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Journal of Experimental Botany, Vol. 53, No. 376, pp. 1847±1856, September 2002 DOI: 10.1093/jxb/erf036 Stimulation of glycolysis in anaerobic elongation of pondweed (Potamogeton distinctus) turions Tatsuhisa Sato, Taro Harada and Kimiharu Ishizawa1 Department of Developmental Biology and Neuroscience, Graduate School of Life Sciences, Tohoku University, 980-8578 Sendai, Japan Received 28 January 2002; Accepted 9 May 2002 Abstract Introduction Stem segments prepared from pondweed (Potamogeton distinctus A. Benn.) turions (overwintering buds) elongate in anaerobic conditions, whereas there is almost no elongation in air. The anaerobic elongation was accompanied by a decrease in dry weights of stem segments, mainly due to consumption of storage starch in the amyloplasts of stem cells. On the other hand, total contents of amino acids increased in stem segments, in which contents of alanine, valine, leucine, and isoleucine increased, but contents of asparatic acid decreased. Moreover, contents of lactate in stem tissues increased at an early stage of anaerobic incubation. In tracer experiments with 14C-glucose, 14C incorporation into stem tissues in anoxia was only half of that in normoxia. However, conversion of 14C to ethanol occurred exclusively in anoxia. 14C-labelled metabolites were analysed by two-dimensional cellulose thin-layer chromatography. 14C incorporation into sucrose and alanine was signi®cantly increased in anoxia. The activity of alanine aminotransferase was enhanced by anoxia, suggesting that pyruvate is a precursor of alanine synthesis. The results suggest that pondweed turions produce energy necessary for anaerobic elongation by activating conversion of storage starch in the amyloplasts to ethanol, alanine and lactate. Some aquatic plants have been found to exhibit strong tolerance to anaerobic conditions. These include rice coleoptiles (Tsuji, 1972), barnyard grass coleoptiles (VanderZee and Kennedy, 1981), Scirpus maritimus rhizomes (Barclay and Crawford, 1982), Trapa natans seedlings (Menegus et al., 1992), Acorus calamus rhizomes (Monk et al., 1984), Potamogeton pectinatus stems (Summers and Jackson, 1994), Potamogeton distinctus stems and Sagittaria pygmaea leaves (Ishizawa et al., 1999). In the last three species, elongation in the absence of oxygen has been reported to be greater than that in air. Strong tolerance to anaerobic conditions is known to be one of the adaptive characteristics of aquatic plants for their survival in anaerobic environments. However, the mechanisms of the tolerance are not clear. Studies on the regulation of energy metabolism in anoxia are important to understand how aquatic seed plants survive when they are exposed to anaerobic conditions. One of the most important issues for the survival of seed plants in anoxic conditions is the mechanism of ATP production, which supports anaerobic growth. Anaerobic conditions stimulate glycolysis, which is a ubiquitous pathway for the production of ATP in anaerobic conditions (Davies, 1980). Crawford (1977, 1978) suggested that anaerobic-tolerant species exhibit a smaller Pasteur effect than do intolerant species. A smaller Pasteur effect would contribute to the preservation of respiratory substrates and to the prevention of over-production of toxic end-products of fermentation. In fact, it has been shown that anaerobictolerant plants can produce various metabolites, such as alanine, succinate, g-aminobutyrate (GABA), and malate, as fermentation end-products (Crawford, 1978). However, it is still obscure whether the accumulation of such metabolites correlates with anaerobic tolerance. In many Key words: Alanine, amyloplast, anoxia, elongation, ethanol, fermentation, glycolysis, lactate, Potamogeton distinctus A. Benn., turion. 1 To whom correspondence should be addressed. Fax: +81 22 217 734. E-mail: [email protected] ã Society for Experimental Biology 2002 1848 Sato et al. plants showing extremely strong tolerance to anoxia, such as Trapa natans (Menegus et al., 1992), Acorus calamus (Monk et al., 1984), Potamogeton pectinatus (Summers et al., 2000), P. distinctus and Sagittaria pygmaea (Ishizawa et al., 1999), alcoholic production is greatly stimulated in anoxia, suggesting that the stimulation of alcoholic fermentation is essential for these plants to survive in anoxia. Kennedy et al. (1992) concluded that the occurrence of a Pasteur effect per se does not correlate with tolerance or intolerance to