8 h, and then were transferred to 50 ml in conical flasks. The solutions were stored at –20 °C prior to measurements of K+, Cl–, and P. Each treatment had three replicates.
Analytical procedures
In all assays, the same spectrophotometer (Shimadzu, Model UV-240, Tokyo, Japan) was used. Biochemicals were purchased from Sigma or CalBiochem.
Ethanol in the treatment solutions and traps was assayed in a 1 ml cuvette containing 100 mol m–3 glycylglycine buffer at pH 9.0 also containing 300 mol m–3 KCl, 1.7 mol m–3 NAD+, and 0.3 units aldehyde dehydrogenase (adapted from Beutler, 1984). The reaction was started by addition of 90 units of alcohol dehydrogenase and monitored at 340 nm.
ATP in the coleoptile tips was extracted in ice-cold 10% perchloric acid. After centrifugation at 15 000 g for 10 min at 4 °C, the supernatant was neutralized to pH 7.6–8.0 by the addition of 3 kmol m–3 KOH and 50 mol m–3 TES, also containing 5 mol m–3 EDTA. ATP in the extract solution was assayed according to Trautschold et al. (1985). A cuvette (1 ml) contained 33 mol m–3 HEPES buffer at pH 7.2, 30 mol m–3 MgCl2, 6 mol m–3 glucose, 1 mol m–3 NADP, and 2 units of glucose-6-phosphate dehydrogenase. The reaction was started by the addition of 2 units of hexokinase and the NADPH produced was determined by measuring the increase in absorbance at 340 nm. At the end of the reaction, 2 µmol of ATP were added to the cuvette to evaluate the efficiency of the ATP measurement.
Glucose in the treatment solutions was assayed in a 1 ml cuvette containing 66 mol m–3 potassium phosphate buffer at pH 7.7, 3.8 mol m–3 Mg2+, 1.5 mol m–3 ATP, and 1.5 mol m–3 NADP (Kunst et al., 1984). The reaction was initiated by adding 2 units of hexokinase and 3.6 units of glucose-6-phosphate dehydrogenase, and measured at 340 nm.
Sugar concentrations in the tips were measured after extraction two times in 4 ml of 80% ethanol boiled with reflux for 20 min. Sugars were measured using anthrone; this reagent is strongly acidic so all polymers of hexoses would be hydrolysed (Yemm and Willis, 1954). To compare the results with ethanol formation the values are expressed as equivalent ‘hexose units’.
Ethanol-insoluble dry weight after extraction with 80% ethanol was measured in a separate experiment for coleoptile tips at 2.5 and 50 mol m–3 exogenous glucose.
Soluble protein concentrations in coleoptile tips extracted with 50 mol m–3 potassium phosphate buffer were measured according to Bradford (1976).
In the culture solutions, P was measured using the molybdate-malachite green method (Motomizu et al., 1983) and K+ by flame photometry (Corning Medical and Scientific, Model 410, Cambridge, UK). Cl– was measured with a Buchler-Cotlove chloridometer (Buchler Instruments, Model 4–2008, Fort Lee, New Jersey, USA).
O2 uptake by coleoptile tips after healing was measured at 30 °C using a Clark-type electrode to monitor the depletion of O2 in a vigorously-stirred solution in a sealed cuvette. The solution was an air-saturated nutrient solution identical in composition to that provided to the tips in the anoxic experiments. The solution contained 20 mol m–3 exogenous glucose and 10 g m–3 carbenicillin.
