Journal of Experimental Botany, Vol. 51, No. 345, pp. 785-796,
April 2000
© 2000 Oxford University Press
Regulation of alcoholic fermentation in coleoptiles of two rice cultivars differing in tolerance to anoxia
1 Faculty of Agriculture (Plant Sciences), The University of Western Australia, Nedlands, WA 6907, Australia
2 Cereal Production, Agriculture Western Australia, South Perth, WA 6151, Australia
Received 26 June 1999; Accepted 22 November 1999
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To investigate regulation of anaerobic carbohydrate catabolism in anoxia-tolerant plant tissue, rate of alcoholic fermentation and maximum catalytic activities of four key enzymes were assessed in coleoptiles of two rice cultivars that differ in tolerance to anoxia. The enzymes were ATP-dependent phosphofructokinase (PFK), pyrophosphate-dependent phosphofructokinase (PFP), pyruvate decarboxylase (PDC), and alcohol dehydrogenase (ADH). During anoxia, rates of coleoptile elongation and ethanol synthesis were faster in the more tolerant variety Calrose than in IR22. Calrose coleoptiles, in contrast to IR22, also showed a sustained Pasteur effect, with the estimated rate of glycolysis during anoxia being 1.4–1.7-fold faster than that of aerobic coleoptiles. In Calrose after 5 d anoxia, maximum catalytic activities of crude enzyme extracts were (in µmol substrate g-1 fresh weight min-1) 170–240 for ADH, 4–6 for PDC and PFP and 0.4–0.7 for PFK. During anoxia, activity per coleoptile of all four enzymes increased 3–5.5-fold, suggesting that PFK and PFP, like PDC and ADH, are synthesised in anoxic rice coleoptiles. Enzyme activities, on a fresh weight basis, were lower in IR22 than in Calrose. In vivo activities of PDC and PFK in anoxic coleoptiles from both cultivars were calculated using in vitro activities, estimated substrate levels, cytoplasmic pH, and S0.5 (the substrate level at which 0.5Vmaxis reached, without inferring Michaelis-Menten kinetics). Data indicated that potential carbon flux through PFK, rather than through PDC, more closely approximated rates of alcoholic fermentation. That PFK is an important site of regulation was supported further for Calrose coleoptiles by a decrease in the concentration of its substrate pool (F-6-P+G-6-P) following the onset of anoxia. By contrast, in IR22, there was little evidence for control by PFK, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.
Key words: Oryza sativa, phosphofructokinases, pyruvate decarboxylase, alcohol dehydrogenase, flooding, anaerobiosis.
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Introduction |
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Anaerobic carbohydrate catabolism, and the concomitant production of energy, is a fundamental component of the adaptation of plant tissue to anoxia. A high rate of glycolysis occurs in some plant tissues and is probably essential for organs that develop during anoxia. In anaerobic plant tissue, glycolysis is linked predominantly to alcoholic fermentation (Davies, 1980
; ap Rees et al., 1987). This also applies to rice coleoptiles (Menegus et al., 1993)
The coleoptiles of several rice cultivars are very tolerant to anoxia. Indeed, rice coleoptiles are one of the few plant organs that can grow under anoxia (Pearce and Jackson, 1991). Even so, coleoptiles from different rice cultivars differ greatly in rates of elongation and alcoholic fermentation under anoxia (Atwell et al., 1982, for elongation; Setter et al., 1994, for elongation and ethanol synthesis). The present study assessed the effects of anoxia on the rate of alcoholic fermentation, concentration of glycolytic intermediates, and activities of the fermentative enzymes, alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC), the glycolytic enzyme, phosphofructokinase (PFK), and pyrophosphate:fructose-6-phosphate-1-phosphotransferase (PFP) in two rice cultivars differing in their tolerance to anoxia. Phosphofructokinase (PFK) catalyses the following irreversible reaction
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). Less information is available for PDC activity, but it increased under anoxia in cereal roots (maize: Bailey-Serres et al., 1988; wheat: Waters et al., 1991) and mRNA transcripts encoding PDC increased in anoxic maize root tips (Drew et al., 1994). Information on the activity of PFP and PFK in anoxic plant tissues is particularly scant. The activity of PFK was the same in anoxic and aerated tissue of maize (Bailey Serres et al., 1988), and cultured cells of rice (Mohanty et al., 1993). In whole rice seedlings, the activity of PFP became 6–11-fold higher than that of PFK during anoxia (Mertens et al., 1990), and in anoxic cultured cells of rice, it was 4–8.6-fold higher than PFK activity (Mohanty et al., 1993). In contrast, in maize root tips, there was only a small increase in PFP activity while the activity of PFK decreased substantially rather than increased during anoxia (Bouny and Saglio, 1996).