anoxia in plants. Pondweed (Potamogeton distinctus A.Benn.), a ¯oating plant, forms yellow banana-shaped overwintering buds (called turions) underground in autumn. Turions can start growing in the following spring when they are exposed to oxygen-de®cient conditions of less than 5 kPa of oxygen (Ishizawa et al., 1999). The stem elongation of pondweed turions is maximal in the absence of oxygen. The anaerobic growth is supported by the maintenance of a large energy charge between 0.8 and 0.9 during an anoxic state lasting over 2 weeks, suggesting the operation of active energy metabolism. The aims of this study were to determine what substances are used for an anaerobic-respiratory substrate and to determine what metabolites are produced when 14Cglucose and 14C-pyruvate are fed to pondweed turions under anaerobic conditions. Materials and methods Plant materials Pondweed (Potamogeton distinctus A.Benn.) was cultured in a small pool constructed in a greenhouse belonging to the department. Banana-shaped turions formed at the top of rhizomes were harvested and stored in the dark at 4 °C until used. Incubation and growth experiments Pondweed turions were placed on moist ®lter paper, pre-incubated overnight at 25 °C in the dark, and then stripped of their scaly leaves. These naked turions were used as experimental material. Ten-mmlong stem segments were prepared from naked turions by cutting the base and the apex with a razor blade. Three segments were stood in an 8310±7 m3 glass tube (5310±3 m in diameter) with incubation medium. Distilled water was usually used as the incubation medium. For 14C-glucose feeding experiments, distilled water containing carbenicillin 50 g m±3 and vancomycin 500 g m±3 was used as the incubation medium, whereas for 14C-pyruvate feeding experiments, 5 mol m±3 2-(N-morpholino)ethanesulphonic acid (MES) buffer (pH 5.5) containing the same antibiotics was used. The glass tubes were set in a 10±4 m3 glass vessel that was connected to a gas-¯ow system (Ishizawa et al., 1999) to supply water-saturated air or nitrogen gas continuously. In the case of the 14C-glucose and 14 C-pyruvate feeding experiments, a vial containing 10±6 m3 of 43103 mol m±3 KOH as an absorbent of carbon dioxide and a bag of Anaeropackâ (Mitsubishi Gas Chemical Co., Tokyo, Japan) as an absorbent of oxygen were put together in the 10±4 m3 glass vessel. Analysis of metabolites in stem segments Extraction: After the incubation, length, fresh weight and dry weight of stem segments were measured. For the dry weight measurement, the segments were heated at 110 °C for 30 min to kill tissues as soon as possible and further dried at 70 °C for 3 h. To determine the contents of sugars and amino acids, three dried segments were serially extracted at 80 °C with 2310±6 m3 of 80% ethanol for 1 h, with the same volume of 80% ethanol for 30 min and ®nally with 10±6 m3 of 80% ethanol for 30 min. The combined extract was dried under reduced pressure and redissoloved in deionized water. The ethanol-insoluble materials were used for analysis of polysaccharides. The water-soluble substances were passed through an ionexchange column packed with Dowex 50W-X8 (NH4+ form) and then through one packed with Dowex 1-X8 (HCOO± form). The fraction passing through both columns was evaporated to dryness, redissolved in deionized water, and used as a neutral fraction for sugar analysis. Substances bound to the Dowex 50W-X8 column were eluted with 23103 mol±3 NH4OH, evaporated to dryness with a rotary evaporator, redissolved in deionized water, and used for amino acid analysis. Substances bound to the Dowex 1-X8 column were eluted with 63103 mol m±3 formic acid and used for the analysis of organic acids. Recoveries of sugars and amino acids eluted from the ion-exchange columns were more than 80% when authentic glucose and leucine were separated with them. Sugar analysis: Total contents of sugars and reducing sugars in the ethanol-soluble fraction were determined by the method using phenol±sulphuric acid (Dubois et al., 1956) and by the Somogyi± Nelson method (Nelson, 1944). To identify sugars in an ethanolsoluble fraction, the neutral substances were applied to a silica gel G plate (Analthech Imc. Newark, USA) and developed in acetone:chloroform:methanol:water (16:2:2:1, by vol.) and detected by heating at 100 °C after spraying with an anisaldehyde±sulphuric acid reagent (1310±7 m3 concentrated H2SO4 plus 5310±6 m3 acetic acid containing 5310±8 m3 anisaldehyde). To determine the contents of