Sugar ‘budget’
Sugar budgets in coleoptile tips after 120 h anoxia at exogenous glucose between 1 and 5 mol m–3 were calculated by integrating three sets of data: sugar consumption via ethanol production, glucose uptake from the medium, and changes in endogenous sugar content. Reserve carbohydrates insoluble in 80% ethanol were not measured, but in whole coleoptiles these polymers, including starch, only decreased by 0.07 µmol hexose units g–1 FW h–1 over 48 h anoxia (Colmer et al., 2001), i.e. less than 2.5% of the ethanol production of coleoptile tips at 1–5 mol m–3 exogenous glucose. For tips at 20 or 50 mol m–3 exogenous glucose, such budgets could not be made, since at these concentrations the measurement of glucose uptake by depletion in the solution had not sufficient reliability. Instead, estimates of glucose uptake were calculated from data on: hexose used to produce ethanol+change in tissue content of hexose equivalents+hexose used in polymer synthesis+hexose used to make amino acids. Polymer synthesis was evaluated as ethanol-insoluble dry weight (assumed to be cellulose for the budgets), while amino acid synthesis in anoxic rice coleoptiles was estimated from Menegus et al. (1984). Menegus et al. (1988) did not detect any substantial amounts of other soluble organic compounds accumulating in anoxic rice coleoptiles.
Statistical analyses of data
Data sets were analysed using the program of general analysis of variance of Genstat 4.2 (5th Edition, Lawes Agricultural Trust, Rothamsted Experimental Station).
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Growth
At 20–50 mol m–3 exogenous glucose, fresh weight and/or dry weight increments by coleoptile tips in anoxia were found in three out of five experiments, while there was never growth at 2.5 mol m–3 glucose or below. In a detailed experiment, relative growth rates (fresh weight basis) of the coleoptile tips were correlated with sugar supply (r2=0.95); there was no growth at 0 mol m–3 exogenous glucose while there was some growth at 20 and 50 mol m–3 glucose, with the highest growth rates at 50 mol m–3 exogenous glucose (Table 1). Furthermore, ethanol-insoluble dry weight at 50 mol m–3 glucose increased by 2.0 mg g–1 FW d–1 during 120 h anoxia, while there were no appreciable changes in ethanol-insoluble dry weight of tips at 2.5 mol m–3 exogenous glucose (Table 1). The reason for the differences in growth response between experiments is unknown.
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Sugar concentrations
Sugar concentrations in coleoptile tips after 120 h in anoxia were dependent on concentrations of exogenous glucose (Table 2). The initial sugar concentration in the tips increased by 13% at 50 mol m–3 exogenous glucose and decreased by 17, 76, 80, and 94%, respectively, at 20, 2.5, 1, and 0 mol m–3 exogenous glucose (Table 2).
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Glucose uptake
Rates of glucose uptake (µmol g–1 FW h–1) by coleoptile tips during 0–120 h in anoxia were: 0.85 (tips at 1 mol m–3 exogenous glucose), 1.5 (2.5 mol m–3), and were estimated at 2.9 (20 mol m–3) and 3.6 (50 mol m–3), implying curvilinearity above 2.5 mol m–3 glucose. At 1 and 2.5 mol m–3 exogenous glucose, the uptake rates were steady with time, despite the large decreases in internal sugar concentrations in these tips (Table 2). The estimated rate of glucose uptake at 2.9 µmol g–1 FW h–1 by coleoptile tips at 20 mol m–3 glucose, was comparable with 2.6 µmol g–1 FW h–1 for 3–5-d-old excised whole rice coleoptiles also supplied with 20 mol m–3 glucose (Colmer et al., 2001
).
In the present study, optimum glucose supply might not have been achieved, however, reassuringly the increase in sugar concentration in tips at 50 mol m–3 (Table 2) indicated that at this exogenous glucose concentration, sugar supply was adequate. Concentrations of glucose higher than 50 mol m–3 were not used, due to possible confounding with water deficits. At 50 mol m–3 glucose, the osmotic pressure is 0.14 MPa, while osmotic pressure in rice coleoptiles ranged between 0.32–0.69 MPa (Atwell et al., 1982; Menegus et al., 1984).
