The catalytic direction of pyrophosphate:fructose 6-phosphate 1-phosphotransferase in rice coleoptiles in anoxia

The catalytic direction of pyrophosphate:fructose-6-phosphate 1-phosphotransferase (PFP; EC 2.7.1.90) in coleoptiles of rice ( Oryza sativa L.) seedlings subjected to anoxia stress is discussed. The stress greatly induced ethanol synthesis and increased activities of alcohol dehydrogenase (ADH; EC 1.1.1.1) and pyruvate decarboxylase (PDC; EC 4.1.1.1) in the coleoptiles, whereas the elevated PDC activity was much lower than the elevated ADH activity, suggesting that PDC may be one of the limiting factors for ethanolic fermentation in rice coleoptiles. Anoxic stress decreased concentrations of fructose 6-phosphate (Fru-6-P) and glucose 6-phosphate, and increased concentration of fructose 1,6-bisphosphate (Fru-1,6-bisP) in the coleoptiles. PFP activity in rice coleoptiles was low in an aerobic condition and increased during the stress, whereas no significant increase was found in ATP:fructose-6-phosphate 1-phosphotransferase (PFK; EC 2.7.1.11) activity in stressed coleoptiles. Fructose 2,6-bisphosphate concentration in rice coleoptiles was increased by the stress and pyrophosphate concentration was above the K_m for the forward direction of PFP and was sufficient to inhibit the reverse direction of PFP. Under stress conditions the potential of carbon flux from Fru-6-P toward ethanol through PFK may be much lower than the potential of carbon flux from pyruvate toward ethanol through PDC. These results suggest that PFP may play an important role in maintaining active glycolysis and ethanolic fermentation in rice coleoptiles in anoxia.     

Ethanolic fermentation and anoxia tolerance in four rice cultivars

The relationship between coleoptile elongation and ethanolic fermentation was investigated in rice (Oryza sativa L.) coleoptiles of four cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress; however, the elongation of cvs Yukihikari and Nipponbare was much greater than that of cvs Leulikelash and Asahimochi. The stress did not significantly increase lactate dehydrogenase (LDH) activity or lactate concentration, but increased alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities, as well as ethanol concentration in the coleoptiles of all cultivars. The elevated ADH and PDC activities and ethanol concentration in cvs Yukihikari and Nipponbare were much greater than those of cvs Leulikelash and Asahimochi, suggesting that ethanolic fermentation is likely more active in cvs Yukihikari and Nipponbare than in cvs Leulikelash and Asahimochi. ATP concentration in cvs Yukihikari and Nipponbare in anoxia was also greater than that in cvs Leulikelash and Asahimochi in anoxia. The ethanol concentration in the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation may be one of the determinants in anoxia tolerance of rice coleoptiles.     

Anoxia tolerance in rice seedlings: exogenous glucose improves growth of an anoxia-'intolerant', but not of a 'tolerant' genotype

This study demonstrated that, in rice seedlings, genotypic differencein tolerance to anoxia only occurred when anoxia was imposedat imbibition, but not at 3 d after imbibition. When seeds wereimbibed and grown in anoxia, IR22 (anoxia-‘intolerant’)grew much slower and had lower soluble sugar concentrationsin coleoptiles and seeds than Amaroo (anoxia-‘tolerant’),while Calrose was intermediate. After 3 d in anoxia, the sugarconcentrations in embryos and endosperms of anoxic seedlingswere nearly 4-fold lower in IR22 than in Amaroo. Sugar deficitin the embryo of IR22 is presumably due to the limitation ofsugar mobilization rather than the capacity of transport asshown by similar sugar accumulation ratios of 1.8 between embryoand endosperm in IR22 and Amaroo at 3 d in anoxia. With 20 molm^–3 exogenous glucose, coleoptile extension and freshweight increments in anoxic seedlings of IR22 were much closerto those in the two other genotypes, nevertheless protein concentrationremained lowest on a fresh weight basis in the coleoptiles ofIR22; indicating that protein synthesis has a lower priorityfor energy apportionment during anoxia than processes crucialto coleoptile extension. In contrast to these responses to anoxiaimposed at imbibition, IR22 had nearly the same high toleranceto anoxia as Calrose and Amaroo, when anoxia was imposed onseedlings subsequent to 48 h aeration followed by 16 h hypoxicpretreatment. In fact, coleoptiles of anoxic IR22 had highersugar concentrations and grew faster than Calrose, and exogenousglucose had no effect on the coleoptile extension of IR22. Excisedcoleoptile tips of IR22 and Amaroo with exogenous glucose hadsimilar rates of ethanol production and were equally tolerantto anoxia. In conclusion, much of the anoxia ‘intolerance’of IR22 when germinated in anoxia could be attributed to limitedsubstrate availability to the embryo and coleoptile, presumablydue to slow starch hydrolysis in the endosperm. Key words: Anoxia, coleoptile, embryo, endosperm, ethanol production, germination, growth, Oryza sativa L., solute net uptake or loss, sugar availability.     