starch in the stem segments, ethanolinsoluble fractions were extracted with 1310±6 m3 of hot water at 100 °C for 10 min and then treated with 1310±6 m3 of 9.23103 mol m±3 perchloric acid at 20 °C for 15 min. After the addition of 33 10±6 m3 deionized water, the supernatant was collected by centrifugation. The precipitate was twice extracted with 2310±6 m3 of 4.83103 mol m±3 perchloric acid. The ®nal precipitate was kept for the analysis of polysaccharides except for starch. The combined extract was hydrolysed at 100 °C for 2 h and then neutralized with 2.53103 mol m±3 NaOH to adjust the pH to around 5.4. The amount of reducing sugar in the hydrolysate was measured by the method of Somogyi±Nelson. The amount of starch is expressed as a value obtained by multiplying the measured amount of reducing sugars by 0.9. To determine the amount of polysaccharides except for starch, the residue obtained by extraction with perchloric acid was hydrolysed with 2310±6 m±3 of 0.53103 mol m±3 HCl at 100 °C for 2.5 h. The hydrolysate was neutralized with 0.5310±3 mol m3 NaOH and centrifuged to collect the supernatant. Reducing sugars in the hydrolysate were measured by the method of Somogyi±Nelson. The amount of polysaccharides, except for starch, is expressed as a value obtained by multiplying the amount of reducing sugars by 0.9. Analysis of amino acids: The amount of amino acids in the fraction eluted from a Dowex 50W-X8 column was determined with ninhydrin after the complete removal of the ammonia used to elute the amino acids. Then each amino acid was separated with a Cosmosil 5C18-MS column (nacalai tesque, Tokyo, Japan) after dabsylation with 4-dimethylaminoazobenzene-4¢-sulphonyl chloride according to the method of Stocchi et al. (1989). Dabsylated amino acids were detected at the absorbance of 436 nm. The amount of each amino acid was determined by using a standard mixture of amino acids (Wako Pure Chemical Industries, Osaka, Japan). Growth and glycolysis in anoxic pondweeds 1849 Total nitrogen contents: To estimate total content of nitrogenous compounds in stem segments, dried segments were degraded by the Kjeldahl method (Hambrñus et al., 1976). The amount of ammonia produced by Kjeldahl degradation was determined using Nessler's reagent (Thompson and Morrison, 1951). Lactate contents: After incubation, the segments (about 120 mg FW) were frozen in liquid nitrogen, powdered with a mortar and pestle, and extracted with 6310±7 m3 of ice-cold 0.63103 mol m±3 perchloric acid for 10 min and then centrifuged to collect the supernatant. The extraction with perchloric acid was repeated again. The combined extract (1.2310±6 m3) was neutralized with 43 103 mol m±3 KOH after the addition of 0.05% methyl orange of 2.5310±9 m3 as a pH indicator. After cooling on ice for 15 min, the extract was centrifuged at 15 000 g for 10 min to collect the supernatant. The amount of lactate in the extract was determined by an enzymatic method with lactate dehydrogenase and alanine aminotransferase (Noll, 1985). 14 C-glucose and 14C-pyruvate feeding experiments After incubation of stem segments in the gas-¯ow system, the stop valve attached to the lid of the growth chamber was closed to shut off the supply of gas. After separation of the growth chamber, D-(U14 C)-glucose of 0.37 MBq (ARC, speci®c activity, 300 mCi mmol±1) or (2-14C)-pyruvate of 0.11 MBq (ARC, speci®c activity, 5 mCi mmol±1) was injected into the small vial with stem segments in the growth chamber by a syringe through a rubber septum (5 mm double-skirted rubber stopper, Sigma±Aldrich Japan, Tokyo, Japan) attached to the lid of the growth chamber. The hole of the rubber septum was sealed with adhesive vinyl tape. The segments treated with 14C-glucose and those treated with 14C-pyruvate were incubated in the dark at 25 °C for a further 8 h and 1.5 h, respectively. After incubation, the segments were washed with deionized water three times, weighed, and plunged into liquid nitrogen. Ethanol (53 10±6 m3) was added to three frozen segments and heated at 80 °C for 2 h. An aliquot of the ethanol-soluble fraction was used to distill ethanol with a small distillatory apparatus. The ethanol-insoluble materials were further extracted with 3310±6 m3 of 50% ethanol and 2310±6 m3 of deionized water at 80 °C for 2 h and 1 h, respectively. The combined ethanol- and water-soluble fraction was dried under reduced pressure and redissolved in deionized water as an ethanolsoluble fraction. An aliquot of the ethanol-soluble fraction