Ethanol production
Rates of ethanol production in rice coleoptile tips were measured to assess the rates of ATP synthesis; this is feasible since, as stated in the Introduction, fermentation in rice coleoptiles consists nearly entirely of ethanol production. At 20 or 50 mol m–3 exogenous glucose, the rates of ethanol production by coleoptile tips decreased substantially after 24 h of anoxia, while further decreases between 48 and 120 h were only slight; the mean rates for this latter period were 5.8 and 7.2 µmol g–1 FW h–1, for 20 and 50 mol m–3 glucose, respectively (Fig. 1). At 0–5 mol m–3 exogenous glucose, rates of ethanol production by tips during the first 24 h in anoxia were already progressively lower the lower the exogenous glucose, and subsequently were much lower than at 20 or 50 mol m–3 exogenous glucose (Fig. 1). Between 24–120 h of anoxia, the average rates of ethanol production at 0 and 5 mol m–3 exogenous glucose, were 0.75 and 3.6 µmol g–1 FW h–1, respectively (Fig. 1).
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Ethanol formation largely depended on glucose uptake. At 1 and 2.5 mol m–3 exogenous glucose, only 40% and 30% of the ethanol formed was derived from the initial endogenous sugars; the remainder would have been derived from absorbed glucose, accounting for almost all the glucose absorbed. For coleoptile tips at 0, 1, or 2.5 mol m–3 exogenous glucose, sugar consumption via ethanol production was 88–98% of the sum of glucose uptake and decreases in endogenous sugar content. At 20 and 50 mol m–3 exogenous glucose, there was flow of hexose to sinks other than ethanol formation, primarily associated with cell expansion; for example, in tips with 50 mol m–3 glucose flow to these other sinks contributed 12% of the known hexose consumption. For tips at 50 mol m–3 exogenous glucose, flow of hexose units (µmol g–1 FW) during 120 h anoxia was: 485 to ethanol, 33 to endogenous sugars, at most 15 to amino acids, 55 to ethanol-insoluble dry weight; giving a total of 588 µmol hexose units.
Energy production and ATP levels
Rates of O2 uptake by coleoptile tips in air-saturated solution were measured to evaluate the rates of energy production during aeration. The rate of O2 uptake was 18 µmol O2 g–1 FW h–1 during the first 3 h after ‘healing’. It was assumed the net yield of ATP in aeration, i.e. during oxidative phosphorylation, is 24–36 mol per mol of glucose respired. The value of 24 assumes a 40% contribution by the alternative oxidase, yielding only 1 rather than 3 mol ATP per mol glucose respired; 40% was the highest value obtained for any plant tissue using the discrimination technique (Lambers et al., 1998). Since 1 mol hexose consumption in glycolysis linked to the TCA cycle requires 6 mol O2, then ATP production in aerated coleoptile tips was estimated at 72–108 µmol g–1 FW h–1.
To assess the rate of ATP production in anoxia, 1 mol hexose consumption in glycolysis linked to ethanol production was assumed to produce 2 mol ethanol and 2 mol ATP. The highest rates of ethanol formation and hence ATP production in anoxic tips were attained during the first 24 h of anoxia at 50 mol m–3 exogenous glucose, but even these rates of 10.5 µmol g–1 FW h–1 were 7–10-fold lower than the assessed ATP production in aerated tips. These large decreases in ATP production in anoxia occur despite hexose being fermented in coleoptile tips during the first 24 h of anoxia at 50 mol m–3 exogenous glucose increased to 175% of the amount respired in aeration (Fig. 1). Similarly, for excised whole rice coleoptiles with 110 mol m–3 exogenous glucose, glycolysis in anoxia was 150% faster than in aeration, and ATP production was 8.5–13-fold lower in anoxia than in air (estimated from data in Menegus et al., 1991). The present study also evaluated longer-term responses, and for tips with 50 mol m–3 exogenous glucose, from 72–120 h anoxia, glycolysis was 125% the rate in aeration (Fig. 1). It is important to establish whether ATP production would have been augmented by any oxidative phosphorylation due to possible contamination by O2. Purified N2 gas and Tygon tubing were used, so any possible O2 contamination would have been very slight. Nevertheless, the degree to which traces of O2 would affect ATP production needed to be evaluated in view of the very high efficiency of oxidative phosphorylation compared with ethanolic fermentation. The O2 concentration in the gas phase of the vessels used in the present study was less than 0.01%, which was the limit of detection of the gas chromatograph used. Taking the worst possible scenario, i.e. an O2 impurity of 0.01%, the maximum contribution to energy production via oxidative phosphorylation in the coleoptile tips in these experiments would still have been only 0.8 µmol ATP g–1 FW h–1; this assessment is based on a curve of O2 uptake rates against external concentrations (S Huang, K Ishizawa, H Greenway, TD Colmer, unpublished data).
