Journal of Experimental Botany, Vol. 52, No. 360, pp. 1507-1517,
July 1, 2001
© 2001 Oxford University Press
Original Papers |
Evidence for down-regulation of ethanolic fermentation and K+ effluxes in the coleoptile of rice seedlings during prolonged anoxia
1 Faculty of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia, 6009, Australia
2 Institute of Agrobiological Genetics and Physiology, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, People's Republic of China
Received 13 November 2000; Accepted 13 March 2001
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Abstract |
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Ethanolic fermentation, the predominant catabolic pathway in anoxia-tolerant rice coleoptiles, was manipulated in excised and ‘aged’ tissues via glucose feeding. Coleoptiles with exogenous glucose survived 60 h of anoxia, as evidenced by vigorous rates of K+ and phosphate net uptake and growth of roots and shoots when re-aerated. In contrast, coleoptiles without exogenous glucose showed net losses of K+ and phosphates starting 12 h after anoxia was imposed and these did not recover fully when re-aerated after 60 h of anoxia. Ethanol production (µmol g-1 FW h-1) declined from about 7.5 during the first 12 h of anoxia to 5 or 2.2 after 48–60 h, in coleoptiles with or without exogenous glucose, respectively. Carbohydrate concentrations changed only slightly in anoxic coleoptiles with exogenous glucose due to net glucose uptake at 2.6 µmol g-1 FW h-1. Ethanolic fermentation, and therefore ATP production, may have been down-regulated after an initial period of acclimation to anoxia in coleoptiles with exogenous glucose. Maintenance requirements for energy were assessed to be 3.4–7.6-fold lower in these anoxic coleoptiles than published estimates for non-growing aerated leaf tissues. A modest part of the required economy in energy consumption would have been derived from diminished ion transport; anoxia reduced K+ and phosphate net uptake by 70–90% in these coleoptiles. K+ efflux was 10-fold lower in anoxic than in aerated coleoptiles with exogenous glucose. Using the unidirectional efflux equation, the membrane permeability to K+ was estimated to be 17-fold lower in anoxic than in aerated coleoptiles, presumably due to predominantly closed K+ channels.
Key words: Energy maintenance requirement, ethanolic fermentation, membrane permeability, potassium, phosphate, solute net uptake or loss.
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Introduction |
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Coleoptiles of rice (Oryza sativa L.) seedlings are regarded as one of the most anoxia-tolerant plant tissues (Menegus et al., 1991
; Pearce and Jackson, 1991). For example, rice seedlings survived 19 d of anoxia initiated on the second day after germination (Couee et al., 1992). During anoxia the coleoptile of rice is able to elongate, mainly by cell expansion, whereas no leaf or root growth occurs (Opik, 1973). Shoot growth in anoxia can also occur in the aquatic species Potamegeton (Summers and Jackson, 1994) and in some marsh species when shoots develop from existing buds of rhizomes (Braendle and Crawford, 1987). Shoot elongation establishes contact with air so that internal O2 diffusion to the submerged organs enables survival and growth in the longer term. These phenomena are presumably crucial for the establishment of rice directly seeded into flooded fields.
Fermentation linked to glycolysis provides some ATP during anoxia; in young rice shoots fermentation consisted nearly entirely of ethanol formation (Menegus et al., 1991). The rice cultivar used in the present study increased in glycolysis in the coleoptile by a factor of 1.4 and 1.7 at the end of 1 or 2 d of anoxia imposed on intact seedlings (Gibbs et al., 2000). Despite the acceleration of glycolysis, ATP production in the anoxic coleoptile must have been 7–13 times lower than in aerated conditions, the factor depending on efficiency of ATP synthesis in aerated conditions and whether hexoses or sucrose are the substrates for fermentation. Yet, when there is growth, part of this scarce energy supply has to be directed to protein synthesis (Mocquot et al., 1981; Mohanty et al., 1993) and solute uptake, for example K+ (Atwell et al., 1982). In anoxic rice coleoptiles, the membrane potential of cells remained more negative than the K+ diffusion potential (Zhang and Greenway, 1995); such ‘energized membranes’ should enable uptake of ions and other solutes to be at least partially maintained under anoxia.
The very limited energy supply in anoxic rice coleoptiles explains why growth consists mainly of elongation and volume increases, while dry mass and protein increments are reduced more severely than elongation (Alpi and Beevers, 1983); biosynthesis of polymers such as cell walls and proteins is energetically demanding. Further economies in energy consumption may be achieved by down-regulation of energy-consuming processes not essential to survival. Such reductions in maintenance requirements for energy during anoxia have been suggested for certain animal cells (Hochachka, 1991) and for storage tissue of beetroot (Zhang and Greenway, 1994).