On the basis of the model of Kacser and Burns for shared control between enzymes within a metabolic pathway (Kacser and Burns, 1979), these observations suggest a dominant contribution by PFK to the control of alcoholic fermentation, provided PFK is the only enzyme catalysing the flux from F-6-P to F-1,6-bisphosphate. Thus, interpretation of the regulation of glycolysis hinges on the question of whether the carbon flux through PFP flows in the direction of PPi synthesis or hydrolysis. If PFP hydrolyses PPi, both PFK and PFP are working in concert to generate the glycolytic flux. However, if PFP generates PPi then the carbon flux through PFK in vivo is the sum of glycolysis and the rate of PPi synthesis. This issue is considered in more detail in the discussion. Further support for a dominant role of PFK in the regulation of anaerobic carbohydrate catabolism comes from changes in glycolytic intermediates in aged carrot tissues (Faiz-ur-Rahman et al., 1974) and potato tissues (Dixon and ap Rees, 1980) during the first 1–1.5 h of anoxia. The work with potato tissues also suggested that pyruvate kinase regulates the exit from glycolysis (Dixon and ap Rees, 1980).
The concept of shared controls was first developed in the model by Kacser and Burns (Kacser and Burns, 1979), who emphasized that the control of flux through a metabolic pathway is shared by all enzymes of the pathway (‘molecular democracy’). Nevertheless, the effect of a change in enzyme activity on the flux will vary between enzymes. The extent to which an individual enzyme exercises control over the flux is expressed as the sensitivity coefficient, dF/F divided by dE/E, with F being the flux through the pathway and E the in vivo enzyme activity (Kacser and Burns, 1979). For individual enzymes this coefficient can range from zero to one and is a measure of its share in the control of the pathway. A high sensitivity coefficient for a particular enzyme indicates that this enzyme exerts a strong control over flux through the pathway.
The principal objectives of the present work were to investigate the contributions of the fermentative enzyme, PDC, and the phosphofructokinases, PFK and PFP, to the regulation of anaerobic carbohydrate catabolism in anoxia-tolerant tissue, and to determine whether the ‘sharing’ of control by these enzymes differed between rice cultivars.
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Materials
Buffers, purified enzymes, coupling enzymes, cofactors, substrates, and Sephadex were obtained from the Sigma Chemical Company (St Louis, Mo, USA). All other reagents were analytical grade. Rice seeds (Oryza sativa cvs Calrose and IR22) were supplied by Dr L Lewin, the Department of Agriculture, Yanco, New South Wales, Australia.
Growth of plant material
Seedlings were germinated and grown in the dark at 30 °C. Ten grams of dehulled rice seeds were surface-sterilized in 0.1% HgCl2 (w/v in 0.1% HCl) for 3 min and rinsed in de-ionized water. Sterilized seeds were submerged under mesh in sealed vessels containing 3 dm3 of the following solution (in mol m-3) CaSO4,0.5; KH2PO4, 0.8 at pH 5.5–6.5. Solutions were bubbled with air (0.25 mol O2 m-3), high purity N2 (‘anoxia’; <0.003 mol O2 m-3) or a mixture of air plus N2 (‘hypoxia’ 0.03 mol O2 m-3) all at 50–60 cm3 min-1 via an inlet tube in the lid of each vessel. Aerated and hypoxic vessels were vented to the atmosphere via a 1.5 cm diameter outlet, plugged with cotton wool, in the lid of each vessel. To prevent leakage of air into anoxic vessels, a tube was connected from the outlet to a water-filled vial through which the outflowing gas passed before venting to the atmosphere. This also served as a check on the sealing of the vessel. The sequence of O2 regimes imposed during germination and growth of seedlings is described in the tables.
In a series of experiments, coleoptiles were excised after exposure of intact seedlings to different O2 regimes for assessment of one or more of the following:- elongation, net ethanol synthesis, CO2 evolution, O2 uptake, concentrations of glycolytic intermediates, and maximum catalytic activities of ADH, PDC, PFK, and PFP. These assessments were carried out at either 25 °C or 30 °C, as shown in captions to the tables.
Assessment of elongation during anoxia
Elongation rates of intact coleoptiles at 30 °C were calculated from the differences in length of coleoptiles before and after the period of anoxia. To assess length, coleoptiles were excised at the seed junction and lengths were measured with a ruler from a random sample of 30 coleoptiles per vessel, with three vessels per O2treatment.
Measurement of O2 uptake, CO2 evolution and net ethanol synthesis
Oxygen uptake and CO2 evolution by coleoptiles were assessed using Warburg manometers (Umbreit et al., 1964). Excised coleoptiles (0.1–0.15 g fresh weight) were transferred to Warburg flasks containing (in mol m-3), CaSO4, 0.5; KH2PO4,0.8; pH 5.5. Oxygen uptake from aerated solution and CO2 evolution under anoxia were assessed at 25 °C over 4 h. Prior to assessment of CO2 evolution, Warburg flasks were made anoxic by flushing with high purity N2 for at least 15 min.
Ethanol was assessed by measuring the amount of ethanol in the tissue and in the incubation solution after 4 h of anoxia. To assay ethanol in coleoptiles, tissues were snap frozen in liquid N2 and killed with 5% (w/v) perchloric acid, before being neutralized with 69% (w/v) K2CO3. Neutralized tissue extracts were centrifuged at top speed in a Clements minifuge (Phoenix) for 5 min at 4 °C and the supernatant removed for ethanol analysis. Ethanol in tissue extracts and in the incubation solution was assayed enzymatically according to Beutler (Beutler, 1983). Recovery of a known amount of ethanol added either to the tissue prior to extraction, or to the bathing solution ranged between 97% and 106%.