ATP concentrations in coleoptile tips at 0, 2.5, or 50 mol m–3 exogenous glucose were measured after 72 h in anoxia, and were found to be similar; in all cases being 30–40% lower than in aerated tips (Table 3). The similar ATP concentrations in anoxic tips without or with 50 mol m–3 glucose is remarkable, in view of the large differences in rates of ethanol production (Fig. 1), and hence, by inference, ATP production. Clearly, in tips without glucose, ATP consumption must have been very much lower than in tips with 50 mol m–3 glucose; for example, between 48 and 72 h, ethanol production (and hence ATP production) by tips was 11-fold lower without exogenous glucose than in 50 mol m–3 glucose (Fig. 1). After 96 h in anoxia, ATP concentrations in tips without exogenous glucose were only 19% and 26% of the values in tips with 2.5 and 50 mol m–3 exogenous glucose, respectively (Table 3).
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Net uptake or loss of K+ and P during anoxia
Taken over the entire experimental period, rates of net uptake or loss of K+ and P were correlated with rates of ethanol production (Fig. 2). The details of net ion uptake and loss are shown in Fig. 3. For coleoptile tips at 20 and 50 mol m–3 exogenous glucose, rates of net uptake were similar during 120 h anoxia for both K+ and P; at both these glucose levels the rates decreased with time from 130 to 50 nmol g–1 FW h–1 for K+, and from 115 to 65 nmol g–1 FW h–1 for P (Fig. 3). At 0, 1.0, or 2.5 mol m–3 exogenous glucose, rates of net uptake of K+ and P between 0–72 h of anoxia were only about one-third of the values for tips at 20 or 50 mol m–3 exogenous glucose (Fig. 3). Between 72–96 h anoxia, net losses of K+ and P commenced from tips without exogenous glucose, and between 96–120 h anoxia net losses commenced from tips at 1.0 mol m–3 glucose (Fig. 3). Between 96–120 h anoxia, the net losses from tips without exogenous glucose reached 167 and 36 nmol g–1 FW h–1, respectively, for K+ and P (Fig. 3).
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Net uptake or loss of K+, Cl–, and P after return from anoxia to aerated solution
Tolerance of coleoptile tips to anoxia was also evaluated by the response of ion uptake or loss during re-aeration following anoxia. This response was tested for coleoptile tips that had previously been in anoxia at 0 or 20 mol m–3 exogenous glucose (Fig. 4). The patterns of net uptake or loss during anoxia were similar to those discussed in the previous section, however, the rate of net loss of K+ was three times larger in the experiment presented in Fig. 4 (cf. Figs 3 and 4A). Tips previously in anoxia for 96 or 120 h without exogenous glucose lost ions during the first hour of re-aeration. These losses were for K+: 2.5 and 3.2 µmol g–1 FW h–1; and for Cl–: 0.26 and 0.35 µmol g–1 FW h–1 (Fig. 4A, C). Rates of net uptake of K+ and Cl– increased with time after re-aeration. Nevertheless, maximum rates of uptake in the tips ‘starved’ of sugars during anoxia were only about 30–35% of those in tips which had received 20 mol m–3 exogenous glucose during anoxia (Table 4), even though all tips were in 20 mol m–3 exogenous glucose since the start of re-aeration.