was applied to a Dowex1-X8 column and a Dowex 50W-X8 column as described in the Extraction section and separated into acidic, basic and neutral substances. The ethanol-soluble fraction and the acidic, basic and neutral substances were analysed by two-dimensional thinlayer chromatography (2-D TLC) with a cellulose thin-layer plate (Avicel SF 20320 cm, Asahi Kasei Co., Tokyo, Japan) and developed according to the method described in a previous paper (Ishizawa and Esashi, 1988). The ®rst solvent was isobutylic acid:water:n-propanol:n-butanol:isopropanol:25% NH4OH:0.053 103 mol m±3 EDTA (50:18:7:1.5:1.5:2:1, by vol.), and the second solvent was sec-butanol:98% formic acid:water (6:1:2, by vol.). Before the second development, three different amounts of 14Cglucose were applied to the plate as a standard for calculating radioactivity incorporated into each separated metabolite. The chromatogram was exposed to X-ray ®lm (Konica Medical ®lm, New A) for 8 d at ±80 °C. To estimate radioactivity of each metabolite separated by 2-D TLC, the density of each spot on the autoradiogram was determined by NIH Image using an Apple Macintosh computer. For identi®cation of metabolites separated by 2-D TLC, the metabolites were eluted from the plate and again analysed together with authentic compounds by cellulose and silica gel G TLC. Radioactivities in distilled ethanol, in the ethanol- soluble fraction and ethanol-insoluble fraction, and in 43103 mol m±3 KOH as an absorbent of carbon dioxide were determined using a liquid scintillation spectrometer (LSC-300, Aloka, Tokyo, Japan). Measurement of alanine aminotransferase activity Fresh weights of stem segments were measured after incubation. The segments were frozen in liquid nitrogen and powdered with a mortar and pestle with cooling by liquid nitrogen. Ten volumes of extract buffer (100 mol m±3 TRIS±HCl, pH 8.0, 15% glycerol and 10 mol m±3 dithiothreitol) were added to the powder. The supernatant was collected by centrifugation at 15 000 g for 15 min and fractionated by the addition of solid (NH4)2SO4. The fraction precipitated with 80% saturated (NH4)2SO4 was dissolved in extraction buffer and desalted with Centricorn (Amicon, USA). All extract procedures were conducted at 4 °C. The activity of alanine aminotransferase was measured by the method of Hùrder and Rej (1983). The reaction mixture was composed of 2310±5 m3 of alanine/TRIS buffer (615 mol m±3 alanine plus 110 mol m±3 TRIS, pH 7.3), 1310±7 m3 of lactate dehydrogenase (speci®c activity:33109 U m±3), 5310±7 m3 of 500 mol m±3 pyridoxal 5-phosphate, and 3.3 mg NADH. A crude enzyme solution of 3310±8 m3 was added to 2310±6 m3 of reaction mixture was pre-incubated at 30 °C for 10 min. After addition of 2310±7 m3 of 2-oxoglutarate/TRIS buffer (180 mol m±3 2-oxoglutarate, 110 mol m±3 TRIS, pH 7.3), the decrease in absorbance at 340 nm was measured at 30 °C. The content of proteins in the sample was measured by the method of Bradford (1976). Preparation of specimens for light microscopic observation Stem segments of pondweed turions were incubated in aerobic and anaerobic conditions and ®xed with 2.5% glutaraldehyde in 10 mol m±3 MES buffer (pH 6.5) overnight, post-®xed with 1% OsO4 in the same buffer for 2 h at 4 °C, dehydrated in a graded ethanol series, and in®ltrated with Spurr's resin (Spurr, 1969). Thin sections were stained with 0.1% toluidine blue O in 100 mol m±3 phosphate buffer (pH 7.4) for observation under an Olympus Vanox light microscope. Results Effects of anaerobic conditions on metabolism in elongating stem segments Effects of anaerobic treatments on growth were examined in stem segments excised from pondweed turions. In the absence of oxygen, length (Fig. 1A) and fresh weight (Fig. 1B) of segments increased, but the dry weight (Fig. 1C) decreased. These results show that some materials in stem tissues are consumed for the stimulated elongation in anaerobic conditions. To examine which components of the stem are used during anaerobic growth, each component was extracted from the growing segments. Over a period of 6 d, neither contents of nitrogenous compounds (Fig. 2A) nor contents of polysaccharides except for starch (Fig. 2B) in segments changed in anaerobic conditions. However, contents of starch (Fig. 2C) decreased in anaerobic conditions. Ethanolsoluble sugars (Fig. 2D) decreased after 1 d of anaerobic incubation and then slightly increased. The amounts of starch that had been consumed after 6 d of anaerobic incubation corresponded to