Pyruvate metabolism in rice coleoptiles under anaerobiosis

Relative importance of ethanolic, lactate and alanine fermentation pathways was estimated in coleoptiles of rice seedlings (Oryza sativa L.) subjected to anoxic stress. The in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and alanine aminotransferase (AlaAT, EC 2.6.1.2) in the coleoptiles increased in anoxia, whereas no significant increase was measured in lactate dehydrogenase (LDH, EC 1.1.1.27) activity. At 48 h, the ADH, PDC and AlaAT activities in anoxic coleoptiles were 62-, 15- and 7.6-fold greater, respectively, than those in the presence of oxygen. Ethanol and alanine in the coleoptiles accumulated rapidly under anoxia, increasing by 48 h, 57- and 5.6-fold compared with those in the presence of oxygen, respectively. However, lactate concentration did not increase and no initial burst of lactate production was detected. The relative ratio of carbon flux from pyruvate to ethanol, lactate and alanine in anoxic coleoptiles was estimated to be 92, 1 and 7% of the total carbon flux, respectively. These results suggest that the potential carbon flux from pyruvate to ethanol may be much greater than the potential flux from pyruvate to lactate and alanine in rice coleoptiles during anoxia.     

Comparative analysis of anoxic coleoptile elongation in rice varieties: relationship between coleoptile length and carbohydrate levels, fermentative metabolism and anaerobic gene expression

Rice ( Oryza sativa L.) seeds can germinate under anoxia and can show coleoptile elongation. The anoxic coleoptile is usually longer than aerobic coleoptiles. Although several hypotheses have been proposed to explain the ability of rice to elongate coleoptiles under anoxia, conclusive experimental evidence explaining this physiological trait is lacking. In order to investigate whether metabolic and molecular markers correlate with anoxic coleoptile length, we screened 141 Italian and 23 Sri Lankan rice cultivars for their ability to elongate coleoptiles under anoxia. Differences in anoxic coleoptile length were used to evaluate whether a correlation exists between coleoptile length and biochemical and molecular parameters. The expression of genes coding for glycolytic and fermentative enzymes showed a very low correlation with anoxic coleoptile length. Although differences were found in carbohydrate content between the varieties tested, this parameter also does not appear to be critical in terms of coleoptile elongation. Efficient ethanol fermentation does, however, correlate well with the elongation of coleoptiles under anoxic conditions.     

Protein synthesis by rice coleoptiles during prolonged anoxia: implications for glycolysis, growth and energy utilization