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Tips, that had been at 20 mol m–3 exogenous glucose during anoxia for 72 or 120 h, showed large increases in the rates of net uptake of K+ and Cl– within 2 h after the start of re-aeration, reached a maximum at 4.5 h, and then declined (Fig. 4A, C). However, even in this glucose treatment, rates of net uptake of K+, Cl–, and P were slow during the first hour of re-aeration (Fig. 4). This lag could be due to: (i) during re-aeration high ion efflux may resume more rapidly than influx. This notion is feasible since, in whole excised rice coleoptiles, K+ efflux is 10-fold lower in anoxia than in continuous aeration (Colmer et al., 2001
). (ii) There might be insufficient energy production during the initial period of re-aeration. After prolonged anoxia, the ability to produce energy via oxidative phosphorylation might be inhibited and recover only gradually. There are no convincing data on this issue. (iii) Membrane injury might occur during sudden re-aeration, due to free radicals of oxygen (Smirnoff, 1995; Blokhina et al., 2003). (iv) Processes other than ion uptake might have higher priority to receive energy upon re-aeration. During the first hour of re-aeration, the patterns of net uptake or loss of P (Fig. 4B) were similar to the patterns of net uptake or loss of K+ and Cl–; net losses of P to the medium from coleoptile tips previously in 96 and 120 h of anoxia at 0 mol m–3 exogenous glucose were 0.35 and 0.7 µmol g–1 FW h–1, respectively (Fig. 4B). However, in contrast to K+ and Cl–, net P uptake after re-aeration of tips that had been at 0 mol m–3 exogenous glucose during anoxia, increased to the same extent as tips that had been at 20 mol m–3 exogenous glucose. In both cases, net P uptake increased gradually and the maximum rates of 1.0–1.3 µmol g–1 FW h–1 were reached between 24–36 h after re-aeration (Fig. 4B). There is no ready explanation for this difference in response of P versus K+ and Cl–.
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Rates of ethanol production manipulated by exogenous glucose determine the tolerance of rice coleoptile tips to anoxia
The present study demonstrated that rates of ethanol production during anoxia by tips of rice coleoptiles, manipulated by exogenous glucose, determined their degree of anoxia tolerance. For example, tips at 20 or 50 mol m–3 exogenous glucose maintained high rates of ethanol production, and these tips survived 120 h anoxia as supported by: (i) growth, albeit slowly, and only in three out of five experiments (Table 1); (ii) net uptake of K+ and P over 120 h anoxia, although this uptake was only a fraction of the maximum rates of uptake in aerated solution (Fig. 3); (iii) maintenance of stable ATP concentrations (Table 3); and (iv) rapid recovery of net uptake of K+, Cl–, and P upon re-aeration after anoxia (measured at 20 mol m–3 glucose only; Fig. 4). By contrast, injury of the coleoptile tips at 0 or 1.0 mol m–3 exogenous glucose after 72 h and 96 h anoxia was indicated by net losses of K+ and P, both during anoxia (Figs 3, 4) and during the first hour after re-aeration (only measured for tips without glucose; Fig. 4). The improvement of anoxia tolerance by high concentrations of exogenous glucose is consistent with previous observations for whole rice coleoptiles and tissues of several other plant species (reviewed by Gibbs and Greenway, 2003
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Pattern of net solute uptake or loss during anoxia, and during re-aeration
Substantial glucose uptake by the rice coleoptile tips is consistent with results using other anoxia-tolerant tissues (reviewed by Gibbs and Greenway, 2003). Nevertheless, the present study is the first unequivocally to establish that anoxic plant tissues can take up substantial amounts of glucose over long periods of anoxia (120 h) from exogenous concentrations as low as 1.0 mol m–3, while at 2.5 mol m–3 this uptake provided sufficient substrate to sustain ethanol formation for at least 120 h (Fig. 1). Tips at 2.5 mol m–3 exogenous glucose did not lose K+ or P, even between 96 h and 120 h anoxia (Fig. 3), showing that these tips did not suffer injury. The dose–response curve, at exogenous glucose between 1.0 and 50 mol m–3, also showed that 50 mol m–3 was sufficient to elicit increases in endogenous sugar concentration. Previously, several authors have used 100–110 mol m–3 glucose (Menegus et al., 1991; Xia and Saglio, 1992), with possible effects of high external osmotic pressures, without evidence that such high concentrations are required to provide sufficient substrate.
