Effects of o(2) concentration on rice seedlings

The ability of rice, wheat, and oat seedlings to germinate and grow as the O₂ concentration was lowered to zero was compared. The germination of rice was completely unaffected by O₂ supply, whereas that of oats and wheat was strongly retarded at levels below 5% O₂. In contrast to the coleoptiles of oats and wheat and to roots of all three species where growth was progressively diminished as the O₂ concentration was lowered, that of the rice coleoptile was progressively increased. However, the dry weight and content of protein, sugars, and cellulose were all depressed in the rice coleoptile in anoxia, and the levels of several respiratory enzymes, particularly those of mitochondria, were also much lower than those of the coleoptiles grown in air. In 1% O₂, the growth of the rice coleoptile was similar to that in air. The effect of ethanol concentration on germination and growth of rice was measured. Coleoptile growth was reduced when the ethanol concentration exceeded 40 millimolarity, and root growth was somewhat more sensitive. Coleoptiles of all three species grown in air were transferred to N₂, and ethanol accumulation was measured over 24 hours. The rate of ethanol accumulation in oats was close to that in rice, and in all three species the amounts of ethanol lost to the surrounding medium were those expected from simple diffusion from the tissue. The ability of the rice coleoptile to grow in anoxia is apparently not due to a particularly low rate of ethanol formation or to unusual ethanol tolerance. Any explanation of the success of rice in anoxia must encompass the much lower rate of ATP synthesis than that in air and account for the biochemical deficiencies of the coleoptile.     

Fatty acids of rice coleoptiles in air and anoxia

The metabolism of lipids, like that of other components, was adversely and strongly affected when rice (Oryza sativa L.) coleoptiles were grown anaerobically. In aerobic coleoptiles, the amounts of total fatty acid, phospholipid, and total lipid per coleoptile increased by 2.5- to 3-fold between days three and seven, whereas under anoxia, the increases were all less than 60%. The total amount of lipid at day seven in anoxia was less than 30% of that in air. In air, the total fatty acid content at day three was 25 nanomoles per coleoptile and this increased to over 71 nanomoles per coleoptile at day seven. All acids except 18:0 showed substantial increases. In anoxia, the corresponding values for total fatty acids were 24 nanomoles and 27 nanomoles. The small increases were confined to the saturated fatty acids; no significant increase occurred in unsaturated fatty acids. A minor fatty acid constituent (16:1) increased from 0.09 to 1.99 nanomoles per coleoptile between days three and seven in air. This component was never observed in any fatty acid preparation from anaerobic coleoptiles. The major phospholipids under all conditions were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidic acid. A small amount of unidentified phosphoester, not present on thin layer chromatography plates from aerobic coleoptiles, was seen in extracts of anaerobic coleoptiles. The fatty acyl substituents of each of the phospholipids were analyzed at days three and seven in coleoptiles grown aerobically and in anoxia. Each phospholipid had its own distinctive fatty acid composition which remained fairly constant under all treatments; 16:0 and 18:2 were the most abundant fatty acids in every phospholipid class. In air, the percentages of total fatty acids that were in the phospholipids were 86% on day three and 87% on day seven. In anoxia, the values at the corresponding ages were 47 and 57%. Since no net synthesis of unsaturated fatty acids occurred in anaerobic conditions, the small increase in total unsaturated acids in the phospholipids between days three and seven must have occurred at the expense of fatty acids preexisting in the neutral lipid. No unusual pathways of biosynthesis or unusual precursors are required to explain the presence of unsaturated fatty acids in the rice coleoptile. The present study and results of experiments where coleoptiles were fed [¹⁴C]acetate (BB Vartapetian et al. 1978 Plant Sci Lett 13:321-328) clearly show that unsaturated fatty acid synthesis in rice coleoptiles requires O₂, as it does in other plants.     

Time requirement for hypoxic acclimation to anoxia tolerance in rice coleoptiles

Rice (Oriza sativa L.) seedlings were subjected tohypoxic pretreatment (H-PT; incubated in 5% O₂ atmosphere) forvarious lengths of time followed by a 24-h anoxic stress. Anoxiatolerance of rice coleoptiles was improved with increasing duration of H-PT, butH-PT longer than 6 h gave no additional improvement. ATP andethanol concentrations in the coleoptiles were increased by H-PT, and the timeand pattern of increase in ATP level and ethanol production rate were similar tothose of increase in the anoxia tolerance. These results suggest that the H-PTmay increase anoxia tolerance due to maintenance of anaerobic glycolysis withinduction of ethanolic fermentation to generate ATP, and hypoxic acclimation toanoxic stress in rice coleoptiles may occur within 6 h.     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

Incubation of 3-d-old seedlings of Oryza sativa L. cv Arborio under anaerobic conditions, leads to a large increase in the titer of free putrescine while aerobic incubation causes a slight decrease. After 2 days, the putrescine level is about 2.5 times greater without oxygen than in air. The rice coleoptile also accumulates a large amount of bound putrescine and, to a lesser extent, spermidine and spermine (mainly as acid-soluble conjugates). Accumulation of conjugates in the roots is severely inhibited by the anaerobic treatment. Feeding experiments with labeled amino acids showed that anoxia stimulates the release of ¹⁴CO₂ from tissues fed with [¹⁴C]arginine and that arginine is the precursor in putrescine biosynthesis. After 2 d of anoxia, the activity of arginine decarboxylase was 42% and 89% greater in coleoptile and root, respectively, than in the aerobic condition. The causes of the differences in polyamine metabolism in anoxic coleoptiles and roots are discussed.     