about 60% of the decreases in dry weights of segments shown in Fig. 1C. Figure 3 1850 Sato et al. Fig. 1. Time-courses of aerobic and anaerobic growth of stem segments excised from pondweed turions. Length (A), fresh weight (B) and dry weight (C) of the segments were measured after incubation in air and nitrogen gas. The fresh and dry weights are means of three segments and the data are means 6SE of nine replicates and six replicates, respectively. The length is the mean 6SE of 27 segments in nine replicates. shows cross-sections of stem segments after 3 d in aerobic (Fig. 3A) and anaerobic (Fig. 3B) conditions. Large aerenchymas developed in stem tissues. Both parenchymatous and epidermal cells contained many amyloplasts. After 3 d of incubation, a number of amyloplasts in the cells had drastically decreased in anaerobic conditions. These results show that storage starch in pondweed stems is the main substrate for anaerobic respiration. On the other hand, ethanol-soluble sugars of pondweed turions were analysed by cellulose TLC and it was found that the main sugar was sucrose (data not shown). Figure 2E shows that total contents of amino acids signi®cantly increase throughout the incubation period in anaerobic conditions. The content of each free amino acid in stem tissues was determined by HPLC analysis Fig. 2. Changes in contents of nitrogenous compounds (A), polysaccharides except for starch (B), starch (C), ethanol-soluble sugars (D), and amino acids (E) in pondweed turions in aerobic and anaerobic conditions. Analytical methods for each substance are described in the Materials and methods. Data are means 6SE of three replicates. Growth and glycolysis in anoxic pondweeds 1851 Fig. 3. Decrease in the number of amyloplasts after anaerobic elongation of the stem in pondweed turions. Cross-sections of stems were prepared from segments that had been incubated in air (A) and nitrogen gas (B) for 3 d and stained with 0.1% toluidine blue O. Bars show 25 mm. Table 1. Changes in contents of amino acids in stem segments incubated in aerobic and anaerobic conditions Amino acids Asp Glu Ser Thr + Arga Gly Ala Pro+GABAa Val Ile Leu Phe Cys Lys His Tyr a Treatment Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Air N2 Contents of amino acids (nmol mg±1 DW) Initial 1d 3d 6d 1.9361.44 5.9860.25 0.0360.03 1.7160.51 1.3160.31 0.4760.07 0.2560.06 1.4660.11 1.3160.31 0.2360.05 0.1660.06 1.4260.14 6.7562.37 0.7560.29 0.7860.19 0.4160.05 0.5660.03 0.1460.02 0.3260.03 0.1660.03 0.3260.03 0.8360.67 1.2661.07 nd nd 0.3260.24 0.3760.17 0.0760.07 0.0460.04 0.1860.24 0.2360.10 2.4261.82 0.8860.88 1.4360.60 0.6960.35 0.4560.12 0.3660.06 2.1160.26 1.4260.33 0.2460.11 0.2560.16 1.2360.24 6.4962.76 0.7760.12 1.8660.34 0.8160.08 1.1360.05 0.3060.03 0.6460.04 0.0960.05 0.7460.01 0.2660.11 1.0460.78 nd 0.3760.22 0.4660.11 0.5160.19 0.1060.10 0.0460.04 0.1860.13 0.4260.13 8.8160.17 2.5862.32 0.5360.12 1.3760.08 0.3260.03 0.4860.02 1.1460.46 2.2360.21 0.1760.04 0.4560.14 0.7860.12 18.363.38 0.5160.21 3.5060.70 0.7560.30 1.2860.05 0.3260.07 1.0060.19 0.1260.04 1.2160.28 0.6960.49 7.6667.08 nd 0.2160.12 0.2860.06 0.5860.10 0.1260.12 0.0960.09 0.1660.06 0.5560.18 0.7460.44 0.3060.15 1.7260.06 0.1460.06 1.4160.70 0.2360.10 0.3860.12 0.1760.09 0.0360.03 0.5060.34 nd 0.1460.14 nd 0.0660.04 Threonine and arginine, and proline and g-aminobutylic acid were not separated. (Table 1). Contents of glutamine, asparagine, methionine, and tryptophan in the tissues were not determined by this method. Moreover, threonine and arginine, and proline and g-aminobutyric acid (GABA) were not completely separated. The content of aspartate in anoxic stems was less than that in normoxic stems. Contents of alanine, valine, isoleucine, and leucine were increased by anaerobic conditions. Contents of GABA plus proline also tended to increase in anaerobic conditions. The increase in alanine content was the most prominent, and its content after 6 d of anaerobic conditions was 10-fold greater than that of initial tissues. In aerobic conditions, contents of amino acids except for that of aspartic acid remained almost constant throughout the incubation period. 