The much lower rates of net uptakes of K+ and P by the coleoptile tips during anoxia than after return from anoxia to air (Fig. 4A, B) are consistent with the pattern for excised ‘whole coleoptiles’ (Colmer et al., 2001). Dramatic reductions in Cl– transport, as documented here for coleoptile tips during anoxia (Fig. 4C), were also found for anoxic maize roots (Gibbs et al., 1998) and for anoxic storage tissue of red beet (Zhang and Greenway, 1995). It would be worthwhile to establish whether, or not, ion transport is inhibited by anoxia in stems of Potamogeton, since, in axoxia, these stems sustain vigorous extension growth (Summers and Jackson, 1996; Ishizawa et al., 1999).
Coleoptile tips which had been in anoxia for 96 h and 120 h without exogenous glucose, showed dramatic net losses of K+, Cl–, and P during the first hour of re-aeration (Fig. 4). These losses of ions upon re-aeration were much larger than the losses during anoxia (Fig. 4), possibly reflecting injury to membranes both during anoxia (cf. Rawyler et al., 1999) and during re-aeration (Smirnoff, 1995; Blokhina et al., 2003).
Assessed ATP production as related to a range of external glucose concentrations
The present study is the first to manipulate ethanol production, in tissues of vascular plants, by a dose–response curve to exogenous glucose, resulting in different rates of ATP production, which were then interpreted in terms of energy requirements for maintenance, as indicated by patterns of net ion uptake or loss. Even in the rice coleoptile tips at 50 mol m–3 exogenous glucose, the estimated rate of ATP production in the quasi steady-state, 48–120 h after the start of anoxia, was only 7.3 µmol g–1 FW h–1, i.e. 10–15-fold lower than the assessed rate in aerated tips, so even at high glucose supply there was a severe reduction in ATP production. Importantly, the present data at 50 mol m–3 exogenous glucose lend further support to the earlier proposed down-regulation of ethanolic fermentation in rice coleoptiles exposed to prolonged anoxia (Colmer et al., 2001); rates of ethanol formation decreased even at 50 mol m–3 exogenous glucose (Fig. 1), despite increases in endogenous sugar concentrations (Table 2). Such down-regulation is consistent with the hypothesis of Felle (2005) that, under an energy-deficit, metabolism is modified. Without exogenous glucose, severe injury occurred when anoxia was prolonged for more than 72 h; deterioration of membrane integrity was indicated by ion losses (Figs 3, 4), a condition that might also have caused the low concentrations of ATP at 96 h anoxia (Table 3). Whether injury is the cause, or the consequence, of the low ATP levels needs to be established with more detailed time-courses.
ATP concentrations in the coleoptile tips in anoxia were lower than in aerated tips, but anoxic tips with 0, 2.5, or 50 mol m–3 exogenous glucose had all shown remarkable homeostasis up till 72 h anoxia (Table 3), despite 2–7-fold differences in rates of ethanol production, as dependent on glucose supply (Fig. 1). ATP consumption must therefore have been reduced to the same degree as the differences in production, so it is concluded that the processes responsible for ATP consumption were not regulated by the ATP concentrations. This is consistent with the conclusion of Xia et al. (1995) that nucleotide levels in maize root tips did not determine their survival. The most likely alternative factor to regulate metabolism in energy-deficient tissues is cytoplasmic pH, which may regulate supply and demand for energy under anoxia, as argued in detail by Greenway and Gibbs (2003). A similar proposition is given by Felle (2005), although he views the pH change more as part of a change in the network of biochemical processes than as the key to regulation.