The experiments described in the present report evaluated the effect of anoxia on net uptake or loss of K+ and phosphate in excised rice coleoptiles. Treatments with or without exogenous glucose provided coleoptiles with substantially different rates of ethanolic fermentation and hence of energy production. Time-courses of net ion uptake or loss in anoxia, and recoveries when resupplied with glucose under anoxia or when re-aerated, were evaluated. Use of excised coleoptiles enabled interpretable measurements on ion net uptake or loss, ethanol production, and effects of substrate supply. Preliminary experiments showed that excised and ‘aged’ coleoptiles with exogenous glucose in aerated solution grew and had fast K+ net uptake rates, while anoxia inhibited these processes to an extent similar to that measured for intact coleoptiles.
The results of the present study will be considered in relation to contrasting modes of acclimation to anoxia, which are best demonstrated by earlier work on animal systems. Mode 1 is based on greatly increased (7–10-fold) glycolytic rates; such increases minimize reductions in ATP production, but seldom can be maintained due to substrate exhaustion (Hochachka, 1991). Mode 2 is based on no change in, or often a down-regulation of, the rate of glycolysis. This response needs to be coupled to a drastic slowing down of energy-requiring processes (i.e. slowed metabolism), that in extreme cases leads to ‘metabolic arrest’ (Hochachka, 1991). Large reductions in ion fluxes have been postulated for anoxic animal cells exhibiting Mode 2 (Hochachka, 1991). The experiments described in the present report showed anoxic rice coleoptiles with exogenous glucose had large reductions both in maintenance requirements for energy and also in K+ and phosphate influx and K+ efflux, compared with aerated coleoptiles.
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Materials and methods |
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Plant culture
Dehulled seeds of rice (Oryza sativa L. cv. Calrose) were surface-sterilized with acidic HgCl2 (0.1% w/v, in 0.1% HCl) for 3 min and then washed thoroughly with deionized water. The seeds (
200 per batch) were transferred to a PVC vessel containing 4.0 l of aerated culture solution of composition (mol m-3): Ca2+, 0.5; Cl-, 0.6; MES, 0.5; the pH had been adjusted to 6.5 using Ca(OH)2. The culture vessels were surface-sterilized, solutions were autoclaved (before adding MES), and all procedures were performed in a lamina flow hood to minimize numbers of bacteria. The seeds germinated and grew in the aerated solution (0.25 mol m-3 O2) for 48 h. The seedlings were then exposed to a 16 h hypoxic pretreatment (0.028 mol m-3 O2), after which the coleoptile of each seedling was excised and ‘aged’ for 5 h in hypoxic (0.028 mol m-3 O2) solution (composition same as growth medium given above plus 20 mol m-3 glucose and 10 g m-3 carbenicillin). Tests showed that carbenicillin at 10 g m-3 did not affect K+ net uptake by the coleoptiles. The 5 h of ‘ageing’ was 3 h longer than the time required for net loss of K+ caused by excision to decrease to zero (data not shown). The importance of an ‘ageing’ period was previously demonstrated for excised segments of maize roots (Gronewald et al., 1979). Tests showed no difference in the rates of K+ net uptake by excised coleoptiles after being ‘aged’ in hypoxia or aerated conditions; coleoptiles were therefore routinely ‘aged’ in hypoxia. All experiments were conducted at 30°C in the dark. After ‘ageing’, samples of coleoptiles were transferred to Thunberg Tubes containing 10 ml of solution with the same composition as used during the ‘ageing’ period. The design of the tubes ensured anoxia in solution in those tubes continuously flushed with humidified high purity N2. A gas-tight syringe was used to withdraw, or add, solution through the outlet of each tube, while maintaining gas flow. At selected times treatments were imposed in individual tubes by withdrawal of the solution and injection of a new solution. The anoxic treatment solutions were pre-flushed for several hours with high purity N2 before being injected into the tubes.
The coleoptile was the only visible shoot tissue at the time of excision. Coleoptiles were excised as close as possible to the seed, and therefore some of the very young leaf tissues were enclosed in the coleoptile base. The excised coleoptiles must have also included the so-called ‘coleoptilar node’ (Hoshikawa, 1993) since depending on the growth condition (see Results) the coleoptiles formed about five small adventitious roots near the base. The emergence of leaves and adventitious roots only occurred in excised coleoptiles in aerated solutions; these organs were not visible in excised coleoptiles in anoxic solution for 84 h (the longest time period used in the present study).
Measurements of K+ net uptake and short-term Rb+ uptake
K+ net uptake was determined by measurements of depletion from the external solution with an initial K+ concentration of 0.25 mol m-3. This concentration was based on preliminary experiments in which K+ net uptake rates by coleoptiles were shown to reach a maximum at an external K+ concentration of 0.2 mol m-3 and higher (data not shown). Solutions were replaced regularly so that depletion never exceeded 11% in the anoxic treatment or 21% in the aerated treatment.