1852 Sato et al. Determination of metabolic fate of glucose incorporated into stem tissues To determine the characteristics of anaerobic metabolism in pondweed stems, 14C-labelled metabolites were analysed after 14C-glucose feeding in an aerobic and an anaerobic closed system for 8 h to pondweed stems that had been incubated for various periods in the gas-¯ow apparatus. The total incorporation of 14C into stem tissues (Fig. 4A) is expressed as the sum of 14C radioactivities in the ethanol-soluble fraction (Fig. 4B), ethanol-insoluble fraction (Fig. 4C), CO2 absorbed by KOH (Fig. 4D), and ethanol extracted from segments (Fig. 4E). Total incorporation of 14C into stem segments gradually decreased in both aerobic and anaerobic conditions. The amount of 14Cglucose incorporated in aerobic conditions was 2.0±3.1fold greater than that in anaerobic conditions. In both aerobic and anaerobic conditions, 14C incorporated into soluble fractions (Fig. 4B) was more than 75% of the total incorporation. Incorporation of 14C into ethanol-insoluble fractions (Fig. 4C) was inhibited by anaerobic conditions, suggesting that anabolism is depressed in anoxia. The amounts of 14C released as CO2 in air were 2.3±3.7-fold higher than those in nitrogen gas (Fig. 4D). On the other hand, over 15% of 14C-glucose incorporated into stem segments in anaerobic conditions was converted into ethanol, but there was almost no 14C incorporation into ethanol in air. The results show that activation of ethanol fermentation continues throughout the anaerobic incubation period. 14 C-labelled metabolites of stem segments were separated by 2-D TLC. Figure 5 shows autoradiograms of 2-D TLC analysis of ethanol-soluble materials extracted from stem segments in air or nitrogen gas for 3 d and 6 d. Sucrose was heavily radiolabelled regardless of the presence or absence of oxygen, indicating that sucrose synthesis is always active in stem tissues. Radioactivity of many metabolites was decreased in anaerobic conditions, but 14C incorporated into alanine was signi®cantly increased. Figure 6 shows changes in the percentages of 14C incorporated into alanine, ethanol and sucrose to total 14C incorporation, which were calculated from the data in Figs 4 and 5. In air, 14C incorporation into ethanol was negligible, and the ratio of 14C incorporation into alanine gradually decreased. In anaerobic conditions, ratios of 14C incorporation into ethanol were high, and they continued to increase with incubation time. The amount of 14C-labelled ethanol released into the gas space of the incubation chamber could not be measured. Therefore, the actual ratio of 14C incorporation into ethanol might be greater than the value shown in Fig. 6. On the other hand, both ratios of 14C incorporated into sucrose and alanine remained at about 10% in anaerobic conditions, and they were always greater than those in aerobic conditions. Fig. 4. Changes in 14C incorporation into the ethanol-soluble fraction (B), ethanol-insoluble fraction (C), CO2 (D), and ethanol (E) in pondweed turions incubated with (U-14C)-glucose in aerobic and anaerobic conditions. Total incorporation of 14C into tissues (A) is the sum of radioactivities in all fractions. Data are means 6SE of three replicates. 14 C incorporation into lactate in the 14C-glucose feeding experiments could not be determined because lactate was not detected by 2-D TLC. Changes in contents of lactate in Growth and glycolysis in anoxic pondweeds 1853 Fig. 5. Autoradiogram of two-dimensional TLC. Ethanol-soluble compounds were extracted from pondweed turions that had been incubated in aerobic and anaerobic conditions for 3 d. Authentic Glc shows spots of 14C-glucose (17, 50 and 170 Bq) that were applied as a standard for quantitative analysis to the TLC plate before the second development. Ala, alanine; Suc, sucrose; Glc, glucose; Fru, fructose. Arrows show the ®rst and second dimensions. stem tissues were therefore measured by enzymatic analysis (Fig. 7). The lactate content increased after 1 d of incubation in anaerobic conditions, and the increase had almost stopped after 3 d of incubation. transferase, which is involved in the conversion of pyruvate to alanine (Fig. 8). Anaerobic conditions activated alanine aminotransferase throughout the incubation period until the sixth day of incubation. Enhancement of alanine aminotransferase activity by anaerobic conditions Table 1 and Fig. 6 clearly show that alanine synthesis is promoted by anaerobic treatments. An experiment was carried out to determine whether the increase in alanine synthesis is accompanied by activation of alanine amino- Metabolism of (2-14C)-pyruvate fed to stem segments 14 C-labelled metabolites in stem segments fed (214 C)pyruvate for 90 min in anaerobic conditions were analysed after anaerobic incubation for 1 d. Ethanolsoluble materials extracted from the segments were applied to a Dowex 50W-X8 column. The metabolites 1854 Sato et al. Fig. 7. Changes in contents of lactate in pondweed turions incubated in aerobic and anaerobic conditions. Data are means 6SE of three replicates. Fig. 6. Distribution of 14C incorporated into pondweed turions among ethanol, sucrose and alanine. Total 14C incorporation and 14C incorporated into ethanol were the same as data shown in Fig. 4. 