Energy requirements for maintenance
Use of the range of external glucose concentrations allows better estimates of energy requirements for maintenance, than the previous ones by Colmer et al. (2001), which were based on a single exogenous glucose level of 20 mol m–3. The energy requirement for maintenance can be assessed from the rates of ethanol production by coleoptile tips at the lowest exogenous glucose that endows survival, without growth. Ethanol production by tips in anoxia will give a reasonable estimate of ATP production; use of PPi as an energy donor would cost equivalent amounts of ATP, unless it was produced during macromolecule synthesis (Greenway and Gibbs, 2003), so this energy consumption will also be revealed in the rates of ethanol production. Moreover, in the excised tips used in the present investigation, glucose was provided as substrate, which rules out a substantial contribution to energy production by sucrose breakdown via sucrose synthase. The criterion of ‘maintenance’ disqualifies use for this assessment of the coleoptile tips at 0 or 1.0 mol m–3 exogenous glucose, as net losses of K+ and P started after 96 h and 120 h of anoxia, respectively (see below and Fig. 3). Tips at 2.5 mol m–3 exogenous glucose did not lose ions even after 120 h anoxia (Fig. 3), so the minimum energy requirement for maintenance during anoxia can be estimated from the ethanol production rate by these tips; which was reasonably stable between 48 h and 120 h anoxia, at 3.3 µmol g–1 FW h–1 (Fig. 1), giving an estimate for energy requirement for maintenance of 3.3 µmol ATP g–1 FW h–1.
An important consideration is the possible energy requirement for glucose uptake by the excised coleoptile tips. Engagement of a glucose-H+ co-transporter, with associated energy-dependent H+ extrusion, was indicated for anoxic rice coleoptiles by transient depolarizations of membrane potential following the addition of 10 mol m–3 exogenous glucose (Zhang and Greenway, 1995). If at 2.5 mol m–3 exogenous glucose the entire uptake would be via a glucose-H+ co-transporter, then either the cell would acidify, or the H+ would have to be exported. The former is unlikely, since at 2.5 mol m–3 glucose, the H+ uptake over 120 h would have been 180 µmol g–1 FW, far in excess of the known buffering capacities of plant cells (Kurkdjian and Guern, 1989). So, substantial H+ extrusion is likely and, if needed for all the glucose uptake, 0.75 µmol ATP g–1 FW h–1 would be consumed to extrude H+ for the 1.5 µmol glucose g–1 FW h–1 absorbed, so as to maintain the membrane potential; provided the H+:ATP stoichiometry during the energy deficit was 2:1, as established for the plasma membrane H+-ATPase of Neurospora (Warncke and Slayman, 1980). Alternatively, with a 1:1 stoichiometry of the H+-ATPase the ATP requirement to extrude H+ associated with glucose uptake would have been 1.5 µmol g–1 FW h–1. The stoichiometry of the H+-ATPase in rice coleoptile tips under the conditions used in the present study is unknown, however, a value of 2:1 was found for leaves of Potamogeton lucens in the dark, alternating with 1:1 in the light (Miedema and Prins, 1993).