Sampling times and other procedures depended on the experiment. In short-term experiments, samples of solution were taken at 0.5–1 h intervals and all treatments were in Thunberg Tubes. In longer term experiments (30–60 h), solutions were sampled every 12 h, or more frequently as indicated in the figures, tables, or text. Samples were stored at 4 °C prior to analyses of K+. In longer term experiments the aerated treatments were in 100 ml of solution in conical flasks, otherwise K+ would have been depleted too much between samplings. Anoxic treatments were always in Thunberg Tubes, depletion in the larger vessels was too small to be measured accurately. Recovery of K+ net uptake by the anoxic coleoptiles when resupplied with glucose (anoxia continued) or when re-aerated (with exogenous glucose) was assessed in some experiments after 30 or 60 h of anoxia. Solution samples were taken every 3 h during this phase of the experiments. The FW of coleoptiles in each replicate was determined before being oven-dried at 70 °C for 2 , and then K+ was extracted by shaking in 500 mol m-3 HCl for 2 d (Hunt, 1982) at room temperature. K+ in the extracts, and also in the samples of external solution, was measured using a flame photometer (Corning Medical and Scientific, Model 410) or an atomic absorption spectrometer (Perkin Elmer, AAnalyst 300).
In some experiments, short-term uptake of Rb+ was measured. After 19 h in aerated or anoxic solutions, the coleoptiles were washed for 3x3 min in incubation solution without KCl (aerated or anoxic, as appropriate) to remove external K+. The influx solution was of the same composition as used previously, except KCl was replaced by 0.25 mol m-3 RbCl. After a 20 or 30 min absorption period (depending on the experiment, see text) the coleoptiles were rinsed for 3x3 min in a solution of the same composition but without Rb+ (or K+). Fresh weights of coleoptiles were determined and tissue K+ and Rb+ analyses were as described above.
Measurements of net loss of phosphates and net uptake of phosphate
Net loss of phosphates from the coleoptiles to the incubation medium was determined, as were changes in total phosphorus concentration in the coleoptiles. In some experiments, 0.05 or 0.10 mol m-3 KH2PO4 was added to the incubation solution (KCl was reduced to maintain K+ at 0.25 mol m-3) so that the net uptake of phosphate could be measured. Coleoptiles assayed for total phosphorus were digested at 180 °C in HNO3/HClO4 (70/30, w/w). Phosphate in the digests and in culture solutions was measured spectrophotometrically (Shimadzu, UV-240) using the molybdate and malachite green method described earlier (Motomizu et al., 1983). Recovery of phosphorus added to the digests was 96.4±1.6%.
Measurements of glucose net uptake, tissue sugars, and tissue starch concentrations
Glucose net uptake by anoxic coleoptiles was measured by depletion. Glucose in samples of solution taken at 0 and 24 h, and then 24 and 48 h of anoxia was assayed using hexokinase/glucose-6-phosphate dehydrogenase (Kunst et al., 1984). Solutions were renewed every 24 h and depletion was at most 14%.
Total sugars (hexose units) in the coleoptiles were extracted from freeze-dried samples in 80% (v/v) ethanol and determined using anthrone (Yemm and Willis, 1954). Starch was assayed by boiling the residue in 3 ml of deionized water for 3 h, after which the solution was incubated at 37 °C. One ml of solution containing 10 units of amyloglucosidase in 100 mol m-3 Na+-acetate buffer at pH 4.6 was added. After 24 h, glucose released from starch into the solution was determined spectrophotometrically using hexokinase/glucose-6-phosphate dehydrogenase (Kunst et al., 1984). Other non-soluble polysaccharides (i.e. other than glucose polymers) in the residue were determined as the total soluble carbohydrates released (determined using anthrone) minus the glucose released (cf. Smouter and Simpson, 1989).
In one experiment, the concentrations of individual sugars extracted in ice-cold 5% (w/v) perchloric acid were determined using HPLC, after neutralisation of the extracts with K2CO3. The liquid chromatograph used consisted of a Waters 600 Pump Delivery System with a Waters 996 Photodiode Array (PDA) detector, equipped with a Waters Sugar-Pak I column (6.5 mm in width, 300 mm in length). The mobile phase and conditions were as described previously (Naidu, 1998).
Measurements of ethanol production rates
In experiments in which ethanol production was evaluated, ethanol from the solution flushed via the outlet of each Thunberg Tube was trapped in two sealed vials in series in an ice bath, each containing 9 ml of deionized water. Ethanol in the incubation medium and the traps was assayed using an enzymatic method (Beutler, 1983). A 1.5 ml cuvette contained 100 mol m-3 glycylglycine buffer at pH 9.0, 1.7 mol m-3 NAD+ and 0.3 units aldehyde dehydrogenase. The reaction was started by addition of 18 units of alcohol dehydrogenase and monitored at 340 nm using a spectrophotometer (Shimadzu, UV-1601). The concentration of ethanol in the incubation medium never exceeded 0.66 mol m-3 and recovery of ethanol from the system was 97–99%.