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.     

Protective effect of exogenous nitrate on the mitochondrial ultrastructure ofOryza sativa coleoptiles under strict anoxia

Summary The effect of exogenous KNO₃, the terminal acceptor of electrons in oxygen-free medium, on mitochondrial ultrastructure and on the growth rate of 4-day-old rice coleoptiles under strictly anoxic conditions was studied. Exogenous nitrate (10 mM) did not exert any significant effect on the growth rate of coleoptiles of intact seedlings compared to their growth in KNO₃-free medium. Anaerobic incubation of detached coleoptiles in KNO₃-free medium for 48 h resulted in the complete destruction of mitochondrial and other cell membranes. In the presence of KNO₃, no mitochondrial-membrane destruction was observed even after 48 h anoxia although the mitochondrial ultrastructure was modifed. Cristae were arranged in parallel rows and elongated dumbbell-shaped mitochondria appeared in some cells. The data obtained indicate a protective role of exogenous nitrate as electron acceptors in oxygen-free medium. The results of the present investigation are discussed and compared with reports of either markedly damaging or favorable effects of exogenous nitrate on the growth, metabolism, and energetics of rice and other plants under hypoxic and anoxic conditions.     

Aerenchyma formation and associated oxygen movement in seminal and nodal roots of wheat

Abstract The present paper describes the effects of growth of roots of wheat (Triticum aestivum cv. Gamenya) in hypoxic nutrient solutions on acrenchyma formation and O₂ movement from shoots to roots. Two types of roots were investigated: (1) seminal roots of 4–7-d-old seedlings, and (2) seminal and nodal roots of 10–28-d-old plants. Gas-filled porosity of seminal and nodal roots increased from 3 to 12% and from 5–7 to 11–15%, respectively, when the roots emerged in stagnant or N₂-flushed solutions (0.003 mol m ^?3 O₂) compared with growth in continuously acrated solutions (0.26 mol m ^?3 O₂). However, neither root type increased in porosity when they were longer than 100–200 mm at the start of the exposure to these stagnant or N₂-flushed treatments. A vernier microscope and cylindrical platinum-electrode were used to examine the relationship between root extension and transport of O₂ from shoots to roots via the gas spaces. Measurements were made when the roots were in an anoxic medium and were dependent solely on O₂ supplied from the shoots. For seminal roots of 5–7-d-old seedlings raised in stagnant solutions (90–100 mm), internal O₂ transport was sufficient to support a rate of root elongation in the O₂-free medium of between 0.03 and 0.17 mm h^?1. When the O₂ pressure around the shoots was increased from 20 to 100 kPa O₂, the O₂ concentrations at the walls of the expanding zone (2–7 mm from the tip) of these roots increased from 0.006 mol m^?3 to between 0.04 and 0.26 mol m^?3, and the rate of root extension increased five-fold. Oxygen transport to roots grown continuously in acrated solutions was considerably less than for roots raised in stagnant solutions; this difference was greater for seminal than for nodal roots. When the acrated seminal roots were longer than 100 mm and transferred to an O₂-free root medium, O₂ concentration became zero at the root tip causing elongation to cease. After 24 h of anoxia, none of these roots were able to resume elongation following a return to acrated solutions.     

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.     

Supply and partitioning of assimilates to roots of Medicago sativa L. and Lotus corniculatus L. under anoxia

Abstract A current explanation of the mechanism of flooding injury to roots suggests that oxygen deficiency depresses the supply of respirable carbohydrates sufficiently to inhibit fermentation. However, even though it has been shown that phloem transport of assimilate is sharply reduced to anaerobic roots, inhibition of assimilate metabolism has also been suggested to be an important factor. This study examines these hypotheses by relating assimilate supply and metabolic activity in anoxic roots of alfalfa (Medicago sativa L.), a flood-intolerant species, and birdsfoot trefoil (Lotus corniculatus L.), a flood-tolerant plant. Roots were made anoxic (severe O₂ deficiency) for 2, 4 or 6 d and shoots were labelled with ¹⁴CO₂. Assimilate transport to the roots and metabolism to structural components were significantly decreased in both species in response to anoxia. Trefoil exhibited significantly greater ¹⁴C incorporation into the residue fraction at 4 d anoxia than did alfalfa, and this was consistent with the greater flooding tolerance of trefoil. When assimilate supply to O₂-deficient roots was decreased by shoot shading, shoot fresh weight was reduced by both anoxia and light treatments. Root-soluble sugars were significantly decreased by shading but were greatly increased in response to anoxia. Root starch concentration also increased under anoxia. Root K⁺ concentration was reduced by anoxia only. The energy status (ATP/ADP) of roots was significantly decreased by shading; however, anoxia reduced the energy status only in unshaded plants. The data indicate that carbohydrate supply to anaerobic roots does not appear to be a limiting factor in the metabolic response of alfalfa roots. Alternatively, metabolism of assimilate in anoxic roots may be an important determinant of survival.     