BACKGROUND AND AIMS: Anoxia-tolerant plant tissues synthesize a number of proteins during anoxia, in addition to the 'classical anaerobic proteins' involved in glycolysis and fermentation. The present study used a model system of rice coleoptile tips to elucidate patterns of protein synthesis in this anoxia-tolerant plant tissue. METHODS: Coleoptile tips 7-11 mm long were excised from intact seedlings exposed to anoxia, or excised from hypoxically pre-treated seedlings and then exposed to anoxia for 72 h. Total proteins or 35S-labelled proteins were extracted, separated using two-dimensional isoelectric focusing/SDS-polyacrylamide gel electrophoresis and analysed using mass spectrometry. KEY RESULTS: The coleoptile tips excised after intact seedlings had been exposed to anoxia for 72 h had a similar proteome to tips that were first excised and then exposed to anoxia. After 72 h anoxia, Bowman-Birk trypsin inhibitors and a glycine-rich RNA-binding protein decreased in abundance, whereas a nucleoside diphosphate kinase and several proteins with unknown functions were strongly enhanced. Using [35S]methionine as label, proteins synthesized at high levels in anoxia, and also in aeration, included a nucleoside diphosphate kinase, a glycine-rich RNA-binding protein, a putative elicitor-inducible protein and a putative actin-depolymerizing factor. Proteins synthesized predominately in anoxia included a pyruvate orthophosphate dikinase (PPDK), alcohol dehydrogenase 1 and 2, fructose 1,6-bisphosphate aldolase and a protein of unknown function. CONCLUSION: The induction of PPDK in anoxic rice coleoptiles might, in combination with pyruvate kinase (PK), enable operation of a 'substrate cycle' producing PPi from ATP. Production of PPi would (a) direct energy to crucial transport processes across the tonoplast (i.e. the H+-PPiase); (b) be required for sucrose hydrolysis via sucrose synthase; and (c) enable acceleration of glycolysis, via pyrophosphate:fructose 6-phosphate 1-phosphotransferase (PFP) acting in parallel with phosphofructokinase (PFK), thus enhancing ATP production in anoxic rice coleoptiles; ATP production would need to be increased if there was a substantial requirement for PPi.     

Genetic and biochemical analysis of anaerobically-induced enzymes during seed germination of Echinochloa crus-galli varieties tolerant and intolerant of anoxia

To compare the regulation of anaerobic metabolism during germination in anoxia-tolerant and intolerant plants, enzymes associated with anaerobic metabolism such as sucrose synthase, aldolase, enolase, pyruvate decarboxylase (PDC), alcohol dehydrogenase (ADH), and aldehyde dehydrogenase (ALDH) were assayed in two varieties of Echinochloa crus-galli, formosensis (tolerant) and praticola (intolerant). The initial and intervening enzymes of the pathway (sucrose synthase and aldolase) and enzymes in the last part of the pathway (PDC, ADH and ALDH) revealed similar changing patterns in activities during germination. This implies that each group of enzymes may be controlled by an identical regulatory mechanism. During anoxia, activities of all enzymes increased 1.5-30-fold in both varieties compared to their activities under aerobic conditions. Activities of sucrose synthase, enolase and ADH exhibited the same induction patterns under anoxia in formosensis and praticola. However, the activities of aldolase, ALDH and PDC were more strongly induced in formosensis under anoxia (1.2-2-fold) than in praticola. These enzymes were also assayed in F(3) families which varied in their anaerobic germinability. For PDC, activities under anoxia in anoxia-tolerant families were similar to those of an anoxia-intolerant family during the whole period although the family did not exhibit anaerobic germinability. This suggests that there is no correlation between PDC activity and anaerobic germinability. For ALDH, activities were more strongly induced under anoxia in anoxia-tolerant families than in anoxia-intolerant families, a trend also exhibited by the parents. This indicates that ALDH may play a role in detoxifying acetaldehyde formed through alcoholic fermentation during anaerobic germination.     