Estimates of membrane permeability to K+ and Cl-
The unidirectional efflux equation (Briggs et al., 1961) was used to compare the calculated product of ‘driving force and permeability of the plasma membrane to K+ (PK+)’, with the assessed efflux for K+. The driving force for K+ efflux, was calculated using the internal K+ concentrations measured in the present experiments and an assessed plasma membrane electrochemical potential. This potential consisted of : (the diffusion potential for K+ calculated using the Nernst equation and K+ data from the present study)+(plasma membrane Em—the diffusion potential for K+ for aerated or anoxic coleoptiles of the same cultivar in an earlier study by Zhang and Greenway, 1995). Direct measurements of Em during the current experiments would have been preferable, but as described in the Results the PK+ in the aerated or anoxic coleoptiles differed by an order of magnitude so that our assessments of PK+ anoxic/PK+ aerated are not in serious doubt.
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Results |
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Growth of excised coleoptiles during anoxia and after return to aerated solution
Average FW increments for individual coleoptiles were: 1.8 mg d-1 in aerated solution with exogenous glucose, 0.2 mg d-1 when anoxic with exogenous glucose, and no change in FW for anoxic coleoptiles without exogenous glucose. For the aerated coleoptiles, leaves emerged from within the coleoptiles during the first 48 h after excision, and the FW increment given for aerated ‘coleoptiles’ includes this new leaf growth. After 60 h of anoxia, re-aeration with exogenous glucose resulted in FW increases at 2.2 and 0.5 mg d-1 over the first 48 h in coleoptiles previously with or without glucose during anoxia, respectively. When re-aerated and supplied with glucose, coleoptiles previously anoxic without exogenous glucose did not grow leaves or roots, whereas those previously anoxic with exogenous glucose produced leaves and 4–5 roots (5 mm in length) during the first 48 h of the recovery period. The FWs given above included the new leaves but not the roots.
Ethanol production rate as affected by exogenous glucose
Over the first 12 h of anoxia, ethanol production in the coleoptiles was 7–8 µmol g-1 FW h-1 (Fig. 1). For anoxic coleoptiles supplied with glucose, a 33% decline in ethanol production during the first 36 h occurred even though total sugar concentrations only declined by 12% in one experiment (Table 1 ) and not at all in another experiment. The coleoptiles had a glucose net uptake rate of 2.6±0.58 µmol g-1 FW h-1 during the first 48 h of anoxia. The rate of glucose absorption was about equal to the demand for glucose in glycolysis, as assessed from the ethanol production data (Fig. 1). Starch in the coleoptiles declined from 5.1±0.17 to 2.7±0.32 µmol hexose units g-1 FW over the first 48 h of anoxia, and other polysaccharides insoluble in 80% ethanol declined from 3.9±0.81 to 2.8±0.46 µmol hexose units g-1 FW during the 48 h. Hydrolysis of polysaccharides therefore made a negligible contribution to ethanolic fermentation over the first 48 h of anoxia, being only 0.05 µmol hexose units g-1 FW h-1. Since total soluble sugars in the anoxic coleoptiles supplied with exogenous glucose declined only slightly, ethanolic fermentation may have been down-regulated after the initial period in anoxia. Ethanolic fermentation remained steady at this slower rate in the subsequent periods (i.e. 36–68 h anoxia; Fig. 1), despite further declines in tissue sugar concentration that also occurred to the same extent in aerated coleoptiles supplied with exogenous glucose (Table 1A).
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) anoxic without glucose; (•) anoxic with glucose; (
) anoxic resupplied with glucose. Seedlings were germinated and grown for 2 d in aerated solution (0.25 mol m-3 O2), then pretreated with 0.028 mol m-3 O2 for 16 h prior to excision of the coleoptile. Excised coleoptiles were ‘aged’ for 5 h in solution of the same composition as the culture medium but also with 20 mol m-3 glucose and O2 at 0.028 mol m-3, prior to the treatments. Rates of ethanol production are plotted at the end of each measurement interval. Data given are means of three replicates±standard errors.
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Ethanol production in anoxic coleoptiles without exogenous glucose showed pronounced, though gradual, declines with time (Fig. 1
). Addition of glucose to anoxic coleoptiles (previously without glucose for 36 h) resulted in complete recovery of ethanol production within 6 h, whereas when glucose was added after 60 h of anoxia the rate of ethanol production only increased to 60% of that in anoxic coleoptiles continuously supplied with glucose (Fig. 1).