In some experiments, net ethanol synthesis by coleoptiles was assessed at 30 °C in continuously flushed plastic vials. Between 0.1–0.15 g fresh weight of excised coleoptiles were incubated in lidded vials in 15 cm3 of the following solution (in mol m-3), CaSO4, 0.5; KH2PO4, 0.8; pH 6.5. During incubation, each vial was flushed with high purity N2 via a plastic inlet tube. To trap ethanol in the outflowing gas, a plastic outlet tube was connected in series to two further vials each containing 10 cm3 of ice-cold de-ionized water. After 4 h, ethanol in the tissue was extracted, and ethanol in the extract and in the incubation solution and ethanol traps was assayed as described in the preceding paragraph. To test the recovery of ethanol in the ethanol traps, a similar amount to that evolved by the tissues was added to the incubation solution without tissues; after 4 h, recovery of ethanol from the incubation solution and solution in the ethanol traps ranged between 98% and 102%.
Extraction and analysis of glycolytic intermediates
To assay glycolytic intermediates, 3-d-old aerated coleoptiles were excised, and samples of 1 g fresh weight were incubated in a moist atmosphere of air or high purity N2for 90 min at 25 °C. Samples were then freeze-clamped, killed with 1 cm3 ice-cold 5% perchloric acid and left at 4 °C for 2 h before neutralizing with 69% (w/v) K2CO3. Neutralized samples were centrifuged at 18 000 g for 30 min at 4 °C (Sorvall) and the supernatants were assayed according to Dixon and ap Rees (Dixon and ap Rees, 1980). The validity of the extraction was tested by extracting a duplicate tissue sample with 5% perchloric acid containing known amounts of intermediates to be assayed. Recoveries of intermediates ranged from 80–104%.
Extraction and assay of PFK, PFP, PDC, and ADH
Extractions were carried out at 2–4 °C. Preliminary tests showed that: (1) both PFK and PFP were unstable during storage in the absence of fructose-2,6-bisphosphate; (2) PFK and PFP were best stored at 10 °C and required 1.5 h storage before optimum activity was reached; (3) the assay for PFK had no reaction till ATP was added as a last ingredient. Similarly, PFP had no reaction till PPi was added. This showed the conversion of NADH by the coupling enzymes was entirely dependent on activities of PFK and PFP, respectively; and (4) glycerol protected PFP but reduced activity of PFK, so two separate extractions were needed; one for PFK, ADH and PDC and a second for PFP.
The extraction buffer used for PFK, PDC and ADH contained (in mol m-3), K-TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulphonic acid), 100; EDTA, 1; MgCl2, 2; DTT, 20; fructose-2,6-bisphosphate, 0.1x10-3; plus 0.1% (w/v) Triton X-100 at pH 7.5. Tissue was ground in a mortar for 2 min in extraction buffer and acid-washed sand, with a ratio of tissue fresh weight to extraction buffer of 1 : 5. The brei was filtered through Miracloth (Calbiochem) and the filtrate centrifuged at top speed in a Clements minifuge for 2 min. Filtration through Miracloth was omitted if the extraction volume was less than 2 cm3. A sample of the supernatant was removed for determination of soluble protein (Lowry et al., 1951). Bovine serum albumin was added to a final concentration of 1% (w/v), and before centrifugation the extract was divided into three aliquots for assay of PFK, PDC and ADH. The cofactor thiamine pyrophosphate (TPP), and MgCl2 were added to the PDC aliquot to give a final concentration of 1 mol m-3 and 2.5 mol m-3, respectively, and the pH was adjusted to 6 at 2–4 °C using unbuffered 200 mol m-3 MES (2-[N-morpholino]- ethane-sulphonic acid). The three aliquots were centrifuged at top speed in a minifuge for a further 2 min, and the supernatants transferred to clean tubes. The PFK aliquot was transferred to 10 °C and the pH adjusted to 7.5 using 500 mol m-3 K-TES pH 8. The PDC aliquot was transferred to 20 °C. The ADH aliquot was stored on ice.
Extraction buffer used for PFP contained (in mol m-3), K-TES, 100; EDTA, 1; MgCl2, 2; dithiothreitol (DTT), 2; fructose-2,6-bisphosphate, 0.1x10-3; plus 20% (v/v) glycerol and 0.1% (v/v) Triton X-100 at pH 7.5. After extraction and addition of 1% (w/v) BSA (final concentration), the PFP aliquot was transferred to 10 °C and the pH adjusted to 7.5 using 500 mol m-3 K-TES, pH 8.
Enzyme assays
All enzyme activities were determined spectrophotometrically in a final volume of 1 cm3. Based on preliminary tests, the assays were carried out between 90 and 210 min after extraction to ensure maximum activities of PDC, PFK and PFP. In some early experiments, activities of PFK, ADH and PDC were assayed at 25 °C. Thereafter, activities of these enzymes and of PFP were assayed at 30 °C.