Alcohol dehydrogenase activity in the roots of marsh plants in naturally waterlogged soils

The aim of this work was to discover whether oxygen tensions in the roots of marsh plants in flooded soils are high enough to allow fully acrobic metabolism. Activity of alcohol dehydrogenase (ADH), a protein synthesised in anoxic plants, was measured in roots of marsh plants growing in habitats where the availability of oxygen to the roots would be expected to differ. Roots of Carex riparia in standing water had ADH activities about 2.5 times higher than those of phosphofructokinase, and comparable to ADH activities of Poa trivialis, Urtica dioica and Ranunculus repens roots in dry soil. Removal of the oxygen supply via aerenchyma to Carex roots caused a 30-fold increase in ADH activity relative to that of phosphofructokinase. There was no change in ADH activity with depth in Carex roots in waterlogged soil, but in Filipendula ulmaria roots activity was 14 times higher below 10 cm depth than near the surface. Urtica roots in waterlogged soil had alcohol dehydrogenase activities 26 times higher than roots in dry soil, but for Poa and Ranunculus roots this figure was only 1.7 and 4.2, respectively. These results indicate that the oxygen tensions in the roots of marsh plants in waterlogged soil differ considerably among species. Ethanol was the major product of fermentation in roots of all species studied. There was no correlation between ADH activity and the rate of ethanol production under anoxia of Urtica roots. The physiological significance of high ADH activities in roots is thus unclear.Abbreviations ADH alcohol dehydrogenase - PFK phosphofructokinase - PFP pyrophosphate:fructose 6-phosphate phosphotransferase     

Purification and characterization of pyrophosphate- and ATP-dependent phosphofructokinases from banana fruit

Pyrophosphate-dependent phosphofructokinase (PFP; EC 2.7.1.90) and two isoforms of ATP-dependent phosphofructokinase (PFK I and PFK II; EC 2.7.1.11) from ripened banana ( Musa cavendishii L. cv. Cavendish) fruits were resolved via hydrophobic interaction fast protein liquid chromatography (FPLC), and further purified using anion-exchange and gel filtration FPLC. PFP was purified 1,158-fold to a final specific activity of 13.9 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Gel filtration FPLC and immunoblot analyses indicated that this PFP exists as a 490-kDa heterooctomer composed of equal amounts of 66- (alpha) and 60-kDa (beta) subunits. PFP displayed hyperbolic saturation kinetics for fructose 6-phosphate (Fru 6-P), PPi, fructose 1,6-bisphosphate, and Pi ( K(m) values = 32, 9.7, 25, and 410 microM, respectively) in the presence of saturating (5 microM) fructose 2,6-bisphosphate, which elicited a 24-fold enhancement of glycolytic PFP activity ( K(a)=8 nM). PFK I and PFK II were each purified about 350-fold to final specific activities of 5.5-6.0 micromol fructose 1,6-bisphosphate produced (mg protein)(-1) x min(-1). Analytical gel filtration yielded respective native molecular masses of 210 and 160 kDa for PFK I and PFK II. Several properties of PFK I and PFK II were consistent with their respective designation as plastid and cytosolic PFK isozymes. PFK I and PFK II exhibited: (i) pH optima of 8.0 and 7.3, respectively; (ii) hyperbolic saturation kinetics for ATP ( K(m)=34 and 21 microM, respectively); and (iii) sigmoidal saturation kinetics for Fru 6-P ( S0.5=540 and 90 microM, respectively). Allosteric effects of phospho enolpyruvate (PEP) and Pi on the activities of PFP, PFK I, and PFK II were characterized. Increasing concentrations of PEP or Pi progressively disrupted fructose 2,6-bisphosphate binding by PFP. PEP potently inhibited PFK I and to a lesser extent PFK II ( I50=2.3 and 900 microM, respectively), while Pi activated PFK I by reducing its sensitivity to PEP inhibition. Our results are consistent with: (i) the respiratory climacteric being regulated by fine (allosteric) control of pre-existing enzymes; and (ii) primary and secondary glycolytic flux control being exerted at the levels of PEP and Fru 6-P metabolism, respectively.     