Effect of sugar supply on net K+ uptake or loss during 60 h exposure to aerated or anoxic solutions
In aerated coleoptiles with exogenous glucose, K+ net uptake was never lower than 1.9 µmol g-1 FW h-1, whereas in coleoptiles without exogenous glucose it declined to 0.22 µmol g-1 FW h-1 between 48 and 60 h after the treatments commenced (Fig. 2). In anoxia, coleoptiles with exogenous glucose only took up K+ at 0.25 µmol g-1 FW h-1 and the rate declined to as little as 0.1 µmol g-1 FW h-1 between 36 and 60 h (Fig. 2). These net uptake rates were 9% and 4%, respectively, of those in aerated coleoptiles. Other experiments showed no enhancement of this net K+ uptake rate in anoxic coleoptiles: (i) during the first 5 h of anoxia, (ii) with SO42- instead of Cl-, (iii) with Ca2+ at 0.5, 1.0, 2.5 or 5.0 mol m-3, or (iv) with 10 mol m-3 sucrose instead of 20 mol m-3 glucose (data not shown). Nevertheless, these results contrasted with net losses of K+ from anoxic coleoptiles without glucose that started between 12 h and 24 h after the commencement of anoxia and increased to 0.9 µmol g-1 FW h-1 between 48 and 60 h (Fig. 2). Addition of glucose after 60 h of anoxia, while the coleoptiles remained anoxic, reversed this K+ net loss to a small net uptake within 9 h (Fig. 3). Re-aeration and supply of glucose to coleoptiles previously with or without glucose for 60 h of anoxia, elicited rapid resumption of K+ net uptake within 3 h and the rates reached 2.0–3.0 µmol g-1 FW h-1 within 6 h of re-aeration (Fig. 3).
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) aerated without glucose; (
) aerated with glucose; (
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Effect of sugar supply on net uptake of phosphate or loss of phosphates during 60 h exposure to aerated or anoxic solutions
The only substantial losses of phosphates occurred in the anoxic coleoptiles without exogenous glucose, for which net losses reached 0.15–0.20 µmol g-1 FW h-1 between 36 and 84 h of anoxia (Fig. 4). Upon addition of glucose after 60 h, while continuing anoxia, loss of phosphates diminished to 0.015 µmol g-1 FW h-1 between 12 and 24 h after the glucose was added. The recovery (i.e. cessation of loss of phosphates) was faster and more complete when the anoxic coleoptiles were not only given exogenous glucose but also re-aerated.
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The losses of phosphates described above were for anoxic coleoptiles in solutions without exogenous phosphate. For anoxic coleoptiles in incubation solution with glucose that also contained 0.050 mol m-3 phosphate, net uptake of phosphate was 0.070±0.003 µmol g-1 FW h-1 between 36–60 h after anoxia was imposed. This phosphate net uptake rate during anoxia was 37% of that when the coleoptiles were re-aerated (see below); thus, the decline in phosphate net uptake in anoxic coleoptiles was not as severe as that for K+ net uptake, the latter being only 9% of the rate in re-aerated solution. Exogenous phosphate during anoxia had no effect on net losses of phosphates from anoxic coleoptiles without exogenous glucose; net loss of phosphates was 0.14±0.018 µmol g-1 FW h-1 whether, or not, coleoptiles were supplied with 0.050 mol m-3 phosphate.
Coleoptiles previously anoxic for 60 h with or without exogenous glucose and then re-aerated with exogenous glucose and 0.10 mol m-3 phosphate, had net phosphate uptake rates of 0.19±0.016 and 0.21±0.015 µmol g-1 FW h-1, respectively, during the first 24 h. Reversion of the substantial net losses of phosphates to fast rates of net uptake in the coleoptiles previously without glucose suggests any deterioration in membrane integrity during anoxia was rapidly repaired after return to aerated conditions.
Net losses of phosphates and K+ from anoxic coleoptiles without exogenous glucose shown in Figs 2, 3 and 4 remained relatively constant when anoxia lasted longer than 24–36 h. However, the endogenous pools were gradually diminishing with time. When losses of phosphates and K+ were expressed as a percentage of the mean total tissue concentrations over each interval, the losses became more severe with time in anoxia (Fig. 5).
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Effect of sugar supply on short-term Rb+ uptake in aerated or anoxic coleoptiles
Rb+ uptake during the first 30 min period following its addition should give a reasonable estimate of influx across plasma membranes (Kochian and Lucas, 1982). Rb+ uptake rates were multiplied by 1.18 to provide an estimate of K+ influx; because net uptake of K+ by rice coleoptiles was 1.18 times faster than that of Rb+ over the first 60 min (data not shown). Subtracting the net uptake of K+ from the assessed K+ influx gives an estimate of K+ efflux (Table 2). In the presence of exogenous glucose, aerated coleoptiles had 12-fold faster K+ influx and 10-fold faster K+ efflux, than anoxic coleoptiles (Table 2). These differences were confirmed in a second experiment in Rb+ uptake over the initial 20 min was evaluated between 20 and 21 h after anoxia was commenced; assessed K+ influx was 18-fold faster and efflux 10-fold faster in aerated, compared with anoxic coleoptiles, both with exogenous glucose (data not shown). Without exogenous glucose the calculated K+ efflux for anoxic coleoptiles was more than 2-fold higher than for coleoptiles with exogenous glucose, while for aerated coleoptiles K+ effluxes were the same with or without exogenous glucose (Table 2).