For PFK, the assay mixture of 1 cm3 contained 0.1 cm3 extract, and (in IU), aldolase, 0.5; glycerol-3-phosphate dehydrogenase, 1; triose phosphate isomerase, 8; and inorganic pyrophosphatase, 0.5 (to prevent possible activity of PFP due to contamination with PPi). It also contained (in mol m-3), K-TES, 100 (pH 7.5); MgCl2, 2; NADH, 0.17; ATP, 0.6; Pi, 3.6; and fructose-6-phosphate, 7.5, to start the reaction. For PDC, the assay mixture contained 0.2 cm3 extract, 10 IU ADH in 100 mol m-3 of DTT, and (in mol m-3), Na-MES (pH 6), 60; Mg2+, 1; TPP, 0.5; NADH 0.17; oxamate, 50; and pyruvate, 10, to start the reaction. For ADH, the assay mixture contained 0.2 cm3 of a 1/20 dilution of extract, and (in mol m-3), Na-TES (pH 7), 50; NADH, 0.17, and acetaldehyde, 10, to start the reaction. For PFP, the assay mixture contained 0.2 cm3 extract, and (in IU), aldolase, 0.5; glycerol-3-phosphate dehydrogenase, 1, and triose phosphate isomerase, 8. It also contained (in mol m-3), K-TES, 100 (pH 7.5); MgCl2, 2; NADH, 0.17; PPi, 1; fructose-2,6-bisphosphate, 0.001; and fructose-6-phosphate, 7.5, to start the reaction.
The reliability of these extraction and assay procedures was checked for each enzyme in two ways (Smith and ap Rees, 1979). First, by recovery of activity of a commercially available purified sample of each enzyme added to the extraction buffer prior to tissue extraction. Second, by recovery of the activity of each enzyme following mixing and extraction of known quantities of anoxic and aerated coleoptiles. Under both these test conditions, recovery of enzyme activities was 87–125% (Table 1).
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Characterization of the response of PFK to a range of substrate concentrations at pH 7.5 and pH 6.8
The response of PFK to a range of substrate concentrations at pH 7.5 and pH 6.8 was assessed using desalted extract. To desalt the PFK extract, 1 cm3 of extract was passed through a 15x50 mm G25 column at 2–4 °C. The column was equilibrated with buffer at pH 7.5 containing (in mol m-3), K-TES, 100; EDTA, 1; MgCl2, 2; KCl, 100; DTT, 20; fructose-2,6-bisphosphate, 0.1x10-3. After desalting, a sample of the extract was removed for determination of soluble protein (Lowry et al., 1951), and BSA was added to a final concentration of 1% (w/v). The extract was transferred to 10 °C, divided into two aliquots and the pH adjusted to 7.5 and 6.8, using unbuffered 100 mol m-3 TES (pH~4.8) containing (in mol m-3), EDTA, 1; MgCl2, 2; KCl, 100; DTT, 20; fructose-2,6-bisphosphate, 0.1x10-3. Aliquots were incubated at pH 7.5 and 6.8 for 2 h. Samples from each of these incubations were then assayed at both pH 7.5 and pH 6.8, varying either the concentration of ATP from 0.01 to 0.6 mol m-3, while maintaining fructose-6-phosphate at 7.5 mol m-3, or varying the concentration of fructose-6-phosphate from 0.01 to 7.5 mol m-3, maintaining ATP at 0.6 mol m-3. In this set of experiments, the assay mixture routinely contained 100 mol m-3 KCl.
Results
Elongation and alcoholic fermentation
Elongation rates of Calrose coleoptiles were 3.7–10-fold faster than IR22 over the first 2 d following exposure to anoxia (Table 2). During this time, rates of ethanol synthesis (µmol g-1 fresh weight min-1) were also 1.4–2.9-fold faster in Calrose than in IR22 (Tables 2, 3). The ratio of ethanol synthesis to CO2 evolution during anoxia was 0.7–1.2 (Table 3) and there were no obvious differences between the two cultivars. In IR22, the ratio of net ethanol synthesis during anoxia to O2 uptake in air was 0.31–0.41 (Table 4). This ratio approximates 0.33, the value expected if there is an equimolar carbon flux through glycolysis under anoxic and aerated conditions, assuming a respiratory quotient of 1.0 under aerated conditions. This reasoning assumes there is no carbon flow from intermediates of glycolysis to anabolic reactions. In the discussion the potential overestimation of the Pasteur effect caused by this assumption is considered. In Calrose, the ratio of net ethanol synthesis during anoxia to O2 uptake in air was 0.68–0.88 (Table 4), suggesting, though not proving, a significant increase in carbon flux through glycolysis in anoxic Calrose coleoptiles.
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When assessed over 5 d of anoxia, the elongation rate of Calrose was only 1.2-fold higher than that of IR22 coleoptiles due, presumably, to a relative decline in elongation rate of Calrose with time (Table 2
). After 5 d anoxia, the rate of net ethanol synthesis was 1.4-fold faster in Calrose than in IR22 coleoptiles.