Regulation of Pyruvate Decarboxylase In Vitro and In Vivo

Results presented in this paper strongly support the view thatregulation of the key enzyme of alcoholic fermentation, pyruvatedecarboxylase (PDC), is achieved in a number of ways, all associatedwith possible lowering of the cytoplasmic pH during anoxia.These mechanisms include not only the well-known acid pH optimumof PDC, but also long-term, reversible changes in characteristicsof the enzyme established both in vitro and in vivo. Following transfer of desalted extracts from pH 6.0 to 7.4,maximal activity of PDC was decreased, while there was a considerableincrease in the lag before maximal activity was reached. Similarchanges in enzyme characteristics were observed when wheat (Triticumaestivum L. cv. Gamenya) roots and rice (Oryza sativa L. cv.Calrose) coleoptiles were transferred from anoxic to aerobicsolutions, provided PDC was assayed within 10 min of the startof maceration. All of the above changes were usually readilyreversible when extracts were returned to pH 6.0, or when plantswere returned to anoxic solutions. Additional regulation of PDC would be achieved by the S_0.5 forpyruvate which is 0.75 mol m^–3 at pH 6.0, 1.0 mol m^–3at pH 6.8, and 2.5 mol m^–3 at pH 7.4; the latter is wellabove estimates for pyruvate concentrations in the cytoplasmof aerated tissues. We assess that the combined effects of the acid pH optimum,the high S_0.5 at pH 7.4 and the long-term decreases in activityobserved during incubation at pH 7.4 would reduce PDC activityin aerobic cells to at most 7% of the activity in anoxic cells.Possible additional controls for the pathway of alcoholic fermentationare briefly considered. Key words: PDC, regulation, anoxia     

Sucrose metabolism in developing endosperm of immature wheat (Triticum aestivum L.) grain

With a view to investigating the role of the enzyme pyrophosphate-fructose-6-phosphate-1-phosphotransferase (PFP) in sucrose breakdown in developing endosperm of wheat grain, the activity of PFP and related enzymes such as phosphofructokinase (PFK), fructose-6-bisphosphatase (FBPase), fructose-6-phosphate-2-kinase (PFK-2) and fructose-2,6-bisphosphatase (F2, 6-P2ase) and the contents of the various intermediates of the pathway serving either the substrate or the effectors of these enzymes such as glu-6-P,glu-1-P,fru-6-P,fru-1,6-P2,DHAP,G3P, UDP-glucose, ADP-glucose, Pi,PPi and fru-2,6-P2 have been determined at 5 days intervals starting from day-5 after anthesis until day-40 after anthesis. These enzymes except PFK-2 had their peak activity at day-25 after anthesis. The activity of PFP was several fold higher than that of PFK at each stage of grain development. PFK-2 exhibited the lowest activity. The various intermediates again had their maximum concentration either at day-20 or day-25 after anthesis. Among hexose phosphates studied, glu-6-P was present in highest concentration at each stage of grain development. The level of Pi was much higher than those of PPi and fru-2,6-P2. Similarly, concentration of UDP-glucose was higher than that of ADP-glucose. Based on these results, it is proposed that the major role of the enzyme PFP in developing wheat grain is to provide PPi for sucrose breakdown via sucrose synthase.     

Anoxia tolerance and α-amylase activity in four rice cultivars

The relationship between anoxia tolerance and reserved carbohydrate catabolism was investigated in four rice (Oryza sativa L.) cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress, however, the elongation of cvs Koshihikari and Awa-akamai was much greater than that of cvs Touzoumochi and Asahimochi. The anoxic coleoptiles of cvs Koshihikari and Awa-akamai contained about 2-fold as much ATP as those of cvs Touzoumochi and Asahimochi. Ethanol production in the anoxic coleoptiles of cvs Koshihikari and Awa-akamai was about 2-fold as much as that of cvs Touzoumochi and Asahimochi, which suggests that ethanolic fermentation is probably more active in cvs Koshihikari and Awa-akamai than in cvs Asahimochi and Touzoumochi. Activity of α-amylase, which catabolizes starch to soluble sugars, in endosperms of cvs Koshihikari and Awa-akamai was about 2-fold that of cvs Touzoumochi and Asahimochi, and soluble sugar concentration in the coleoptiles of cvs Koshihikari and Awa-akamai was about 3-fold greater than that of cvs Touzoumochi and Asahimochi. Soluble sugar concentration and ethanol production rate in the coleoptiles of rice seedlings were correlated well with α-amylase activity in their endosperms, which were also correlated well with anoxia tolerance with respect to the coleoptile elongation and ATP concentration in the coleoptiles. These results suggest that the ability to degrade starch to soluble sugar by α-amylase in endosperm may be important for the anoxia tolerance in rice coleoptiles and it may serve to distinguish the anoxia tolerance of rice coleoptiles.     