14C incorporation into sucrose and alanine was estimated from the density and size of each spot on the autoradiogram shown in Fig. 5. Data are means 6SE of three replicates. bound to the resins were eluted with ammonia and separated by 2-D TLC analysis. Only two radioactive metabolites were detected on the autoradiogram. The two compounds eluted from the plate were identi®ed as glutamic acid and GABA by cellulose TLC with a solvent, phenol:water (3:1, w/w), and by silica gel G TLC with a solvent, ethanol:28% ammonia water (77:23, v/v), respectively (data not shown). Discussion Anaerobic elongation of pondweed turions is supported by a metabolic system to keep the energy status at the same level as that in air (Ishizawa et al., 1999). Findings in this study show that storage starch is the main substrate for anaerobic respiration. Consumption of starch was promoted in anaerobic conditions, but inhibited in aerobic conditions (Figs 2C, 3). In a previous paper (Ishizawa et al., 1999), it was reported that the rate of ethanol production is greatly enhanced by anaerobic conditions. 14C-glucose incorporated into turion tissues was ef®ciently converted into ethanol (Figs 4, 6). These results show that ethanol fermentation is the main metabolic pathway for production of ATP which is necessary for anaerobic growth of pondweed turions. Summers et al. (2000) reported that anaerobic elongation of Potamogeton pectinatus stems is associated with an unusually large Pasteur effect. They suggested that starch is a source of energy in anaerobic conditions. Both Potamogeton distinctus and P. pectinatus seem to have a similar mechanism for regulation of energy production through glycolysis and ethanol fermentation in anaerobic growth. In the case of Potamogeton distinctus, the consumption of starch was clearly prevented under aerobic Fig. 8. Changes in the activities of alanine aminotransferase in pondweed turions incubated in aerobic and anaerobic conditions. Data are means 6SE of three replicates. conditions compared with anaerobic conditions (Fig. 2C), suggesting a manifestation of the Pasteur effect. The occurrence of the Pasteur effect should show an enhancement of CO2 released under anaerobic conditions. However, the ratios of 14C incorporation into CO2 in aerobic conditions to that in anaerobic conditions remained around 3.0 (Fig. 4D), suggesting that a Pasteur effect does not occur. The incompatible result seemed to be attributed to potential problems of the method to estimate a rate of CO2 production. Since the uptake of 14C-glucose by turions was signi®cantly inhibited in anoxia (Fig. 4A), it was unsuitable to compare the amounts of 14C incorporation into CO2 in anaerobic conditions directly with those in aerobic conditions. On the other hand, SchluÈter and Crawford (2001) reported that long-term anoxia tolerance in leaves of Acorus calamus and Iris pseudacorus is associated with down-regulation of glycolysis. Whether a Pasteur effect is brought about in the growth of anoxic pondweed turions warrants further investigation. Figures 5 and 6 show that the active biosynthesis of sucrose continues in anoxic pondweed turions. Sucrose is the main sugar in ethanol-soluble fractions of pondweed stems. Figure 2D shows that ethanol-soluble sugars decrease at an early stage of anaerobic incubation, Growth and glycolysis in anoxic pondweeds 1855 suggesting that sucrose is actively used. These results suggested that sucrose biosynthesis is enhanced by anaerobic conditions. Also, in the case of Potamogeton pectinatus stems (Summers et al., 2000), sucrose accumulates in anaerobic conditions. In both cases, the conversion from starch to sucrose may be necessary in order to use it as a substrate for glycolysis. The meaning of sucrose conversion remains unclear. Sugars produced by starch degradation may be transported to a site of glycolysis after the conversion of sucrose. Alternatively, the conversion to sucrose may be involved in the regulation of carbohydrate metabolism just as the futile cycle of sucrose metabolism operates in sink organs (Nguyen-Quoc