Maximum catalytic activities of enzymes
On a fresh weight basis, the maximum catalytic activity of PDC and ADH in coleoptiles from both cultivars increased over 2 d following imposition of anoxia (Experiment 4; Table 5a). Under these conditions, the activity of PFK on a fresh weight basis increased only marginally in Calrose and was unchanged in IR22. (Experiment 4; Table 5a). Data from a subsequent experiment showed that in Calrose coleoptiles, maximum catalytic activities of PDC, ADH, PFK, and PFP, assessed on a fresh weight basis, either did not change or decreased between day 3 and day 5 of anoxia (Table 6). However, coleoptile expansion during this period of anoxia (data not presented) suggested that net enzyme synthesis might occur in these anoxic tissues. Indeed, when expressed on a coleoptile basis, activity of all four enzymes increased by 3–5.5-fold (Table 6), suggesting that PFK and PFP, like PDC and ADH, are synthesized in anoxic rice coleoptiles.
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During anoxia the maximum catalytic activities (in µmol g-1 FW min-1) of PDC and PFK were generally smaller in IR22 than in Calrose (Table 5a
; Table 9).
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Reconciling in vitro and in vivo enzyme activities
Preliminary comparisons between net rates of ethanol synthesis (i.e. in vivo rates of alcoholic fermentation) and maximum catalytic enzyme activities, assessed in vitro under conditions of optimum pH, showed that maximum ADH activity far exceeded the rate of net ethanol synthesis in both cultivars (Table 5a). Moreover, maximum PDC activity was 8–52-fold higher and 22–36-fold higher than the rate of net ethanol synthesis in Calrose and IR22, respectively (Table 5b). When comparing maximum PFK and PFP activities with ethanol synthesis, enzyme activities were doubled to account for the generation of two ethanol molecules per hexose phosphate. The data suggest that the potential carbon flux through PFP was 36–96-fold greater than the observed rate of net ethanol synthesis in Calrose and around 60-fold greater in IR22 (Table 5b). In contrast, carbon flux through PFK only exceeds ethanol synthesis by 2–6.4-fold and 3–6-fold in Calrose and IR22, respectively (Table 5b).
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To calculate the activity in vivo of these key enzymes, estimates of the pH response characteristics and S0.5 of the enzymes are needed. These data are known for PDC (Morrell et al., 1990
) and are shown in Table 7 for PFK at pH 6.8 and 7.5. A pH within this range is likely to prevail in vivo, as the cytoplasmic pH of anoxic rice shoots, assessed using NMR, was 7.0–7.15 (Menegus et al., 1991). The in vivo activities of (a) PDC and (b) PFK were estimated using the following observations and assumptions:(a) PDC: (1) 5–8% lower activity at pH 6.8 than at pH 6 (Morrell et al., 1990). (2) S0.5 (pyruvate) at pH 6.8 of 1 mol m-3 (Morrell et al., 1990), compared with estimated cytoplasmic concentration of 0.36 mol m-3 and 0.28 mol m-3 for anoxic Calrose and IR22 coleoptiles, respectively (Table 8, assuming cytoplasmic volume=10% of tissue volume). Cytoplasmic pyruvate concentrations estimated for Calrose and IR22 are consistent with assessments of cytoplasmic pyruvate ranging from 0.23–0.3 mol m-3 in three other investigations (see Morrell et al., 1990). To estimate in vivo PDC activity in Calrose coleoptiles, the maximum catalytic activity of PDC is multiplied by (the proportional activity at pH 6.8)x([pyr]cyto/S0.5 at pH 6.8)x0.5=0.94x 0.4/1x0.5, where the factor 0.5 is introduced to adjust maximum enzyme velocity to that occurring at S0.5.(b) PFK: (1) 14% lower activity of PFK, extracted from Calrose coleoptiles, when incubated and assayed at pH 6.8 compared to pH 7.5 (anoxic sample in Table 7). (2) S0.5 (F-6-P) of 0.35 mol m-3 for PFK extracted from anoxic tissues incubated and assayed at pH 6.8 (Table 7), compared with estimated cytoplasmic concentration of F-6-P of 0.54 mol m-3 and 0.64 mol m-3 for anoxic Calrose and IR22 coleoptiles, respectively (Table 8).
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Estimated in vivo activities of PDC and PFK are given in Table 9
. When comparing in vivo PFK activity with ethanol synthesis, enzyme activity was doubled to account for the generation of two ethanol molecules per hexose phosphate. The carbon flux estimated from in vivo activity of PFK exceeded net ethanol synthesis by 1.1–3.8 in Calrose and by 1.8–4 in IR22 coleoptiles (Table 9). In vivo PDC activity was 1.5–9.7-fold higher and 3–5.1-fold higher than the rate of net ethanol synthesis in Calrose and IR22, respectively (Table 9).
Glycolytic intermediates
Concentrations of hexose phosphates, fructose-1,6-bisphoshate, PEP, and pyruvate are presented in Table 8. Glycolytic intermediates were measured 90 min after exposing 3-d-old coleoptiles to anoxia. In Calrose, glucose-6-phosphate, fructose-6-phosphate and PEP were significantly lower following exposure to anoxia than in air, but fructose-1,6-bisphosphate and pyruvate did not change significantly (Table 8). In contrast, in IR22 neither fructose-6-phosphate nor fructose-1,6-bisphosphate changed significantly in anoxia, while the concentration of PEP and pyruvate were significantly lower than in air (Table 8).