Activity of biochemical pH-stat enzymes in cereal root tips under oxygen deficiency

The activity of the enzymes of alcoholic and lactic-acid fermentation: pyruvate decarboxylase (PDC, EC 4.1.1.1), alcohol dehydrogenase (ADH, EC 1.1.1.1), lactate dehydrogenase (LDH, EC 1.1.1.27) and the enzymes of malic acid metabolism: phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.23), NAD-dependent malate dehydrogenase (NAD-MDH, EC 1.1.1.37), and NADP-dependent malic enzyme (NADP-ME, EC 1.1.1.40) involved in the operation of biochemical pH-stat was investigated in the root tips of wheat (Triticum aestivum L.) and rice (Oryza sativa L.) under hypoxia and anoxia. Exposures lasted for 6, 12, and 18 h. The most pronounced response was detected for the enzymes of alcoholic fermentation. The activation of ADH and PDC in wheat occurred only under hypoxia, whereas in rice it was detected both under hypoxia and anoxia. The activation of LDH in wheat occurred under hypoxia, and in rice, the activity of this enzyme was slightly enhanced. The activity of the enzymes of malic acid metabolism did not change except in wheat root tips under hypoxia when PEPC activity decreased and NADP-ME activity simultaneously rose. The role of biochemical pH-stat in the regulation of cytoplasmic pH in plant cells under oxygen deficit and the mechanisms for regulating the activities of enzymes involved in biochemical pH-stat are discussed as well as the interaction between biochemical pH-stat and other mechanisms maintaining pH of plant cells. The results are analyzed within a context of intracellular pH regulation.     

Effects of Anoxia on Wheat Seedlings: II. INFLUENCE OF O2 SUPPLY PRIOR TO ANOXIA ON TOLERANCE TO ANOXIA, ALCOHOLIC FERMENTATION, AND SUGAR LEVELS

Prior exposure of roots of intact wheat seedlings for 15–30h to hypoxia (0016-006 mol m 02) greatly increased their toleranceto subsequent anoxia, as assessed by the ability of the rootsto elongate upon return to air. Such hypoxically pretreatedroots had 2–4-fold higher activities of pyruvate decarboxylase(PDC) and 35–l7-fold higher activities of alcohol dehydrogenase(ADH) in their 0–1 mm apices and 0–5 mm root tipsthan in apices and tips of roots pretreated in air (026–031mol m3 02). The ADH/ PDC ratio increased I 3–5-fold duringhypoxic pretreatment. Furthermore, the rate of alcoholic fermentationby 0–5 mm tips of the hypoxically pretreated roots was14-4-fold faster than in tips from aerobically pretreated roots.No consistent difference between 02 pretreatment was found foralcoholic fermentation by tissues taken between 10 and 20 mmfrom the root tip. The observed activities of PDC and rates of alcoholic fermentationindicate that alcoholic fermentation is usually rate-limitedby PDC in 0–1 mm apices and 0–5 mm tips of wheatroots. Comparisons with data in the literature indicate thatwheat has at most a small Pasteur effect, which may explainwhy wheat is more intolerant to anoxia than rice. Exogenous glucose delayed the loss of elongation potential inboth aerobically and hypoxically pretreated roots. In the absenceof glucose, more than 85% of aerobically pretreated roots hadlost their elongation potential after 9 h anoxia, compared with30% in the presence of glucose. After 21 h anoxia nearly allaerobically pretreated roots had lost their elongation potential,compared with 10% and 0% of hypoxically pretreated roots inthe absence and presence, of glucose, respectively. The protective effect of glucose was presumably not due to anendogenous sugar deficiency; at the start of anoxia, 0–1mm apices of aerobically pretreated roots contained sufficientsugar for 23 h of their measured rate of ethanol synthesis yet,85% of these apices had lost their elongation potential afteronly 9 h of anoxia. It is suggested that in wheat roots, lowrates of synthesis of ethanol and hence of ATP, lead to injuryof cells, in turn generating a requirement for exogenous glucose,despite high endogenous sugar concentrations. Key words: Wheat seedlings, anoxia, glucose, O₂ pretreatment, alcoholic fermentation     