and Foyer, 2001). Further study on the degradation process of storage starch in amyloplasts in anaerobic conditions (Fig. 3) is needed to determine the meaning of sucrose biosynthesis. Many plant tissues in anaerobic conditions produce lactate in addition to ethanol (Davies, 1980). In the case of pondweed turions, lactate accumulation almost stopped at 1 d after anaerobic incubation (Fig. 7), whereas the conversion from glucose to ethanol increased after 3 d of anaerobic incubation (Fig. 6). These results suggest that lactate fermentation precedes alcoholic fermentation in pondweed turions exposed to anaerobic conditions. Davies et al. (1974) and Roberts et al. (1984) proposed that a shift from lactate fermentation to alcoholic fermentation occurs by the operation of a biochemical pH-stat. This metabolic change in pondweed turions may be regulated by the biochemical pH-stat mechanism. However, it should be noted that the pH-stat hypothesis is debatable (Ratcliffe, 1997; Vartapetian and Jackson, 1997). In addition to ethanol and lactate, there was a signi®cant accumulation of alanine as an end-product of anaerobic metabolism in pondweed turions (Figs 5, 6; Table 1). Several plants subjected to anaerobic stress are known to produce alanine (Streeter and Thompson, 1972; Smith and ap Rees, 1979; Menegus et al., 1988). These alanine accumulators are not always anaerobic-tolerant plants. Therefore, the degree of anaerobic tolerance is unlikely to be determined by the accumulation of alanine itself. In maize root tips (Roberts et al., 1992), alanine as well as lactate is produced at the early stage of hypoxia, but alanine itself does not seem to contribute to cell acidosis. In pondweed turions, however, a high rate of alanine conversion from glucose was maintained throughout the period of anaerobic elongation (Fig. 6). Unlike ethanol fermentation, the rate of alanine production did not change during the 6-d anaerobic period (Fig. 6). Crawford (1978) pointed out that diversity of end-products in anaerobic metabolism is important to prevent the accumulation of a toxic metabolite, such as lactate or ethanol. Alanine production may contribute to the ef®ciency of glycolysis by dispersing the end product. The metabolic fate of alanine, which accumulated in anaerobic conditions, was not examined after anoxic turions had been transferred to aerobic conditions. Lactate and ethanol, but not alanine, are wasteful end-products of anaerobic metabolism. The physiological signi®cance of the accumulation of alanine may be a temporary store of carbon and nitrogen in a state of anoxia. The activity of alanine aminotransferase was enhanced in pondweed turions in anaerobic conditions (Fig. 8). The activities of aminotransferase in barley roots (Muench and Good, 1994) and in corn roots (Muench et al., 1998) were reported to be greatly enhanced by anaerobic treatments. In the case of barley (Muench and Good, 1994), a speci®c isozyme of alanine aminotransferases is increased in anaerobic conditions. On the other hand, valine and leucine, which are usually produced from pyruvate, signi®cantly increased in anoxic turions, whereas aspartate signi®cantly decreased (Table 1). Such a signi®cant decrease in asparatate content by anaerobic treatment has been found in maize root tips (Roberts et al., 1992), pea roots (Smith and ap Rees, 1979) and radish leaves (Streeter and Thompson, 1972). In these cases, aspartate is thought to be the main source of the amino group of alanine. All these facts suggest that alanine is produced from pyruvate by an action of alanine aminotransferase. In this study, however, 14C incorporation into alanine could not be detected when 14C-pyruvate was fed to pondweed turions in anaerobic conditions, but 14C incorporation into GABA and glutamate was detected. Table 1 suggests the increase in GABA contents by anaerobic treatments. Stimulation of GABA production by anaerobic conditions has been reported in many plants (Shelp et al., 1999). GABA synthesis is thought to contribute to the prevention of cytoplasmic acidosis in anaerobic conditions because it occurs through the H+consuming a-decarboxylation of glutamate by an action of glutamine decarboxylase (EC 4.1.1.15). Pyruvatedependent 4-aminobutylate transaminase has been reported to be involved in GABA synthesis (Van Cauwenberghe and Shelp, 1999). Therefore, 14C-pyruvate may be exclusively used for GABA synthesis rather than alanine synthesis. 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