Discussion
Rice coleoptiles are among the few plant organs that grow under anoxia. Coleoptiles of Calrose elongate more rapidly than IR22 under anoxia (Table 2; Atwell et al., 1982; Setter et al., 1994). This study shows that the faster elongation rate of Calrose coleoptiles was coincident with a 1.4–2.9-fold higher rate of alcoholic fermentation in Calrose than in IR22. Similarly, in a comparison of four rice cultivars, the cultivar with the fastest elongation also had the highest rate of alcoholic fermentation (Setter et al., 1994).
Calrose coleoptiles apparently exhibit a Pasteur effect, at least over the first 2 d of anoxia, with the estimated glycolytic flux in anoxia being 2.0–2.7-fold greater than in air, based on the ratio of net ethanol synthesis in anoxia over O2 uptake in air (Table 4). The rate of net ethanol synthesis (i.e. in vivo rate of alcoholic fermentation) provides a good estimate of the net anaerobic glycolytic carbon flux because (1) ethanol is the principal end-product of anaerobic carbohydrate catabolism in rice coleoptiles (Menegus et al., 1993). Indeed, rice shoots produced very little lactate under anoxia (Menegus et al., 1991), and (2) the contribution to ethanol synthesis from decarboxylation of malate to pyruvate is likely to be small. For example, in maize root tips, during the first 60 min of anoxia, malate decarboxylation was only 0.03 times the rate of ethanol synthesis (Roberts et al., 1992).
The ratio of net ethanol synthesis in anoxia over O2 uptake in air probably overestimates the Pasteur effect because the glycolytic flux also provides carbon for protein synthesis, particularly in aerobic tissues. Rates of protein synthesis in excised rice coleoptiles have not been reported. However, allowing for the rates of protein synthesis observed for intact coleoptiles exposed to anoxia and air (Alpi and Beevers, 1983), the Pasteur effect in Calrose was estimated to be 1.4–1.7. No Pasteur effect was observed in IR22 (Table 4). Consistently higher rates of alcoholic fermentation were found in anoxia-tolerant than in anoxia-intolerant maize root tips (Hole et al., 1992). In the study by Hole et al., anoxia tolerance was induced in maize roots by hypoxic pretreatment (Hole et al., 1992).
In Calrose, the maximum catalytic activity per coleoptile, of four key enzymes of anaerobic metabolism increased under anoxia (Table 6). These increases presumably represent de novo protein synthesis, since the coleoptile expanded in volume under anoxia, and suggest that, at least in the rice cultivar Calrose, ADH, PDC, PFP, and PFK, are ‘anaerobic proteins’. This is consistent with the large number of proteins synthesized in anoxic rice tissue (embryos, Mocquot et al., 1981; rice coleoptiles, between day 3 and 4 of anoxia, Atwell and ap Rees, 1986; cultured rice cells, Mohanty et al., 1993). Alternatively, the observed increase in maximum catalytic activity per coleoptile represents activation of pre-existing enzymes. This question can be resolved by measuring de novo protein synthesis.
While increases in activity of ADH and PDC are common in plant tissues under anoxia, elevated PFK activity has not been widely observed (see Introduction). Activity of PFK sometimes even decreases (e.g. in maize, Bouny and Saglio, 1996), and PFK is not generally included in the suite of anaerobic proteins. By contrast, the observation that PFK is probably synthesized in anoxic Calrose coleoptiles is compatible with the increase of mRNA of PFK when 7-d-old rice seedlings were submerged without aeration (Umeda and Uchimiya, 1994), and suggests a direct response to anoxia. Further elucidation of the role of PFK in anoxic rice coleoptiles requires analyses to establish levels of gene expression.
Potential for glycolysis
The significance of PFK as a control point for glycolytic flux under anoxia depends on the function of PFP. PFP catalyses a reversible reaction and the direction of the net flux through this step depends on whether other metabolism produces or requires PPi (Stitt, 1990). For example, if PFP catalysed phosphorylation of fructose-6-phosphate under anoxia a substantial proportion of the carbon flux through glycolysis would bypass PFK. Consequently, the role of PFK as a control point in glycolysis would be diminished. On the other hand, if PFP catalyses a net flux from fructose-1,6-bisphosphate to fructose-6-P, it would convert ATP to PPi in a so-called substrate cycle (Stitt, 1990). Under this condition, the maximum catalytic activity of PFK becomes very important, since PFK would have to catalyse a carbon flux which is the sum of the glycolytic flux and the flux required to sustain conversion of ATP to PPi in the substrate cycle (Stitt, 1990). The latter requirement consists of (1) a portion of the flux through PFK and thence glycolysis to provide the ATP necessary to drive the production of PPi in the substrate cycle, and (2) a portion of the flux through PFK to provide F-1,6-bisP, the substrate, along with Pi, involved in the synthesis of PPi in a reaction catalysed by PFP (Fig. 1).