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Membrane permeability to K+ in aerated or anoxic coleoptiles
The driving forces for K+ efflux, calculated as described in the Materials and methods, were 0.486 and 0.786 mol m-3 for coleoptiles supplied with glucose in aerated or anoxic solutions, respectively. Yet, K+ efflux was found to be 10 times slower in anoxic than in aerated coleoptiles (Table 2), so PK+ would have been 16 times lower for anoxic than for aerated coleoptiles. This large decrease in PK+ was confirmed in a second Rb+ influx experiment, which gave 18 times lower PK+ in anoxic than in aerated coleoptiles (data not shown). In contrast to the large decreases in PK+ in anoxic coleoptiles supplied with glucose, PK+ was only 2.5-fold lower in anoxia than in aerated coleoptiles when there was no exogenous glucose.
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Discussion |
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Anoxia tolerance in the coleoptile of rice as influenced by sugar supply
Excised rice coleoptiles (cv. Calrose) were very tolerant to anoxia, when supplied with exogenous glucose, as evidenced by some net uptake of K+, phosphate, and substantial amounts of glucose during 60 h of anoxia. Furthermore, when re-aerated the coleoptiles resumed fast rates of ion net uptake, and those that received glucose during anoxia also grew leaf and root tissues during the recovery phase.
Ethanol production in anoxic rice coleoptiles declined from about 7.5 µmol g-1 FW h-1 during the first 12 h to 5 and to 2.2 µmol g-1 FW h-1 after 48 h in coleoptiles with or without exogenous glucose, respectively (Fig. 1). The declines in coleoptiles without glucose were undoubtedly due to substrate limitation (Table 1), whereas total sugars changed only slightly in coleoptiles with exogenous glucose due to net uptake of glucose at 2.6 µmol g-1 FW h-1. As discussed below, ethanolic fermentation in these coleoptiles with exogenous glucose may have been down-regulated subsequent to a period of acclimation to anoxia. The slowing down of ethanolic fermentation in the excised rice coleoptiles with exogenous glucose during anoxia (present study) contrasts with the apparent maintenance of rates of ethanolic fermentation in rice coleoptiles that remained intact for 2 d of anoxia before being excised immediately prior to measurements that lasted 4 h (Gibbs et al., 2000). This difference may be real, or it could be associated with continued expansion in some tissues of recently excised coleoptiles, wounding effects, or due to exposure to air during the excision process; all may result in an increased energy demand, and hence ethanolic fermentation rate.
Solute transport in rice coleoptiles as influenced by sugar supply
The coleoptiles showed a sustained, albeit slow, net uptake of K+, and phosphate over 60 h of anoxia. In a previous study of anoxic coleoptiles of the same cultivar used here, the plasma membrane Em was more negative than the K+ diffusion potential (Zhang and Greenway, 1995). The present data indicate that despite this ‘energized membrane’, anoxic rice coleoptiles do not maintain vigorous ion fluxes. Even so, the phosphate net uptake implies maintenance of some energy-dependent ion transport, since it would have been against an electrochemical gradient across the plasma membrane. Nevertheless, the principal solute transport maintained in anoxic rice coleoptiles may be that of sugars to provide substrate for glycolysis; at an external concentration of 20 mol m-3 glucose, net uptake by rice coleoptiles was 2.6 µmol g-1 FW h-1 (present study), a rate similar to the 2.3 µmol g-1 FW h-1 by 3 mm maize root tips (Xia and Saglio, 1988). Transport processes associated with the observed pH regulation of the cytosol in anoxic rice shoots (Menegus et al., 1991) were presumably also maintained.
Excised coleoptiles exposed to anoxia with or without glucose were used to evaluate the effects of limiting substrate supply for glycolysis, and hence energy production, on ion net uptake or loss. Net losses of K+ and phosphates only occurred from anoxic coleoptiles without exogenous glucose; demonstrating that anoxia tolerance was decreased in the absence of a sugar supply, a finding consistent with earlier work on rice and some other species (Vartapetian et al., 1976; Webb and Armstrong, 1983; Waters et al., 1991b). Losses of phosphates under anoxia also occurred in the presence of glucose and amino acids for excised young shoots of wheat, but not of rice (Menegus et al., 1991). In the anoxic rice coleoptiles without exogenous glucose, losses of phosphates remained high (0.15–0.20 µmol g-1 FW h-1) with time, despite decreasing driving forces for phosphate efflux; being diminished internal phosphorus concentrations and less negative Em expected from decreases in internal K+. Such losses were an order of magnitude faster than phosphate effluxes of 0.043 µmol g-1 FW h-1 in phosphorus-sufficient tissues of Spirodela and Lemna in solutions containing 1 mol m-3 phosphate (McPharlin and Bieleski, 1989). The continued losses of phosphates despite decreases in driving forces imply that the coleoptiles without exogenous glucose deteriorated in health with time in anoxia.