Evaluation of the importance of lactate for the activation of ethanolic fermentation in lettuce roots in anoxia

According to the Davies–Roberts hypothesis, plants primarily respond to oxygen limitation by a burst of lactate production and the resulting pH drop in the cytoplasm activates ethanolic fermentation. To evaluate this system in lettuce ( Lactuca sativa L.), seedlings were subjected to anoxia and in vitro activities of alcohol dehydrogenase (ADH, EC 1.1.1.1), pyruvate decarboxylase (PDC, EC 4.1.1.1) and lactate dehydrogenase (LDH, EC 1.1.1.27) and concentrations of ethanol, acetaldehyde and lactate were determined in roots of the seedlings. The in vitro activities of ADH and PDC in the roots increase in anoxia, whereas no significant increase was measured in LDH activity. At 6 h, the ADH and PDC activities in the roots kept in anoxia were 2.8- and 2.9-fold greater than those in air, respectively. Ethanol and acetaldehyde in the roots accumulated rapidly in anoxia and increased 8- and 4-fold compared with those in air by 6 h, respectively. However, lactate concentration did not increase and an initial burst of lactate production was not found. Thus, ethanol and acetaldehyde production occurred without an increase in lactate synthesis. Treatments with antimycin A and salicylhydroxamic acid, which are respiratory inhibitors, to the lettuce seedlings in the presence of oxygen increased the concentrations of ethanol and acetaldehyde but not of lactate. These results suggest that ethanolic fermentation may be activated without preceding activation of lactate fermentation and may be not regulated by oxygen concentration directly.     

Evidence for down-regulation of ethanolic fermentation and K+ effluxes in the coleoptile of rice seedlings during prolonged anoxia.

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 (micromol x g(-1) FW x 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 micromol x g(-1) FW x 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.     

Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds

The distribution of pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles·min·milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K_m values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P₂. The activation is caused by lowering the K_m for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P₂ and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K_m of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K_m for fructose 6-P. The ATP content appears to be at saturating levels for PFK.     

Salt stress and glycolytic regulation in suspension-cultured cells of the mangrove tree, Bruguiera sexangula

Suspension-cultured cells derived from seedlings of Bruguiera sexangula are tolerant to NaCl. To examine the influence of long-term salt stress on glycolysis, we determined the effect of 100 m M NaCl on the activities of two key enzymes, phosphofructokinase (PFK, EC 2.7.1.11) and pyruvate kinase (PK, EC 2.7.1.40), and on the bypass enzymes, pyrophosphate: fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.49) and phosphoenolpyruvate phosphatase (PEPase, EC 3.1.3.60). From 10 days after NaCl treatment, increases were found in the activities of PFK, PK and PEPC. In contrast, there was little or no difference in the activities of PFP or PEPase. The short-term effect of salt stress was also investigated. NaCl (150 m M ) caused a 1.4-fold increase in respiratory O₂ uptake at 24 h after treatment. Alongside this respiratory rise, drastic changes in the levels of glycolytic metabolites were found: a decrease in the levels of glucose, glucose-6-phosphate and fructose-6-phosphate, and an increase in the levels of fructose-1, 6-bisphosphate and metabolites of the later steps of the glycolytic pathway. The crossover diagram of metabolites suggests that NaCl stimulates those steps catalysed by PFK and/or PFP. The in vitro activities of partially purified PFK and PFP were increased by the addition of 150 m M NaCl. The effect of salt on the kinetic properties of PFK and PFP was studied, and possible control mechanisms of glycolysis on salt stress are discussed.