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To illustrate this point, a system is used that has a flux of 10 mol C6 through PFK in the absence of PPi synthesis. Two scenarios are considered that might occur following a diversion of 1 mol of F-1,6-bisP to PPi synthesis. (1) If PFK activity was maximal before PPi synthesis began then, following the diversion of 1 mol of F-1,6-bisP, the net ATP yield would be reduced from 20 mol to 17 mol; a reduction of 2 mol as a result of the decreased flux through glycolysis, plus a further reduction in the net yield of ATP by 1 mol, as ATP produced in glycolysis is used in the substrate cycle. That is, if PFK cannot be further stimulated, the production of 1 mol of PPi would reduce net ATP yield by 3 mol. (2) If PFK is below maximum activity before diversion of F-1,6-bisP to PPi synthesis, then PFK activity is likely to be stimulated in the usual way by the reduction in levels of PEP (Stitt, 1990
). At its maximum, this stimulation would return net ATP synthesis to its previous level. Under these conditions, the flux through PFK would be 11.5 mol of F-6-P, and the combined system would produce 20 mol ATP plus 1 mol PPi.
Regulation of PFP has been discussed in detail (Stitt, 1989), and the inhibition of potato tuber PFP by pyrophosphate and phosphate is reported. Changes in the levels of pyrophosphate and phosphate will influence the direction of net flux through PFP and hence may be involved in the regulation of the substrate cycle. However, as there are no values of PPi and Pi in the cytoplasm of anoxic rice coleoptiles in the present work, regulation of the system remains unclear.
Inferences that PFP catalyses PPi synthesis under anoxia can be drawn from the 18-fold and 60-fold increase in the PPi-ase in the tonoplast fraction of rice seedlings after 2 d and 6 d of anoxia, respectively (Carystinos et al., 1995). These authors speculate that PPi hydrolysis energizes H+ transport, maintaining the pH gradient across the tonoplast, even in anoxia, while providing energy for sequestering anaerobic end-products, other than ethanol, into the vacuole. It is feasible that, in anoxic plant tissue, PPi for this reaction is produced in the substrate cycle. That is, during anoxia, the substrate cycle would preferentially direct energy from ATP to PPi and, hence, to PPi consuming reactions. Other PPi consuming reactions are likely to include sucrose mobilization via sucrose synthase (Stitt, 1990). A 3-fold increase in activity of sucrose synthase in tomato roots after 20 h hypoxic pretreatment has been observed (Germain et al., 1997). Moreover, during anoxia, sucrose, but not glucose or fructose, sustained glycolytic flux, sucrose being mobilized presumably via the sucrose synthase pathway, and this was associated with increased anoxia tolerance in these tissues (Germain et al., 1997). In conclusion, PFP may not contribute to the glycolytic flux during anoxia; instead it might maintain PPi levels in the anoxic tissue.
Calrose—the anoxia-tolerant cultivar
In general, comparison of maximum catalytic activities of the enzymes and in vivo rates of alcoholic fermentation in Calrose are consistent with high sensitivity coefficients for PFK and to a lesser extent for PDC (Table 5b), and a much lower sensitivity coefficient for ADH. The latter is in keeping with the finding that large differences in ADH activities in crosses between inbred lines and ADH1 null mutants of maize had no effect on alcoholic fermentation until the ADH activity was 6% or lower than the maximum catalytic activity (Roberts et al., 1989). Of the enzymes assayed, the estimated activity of PFK in vivo most closely approximates the in vivo flux through glycolysis, assessed by the rate of alcoholic fermentation. If the hypothesis that, under anoxia, PFP catalyses the production of PPi is correct, then activity of PFK would need to provide carbon for the substrate cycle, so generating PPi, as well as for glycolysis. If so, the total carbon flux through PFK would approximate even more closely the estimated activity of PFK in vivo.
Changes in the amounts of glycolytic intermediates following the onset of anoxia in the present study provide supporting evidence that PFK has a regulatory role in the glycolytic carbon flux in Calrose coleoptiles. Assessment of changes in glycolytic intermediates following the onset of anoxia were based on the study of regulatory steps in glycolysis in potato tubers (Dixon and ap Rees, 1980). The amounts of hexose phosphates, fructose-1,6-bisphosphate, PEP, and pyruvate measured in aerated 3-d-old rice coleoptiles were 1.5–6-fold higher than in aerated potato tuber (Dixon and ap Rees, 1980). These differences are likely to reflect variation between species rather than losses during extraction because, in both studies, percentage recoveries of measured amounts of intermediates following extraction confirmed the validity of the extraction method (89–122% for potato tubers; Dixon and ap Rees, 1980; 80–104% for rice coleoptiles in this study).
The decline in both glucose-6-phosphate and fructose-6-phosphate, which form the substrate pool for PFK (Table 8) is consistent with that observed in anoxic potato tubers (Dixon and ap Rees, 1980), and is in keeping with an acceleration of glycolysis in anoxia, controlled by PFK (Dixon and ap Rees, 1980). The decline in PEP when Calrose coleoptiles are made anoxic was similar to the decline observed in anoxic potato tubers (Dixon and ap Rees, 1980), and suggests that pyruvate kinase also contributes to regulation of the flux through glycolysis. However, it is unlikely to control the entry of hexose phosphates into glycolyis directly, but rather to regulate the exit of intermediates from glycolysis (Dixon and ap Rees, 1980). In the present case, increased flow to pyruvate and ethanol would lower PEP, thus mitigating the inhibition of PFK by PEP (Stitt, 1990) and hence up-regulating PFK activity in anoxic Calrose coleoptiles.