Despite their deterioration, coleoptiles in anoxia without exogenous glucose for 60 h showed fast rates of phosphate and K+ net uptake when re-aerated and supplied with glucose. Furthermore, when glucose was added after 60 h, while anoxia was maintained, net K+ losses reverted to a small net uptake, and net losses of phosphates were also reduced to very low levels (Figs 3 and 4, respectively). Surprisingly, at 6–9 h after glucose was supplied to anoxic coleoptiles without it for 60 h, the rate of ethanol production was only 3 µmol g-1 FW h-1, compared with 4 µmol g-1 FW h-1 in coleoptiles after only 20 h of anoxia without exogenous glucose (Fig. 1); yet only the latter coleoptiles showed substantial net losses of K+ and phosphorus (Figs 3, 4). One possible reason for this discrepancy may be that certain cells had degenerated in the coleoptiles in anoxia for 60 h without exogenous glucose. For example, the cells at the base of the coleoptile in the meristematic regions may have died since development of leaves or roots did not occur in these coleoptiles when re-aerated. Deterioration of the expanding zone would have a disproportionate effect on ethanolic fermentation of the coleoptile as a whole when expressed on a FW basis, since the rates in expanding zones can be 3-fold higher than in the other zones (Setter and Ella, 1994). If this was the case, then the recuperating cells would have had a faster rate of ethanol production than measured for the sample as a whole, which in turn could have provided the additional energy required for net uptake of ions by these cells.
Membrane permeability to K+ and Cl- in anoxic rice coleoptiles
Estimates of membrane permeability indicated that PK+ was reduced about 17-fold in anoxic compared to aerated rice coleoptiles (with exogenous glucose). Thus, the declines in net K+ fluxes under anoxia were due to greatly reduced influx rates, while loss was avoided by a large decrease in permeability of the plasma membrane to K+. Large decreases in ion fluxes have previously been described for certain animal cells when anoxic, the decrease in permeability being attributed to closure of ion channels (Hochachka, 1991). For certain animal cells exposed to O2 concentrations at 45% of aerated levels, a large decrease in the probability of opening of the K+-out channel, but no change in single channel conductance, has been measured using the patch clamp technique (Lopez-Barneo, 1994). In contrast to the large decreases in PK+ in anoxic coleoptiles supplied with glucose, those without exogenous glucose only had a 2.5-fold lower PK+ than in aerated coleoptiles. This probably does not result from these coleoptiles having more open K+ efflux channels than anoxic coleoptiles with glucose, rather the higher PK+ was presumably related to the same deleterious changes which caused the substantial phosphorus loss from the anoxic coleoptiles without exogenous sugars. Data on the membrane permeability for other solutes in anoxic rice coleoptiles supplied with glucose are required to determine if the decreased permeability is specific for K+ or a more general response.
Assessment of energy requirements for maintenance in anoxic rice coleoptiles
Coleoptiles with exogenous sugars:
Energy produced (i.e. net rate of ATP synthesis) in anoxic rice coleoptiles can be estimated from data on rates of ethanol production as described in Table 3A. The assessed ATP production (µmol of ATP g-1 FW h-1) in anoxic rice coleoptiles was 3.4–7.6 times lower than the medium of the summed values of energy requirements in aerated tissues listed in Table 3B. These are minimum estimates since energy requirements for maintenance in aerated tissues listed in Table 3B were at most only 75% of the measured rates of dark respiration in fully grown bean leaves (de Visser et al., 1992). These data strongly support the notion of reductions in maintenance requirements for energy in anoxic rice coleoptiles. Reductions in maintenance requirements during anoxia have also been suggested for certain animal cells (Hochachka, 1991) and for root storage tissue of red beet (Zhang and Greenway, 1994).
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The assessed energy requirements for maintenance of 3.8–5.1 µmol g-1 FW h-1 in anoxic rice coleoptiles (present study) compare with an estimate of 12.5 µmol g-1 FW h-1 in hypoxically pretreated 5 mm maize root tips exposed to anoxia (calculated from Xia et al., 1995
). This seemingly much higher value for maize root tips, as compared to rice coleoptiles, is mainly due to differences in protein content, being (protein g-1 FW): 22 mg (rice coleoptiles, Gibbs et al., 2000) versus 58 mg (maize root tips, Spicket et al., 1993), presumably due to the different cytosol:vacuolar ratios in these tissues. When expressed on a per unit protein basis (µmol mg-1 protein h-1), the ATP production rates were 0.17–0.23 in the anoxic rice coleoptiles and 0.22 in the maize root tips. These very similar rates are remarkable, but need to be confirmed side-by-side in the same experiment.