Comparison of energy requirements for maintenance under anoxia in various tissues
The present data further support the hypothesis that energy requirements for maintenance are reduced during an energy crisis, as proposed by Greenway and Gibbs (2003). In this earlier paper one of the present authors suggested that energy requirements for the maintenance of plant tissues can best be expressed on a protein basis, rather than on a fresh weight basis; since different tissues have various ratios of cytoplasm:vacuole. The data presented here show that the suggestion by Greenway and Gibbs (2003) needs qualification. Data on energy requirements for maintenance in anoxia for four plant systems are given in Table 5 on both a fresh weight and a soluble protein basis. The rice coleoptile tips are distinguished by a low energy requirement in anoxia when expressed on a fresh weight basis. However, the tips have a 2.6–5.4-fold higher requirement than the other anoxic tissues/cells when expressed on a soluble protein basis (Table 5). All the same, the assessed requirements were 58–88% of those required for the maintenance of leaves in air (Table 5). So, the data presented here are consistent with a considerable reduction in energy requirement for maintenance during anoxia, particularly since the coleoptiles were at 30 °C, while the leaves were at 18–21 °C. Higher temperatures tend to increase the energy cost of cell maintenance (Penning de Vries, 1975).
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Further studies are required to elucidate why, under anoxia, tips of rice coleoptiles have so much higher energy requirement for maintenance than other tissues, when expressed on a soluble protein basis. Moreover, the best approach for expressing energy requirements of maintenance under an energy deficit is to use both a fresh weight and a protein basis, while the resulting values then need to be considered further.
Energy apportionment to different metabolic processes
Possible reductions in energy consumption during anoxia, as compared with aerobic tissues, may include: slower growth, including less protein synthesis (Mocquot et al., 1981; Alpi and Beevers, 1983; Ricard and Pradet, 1989) and much lower K+ effluxes (Colmer et al., 2001). At the lower rates of ethanol production, i.e. at low exogenous glucose, further reductions in energy consumption would be required and these may include: further reductions in growth, as shown by less or no net increase in fresh weight or dry weight (Table 1), and reduction in ion uptake rates (Fig. 3).
Coleoptile tips at 50 mol m–3 exogenous glucose produced an additional 2.9–3.7 µmol ATP g–1 FW h–1 than the energy requirement for maintenance, as deduced from tips at 2.5 mol m–3 exogenous glucose (calculated from Fig. 1 and another experiment for which data are not shown). The processes on which the additional energy in tips at 50 mol m–3 glucose was spent have not been accounted for fully. The increase in ethanol-insoluble dry weight in excised rice coleoptile tips at 50 mol m–3 glucose was 2 mg g–1 FW d–1 (Table 1), which may cost 0.46 µmol ATP g–1 FW h–1, if 1 mg of cell wall synthesis from glucose required 5.6 µmol ATP (Stitt, 1998). In this calculation, the second mol of ATP required for incorporation of 1 mol of hexose into a polymer (Stitt, 1998) was not included, since PPi is formed, and it can be used for essential processes during an energy-deficit (reviewed by Greenway and Gibbs, 2003). Net protein synthesis associated with tissue expansion might be one other requirement for the additional energy produced by coleoptile tips at 50 mol m–3 exogenous glucose. Another possibility is consumption of ATP for H+-extrusion if glucose uptake occurred via a H+-glucose co-transporter, but there is insufficient information on the mechanism(s) for glucose uptake from high (20–50 mol m–3) external concentrations (discussed in Greenway and Gibbs, 2003), to consider this further. Summing up, between 10–59% of the higher energy production for tips at 50 mol m–3, compared with 2.5 mol m–3 exogenous glucose, can not yet be accounted for; the range reflects the uncertainty of energy expenditure on glucose uptake at high exogenous glucose.
In conclusion, the model system of tips of anoxia-tolerant rice coleoptiles, combined with a dose–response curve to exogenous glucose, contributed substantially to the elucidation of energy requirements for maintenance during an energy crisis; while delineating when energy supply becomes insufficient to retain endogenous solutes and an ability to survive.
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Acknowledgements |
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Brian Atwell and Malcolm Drew for constructive comments on a draft of this manuscript. Shaobai Huang is grateful to the UWA for a University Postgraduate Award and International Postgraduate Fee Waiver Scholarship. We thank the Grains Research and Development Corporation in Australia for funding the research visit by K Ishizawa to UWA.
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Footnotes |
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* Present address: Department of Biology, Miyagi University of Education, Sendai 980-0845, Japan.
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