Anoxia-tolerant versus intolerant
The basis for differences in rate of alcoholic fermentation between cultivars cannot be fully elucidated from this study because the widely different growth rates in these cultivars raises difficulties in interpreting energy metabolism. Even so, the maximum catalytic activities of PDC and PFK are, on average, 20% and 37% lower, respectively, in IR22 than in Calrose (Table 5a). These differences in enzyme activity are not as pronounced as the average difference in the rate of alcoholic fermentation, which is 49% lower in IR22 than in Calrose. Nevertheless, the ratios of maximum catalytic activity of PFK, and of PDC, to the carbon flux to ethanol are of the same order of magnitude in the two cultivars (Table 5b), and are consistent with high sensitivity coefficients for PFK and to a lesser extent for PDC in both Calrose and IR22. Thus, the combination of lower maximum catalytic activities in PDC and PFK in IR22 may contribute to the cultivar difference in alcoholic fermentation. However, this interpretation remains tenuous for the following reasons. When net ethanol synthesis and maximum catalytic activity of PDC and PFK in IR22 are expressed as a percentage of that observed in Calrose, there is, between experiments, (1) no correlation between PDC activity (IR22 as a percentage of Calrose) and net ethanol synthesis (IR22 as a percentage of Calrose), and (2) negative correlation between PFK activity (IR22 as a percentage of Calrose) and net ethanol synthesis (IR22 as a percentage of Calrose). That is, in this study, the more the maximum catalytic activity of PFK in Calrose exceeded IR22, the smaller the cultivar difference in net ethanol synthesis.
Furthermore, changes in the concentrations of glycolytic intermediates in IR22 following the onset of anoxia do not support the view that lower maximum catalytic activity in PDC and PFK in IR22 are responsible for the cultivar difference in alcoholic fermentation. Firstly, in this cultivar, the concentration of fructose-6-phosphate, the substrate of PFK, remains unchanged following the onset of anoxia (Table 8). This is consistent with the lack of a Pasteur effect observed in IR22, and suggests that PFK does not have a major regulatory role in glycolytic carbon flux in this cultivar.
Secondly, the significant decrease in pyruvate in anoxic IR22 coleoptiles may adversely affect flux through PDC in this cultivar. By contrast, pyruvate concentration was maintained following the onset of anoxia in Calrose coleoptiles (Table 8). A case for pyruvate concentration influencing flux through PDC rests on S0.5 (pyruvate) of PDC. Half maximal velocities of PDC are achieved at pyruvate concentrations of 1.0 mol m-3 at pH 6.8 and 2.5 mol m-3 at pH 7.5 (Morrell et al., 1990), well above the estimated cytoplasmic pyruvate levels of 0.36 mol m-3 in Calrose and 0.28 mol m-3 in IR22 (calculated assuming 10% cytoplasm; Table 8). Thus, the low level of pyruvate in anoxic IR22 indicates that low substrate supply to PDC rather than the PDC activity, is associated with the low rate of ethanol synthesis.
Consistent with this view, recent evidence suggests that in IR22 substrate availability, rather than enzyme activity, limits carbon flux through glycolysis to ethanol. For example: (1) ethanol synthesis increases 3-fold upon addition of glucose to excised IR22 coleoptiles (Setter et al., 1994), strongly suggesting that substrate availability is a principal limiting factor in this slowly elongating genotype. In Calrose, the rate of ethanol synthesis was only 1.6-fold higher with exogenous glucose than without (Setter et al., 1994). The relatively small increase observed in Calrose does not necessarily contradict the conclusion that PFK largely regulates the carbon flux from hexose-phosphates to ethanol in this cultivar. The coleoptile is quite heterogeneous with expanding cells at the base and fully expanded cells at the tip. Hence a strong stimulation of ethanol synthesis by glucose in, for example, some of the expanding cells could account for the stimulatory effect of exogenous glucose, while data on level of glycolytic intermediates and enzyme activities are dominated by the larger proportion of expanded cells. (2) Glucose-feeding increased the elongation rate of coleoptiles of intact IR22 seedlings by over 2-fold between 3 d and 6 d of anoxia, while during the same period, the rate of elongation of intact Calrose coleoptiles remained unchanged (Shaobai Huang, personal communication). Total soluble sugars were 50% higher in IR22 supplied with glucose over 6 d of anoxia than in coleoptiles grown without exogenous glucose. In contrast, total soluble sugars were only 10% higher in glucose-fed Calrose coleoptiles exposed to anoxia for 6 d. (Shaobai Huang, personal communication).
Data from both these investigations are particularly relevant to the present study, which focuses primarily on the response of seedlings between 3 d and 5 d of anoxia.
In conclusion, this study suggests that PFK is an important site of regulation of anaerobic carbohydrate catabolism in anoxia tolerant Calrose rice coleoptiles. By contrast, in IR22, control by PFK was not evident, consistent with recent evidence that suggests substrate supply limits alcoholic fermentation in this cultivar.
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The authors thank Dr Shaobai Huang for kindly providing data from recent substrate feeding experiments, and Drs Brian Atwell and Bijayalashkmi Mohanty for their useful criticism of the manuscript.
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3 To whom correspondence should be addressed. Fax: +91 8 9380 1140. E-mail:djgibbs{at}cyllene.uwa.edu.au
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