Reductions in K+ influx and efflux (Table 2) would have contributed to the reduction in maintenance energy requirements in anoxic rice coleoptiles. Assuming such reductions in fluxes also occur for some other ions, and not only in the plasma membrane but for all membranes, as demonstrated for mitochondrial membranes in anoxic animal cells (Hochachka, 1991), reduced ion fluxes could decrease maintenance energy requirements from 23.2 to about 18.5 µmol of ATP g-1 FW h-1 (based on medium values, Table 3). Since the assessed ATP production in the anoxic rice coleoptiles with exogenous glucose was only 3.8–5.1 µmol of ATP g-1 FW h-1, further, substantial, reductions in energy requirements for maintenance would still be necessary. These economies probably include a reduction in the rate of protein turnover, since this turnover consumes on average about 45% of the assessed ATP production in bean leaves (de Visser et al., 1992). Evidence for this economy under anoxia consists of (i) reduced incorporation of 14C-labelled amino acids into proteins of rice embryos by about 40% (Mocquot et al., 1981), and (ii) a 77-fold increase in the half-life of cytochrome oxidase in anoxic Artemia franciscana embryos (Anchordoguy et al., 1993).
Coleoptiles without exogenous glucose:
ATP production in these coleoptiles between 36–60 h of anoxia would have been only 2.15 µmol g-1 FW h-1 if all the substrates had been sugars. However, the assumption that glucose was the main substrate used in catabolism is no longer valid for the coleoptiles without exogenous glucose. For example, between 36 and 60 h of anoxia the total ethanol produced was 51 µmol g-1 FW (Fig. 1), yet tissue sugars were already relatively low in these coleoptiles after 36 h of anoxia and provided at most only 35% of the carbon evolved in ethanolic fermentation (calculated from Table 1A). Polymers of hexose (e.g. starch) in the coleoptiles were low relative to those of soluble sugars (see Results), so during sugar starvation ethanol was presumably derived also from catabolism of amino acids, organic acids and perhaps proteins. Thus, the ATP production rate in these coleoptiles would have been even lower than the estimate provided above.
Rice coleoptiles exposed to anoxia for 60 h without exogenous glucose did not form roots or leaves when re-aerated, indicating ATP production was insufficient for maintenance of at least some of the cells. This situation for rice coleoptiles may be similar to that for roots in which expanded zones are often more anoxia tolerant than the apices. For examples, death of tips of anoxic wheat and maize roots contrasted with survival of the expanded tissues during anoxia (Waters et al., 1991a; Andrews et al., 1994, respectively).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Anoxic rice coleoptiles with exogenous glucose were clearly maintained in a healthy state, despite reductions in ethanolic fermentation with time in anoxia that would have resulted in a relatively low rate of ATP production (i.e. 3.8–5.1 µmol ATP g-1 FW h-1). Such declines in fermentation rates in plant tissue during long-term anoxia have been documented previously in aged storage root tissue of red beet (Zhang and Greenway, 1994
). The present study demonstrated that the decline in ethanolic fermentation occurred in anoxia-tolerant rice coleoptiles which (i) were only slightly decreased in sugar levels, and (ii) were capable of rapid resumption of growth and ion uptake even after 60 h in anoxia. These findings reinforce the suggestion (by Zhang and Greenway, 1994) that rates of ethanolic fermentation in some anoxia-tolerant plant tissues are down-regulated subsequent to a period of acclimation to anoxia. Additional experimental work is required since ‘down-regulation’ implies that the potential capacity for ethanolic fermentation was greater than the actual value. Whether these anoxic tissues can increase rates of ethanolic fermentation when exposed to an imposed increase in energy demand should be determined.
The slowed ethanolic fermentation in anoxic rice coleoptiles makes them analogous to those anoxic animal cells which down-regulate glycolysis and are assumed to have channel arrest, thus reducing requirements for energy (Hochachka, 1986). It remains to be determined whether cells of some other anoxia-tolerant plant species also show large decreases in membrane permeability or are capable of retaining vigorous ion exchange during anoxia.
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Acknowledgments |
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We thank Brian Atwell, Jane Gibbs, Cornelia Ullrich, and Wolfram Ullrich, for incisive criticism on a draft manuscript. This work was supported by the Australian Research Council and by Plant Sciences, Faculty of Agriculture, UWA. Shaobai Huang is grateful to the Crawford Fund for International Agricultural Research for a training award held at UWA. We thank Leon Miguel for his assistance with some preliminary experiments.
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Notes |
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3 To whom correspondence should be addressed. Fax: +61 8 9380 1108. E-mail: tdcolmer{at}cyllene.uwa.edu.au
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