Anaerobiosis-induced differentiation of Acanthamoeba castellanii

Acanthamoeba castellanii is a free living amoeba ubiquitous in soil and also commonly found in aquatic environments. In waterlogged soils, anoxia is quickly established as the dissolved oxygen is consumed by the organisms present. We were interested in the effects of anoxic conditions upon this organism. Batch cultures degassed with N₂ during mid-exponential growth, induced encystation within 12 h of anoxia, and mature cysts were formed within 2–3 days. Excystation (99%) was achieved by subsequent aeration of these cultures after 3–6 days. Anoxia-induced cysts, maintained in anoxic conditions for up to four months, remained viable. Difference spectra, during anaerobiosis, revealed that cytochromes were not lost, suggesting that the organism retains its respiratory components. The growth rate of trophozoites, grown in a chemostat, was dependent on the concentration of O₂ in the head space and glucose uptake increased at lower dissolved O₂ tensions. The results obtained suggest that A. castellanii has a complex adaptive strategy enabling it to cope with microaerobic and anoxic conditions which may be experienced in the environment.     

Exogenous Nitrate as a Terminal Acceptor of Electrons in Rice (Oryza sativa) Coleoptiles and Wheat (Triticum aestivum) Roots under Strict Anoxia

To elucidate the physiological role of exogenous nitrate under anaerobic conditions, we studied the effect of 10 mM KNO₃ on the mitochondrial ultrastructure in rice (Oryza sativa L.) coleoptiles and in wheat (Triticum aestivum L.) roots, detached from four-day-old seedlings, under strict anoxia. In wheat roots, following 6-h-long anoxia in the absence of exogenous nitrate, the mitochondrial membranes were partially degraded and, after 9 h under anoxia, the mitochondrial membranes and the membranes of other organelles were completely destroyed. In rice coleoptiles, the partial membrane degradation was observed only after 24 h and their complete breakdown after 48 h of anaerobiosis. In the presence of exogenous nitrate, no membrane destruction was noticed even after 9 and 48 h of anaerobiosis in wheat roots and rice coleoptiles, respectively. These results indicate that exogenous nitrate exerts protective action as a terminal electron acceptor, alternative to the molecular oxygen. Our findings are compared with the results of other researchers concerning the adverse or favorable nitrate action on plant growth, metabolism, and energy status under anaerobic stress.     

Role of covalent modification in the control of glycolytic enzymes in response to environmental anoxia in goldfish

Summary The effects of environmental anoxia (24 h at 7°C in N₂/CO bubbled water) on the maximal activities, selected kinetic properties, and isoelectric points of phosphofructokinase and pyruvate kinase were measured in eight tissues of the goldfish,Carassius auratus, in order to evaluate the role of possible covalent modification of enzymes in glycolytic rate control and metabolic depression during facultative anaerobiosis. Both enzymes showed modified kinetic properties as a result of anoxia in liver, kidney, brain, spleen, gill, and heart. Effects of anoxia on properties of pyruvate kinase included reducedV _max, increased S_0.5 for phosphoenolpyruvate, increasedK _a for fructose-1,6-bisphosphate, and strongly reduced I₅₀ for alanine; all these effects are consistent with an anoxia-induced phosphorylation of pyruvate kinase to produce a less active enzyme form. Anoxia-induced alterations in phosphofructokinase kinetics included tissue-specific changes in S_0.5 for fructose-6-phosphate, Hill coefficient,K _a values for fructose-2,6-bisphosphate, AMP, and NH ₄ ⁺ , and I₅₀ values for ATP and citrate, the direction of changes being generally consistent with the production of a less active enzyme form in the anoxic tissue. Enzymes from aerobic versus anoxic skeletal muscle (both red and white) did not differ in kinetic properties but anoxic enzyme forms had significantly different pI values than the corresponding aerobic forms. Enzyme phosphorylation-dephosphorylation as the basis of the anoxia-induced changes in the kinetic properties of PFK and PK was further tested in liver: treatment of the aerobic forms of both enzymes with cAMP dependent protein kinase altered enzyme kinetic properties to those typical of the anoxic enzymes while alkaline phosphatase treatment of the anoxic enzyme forms had the opposite effect. The data provide strong evidence that coordinated glycolytic rate control, as part of an overall metabolic rate depression during anoxia, is mediated via anoxia-induced covalent modification of regulatory enzymes.Abbreviations cAMP cyclic 35 adenosine monophosphate - F16P ₂ fructose-1,6-bisphosphate - F26P ₂ fructose-2,6-bisphosphate - F6P fructose-6-phosphate - PEP phosphoenolpyruvate - PFK phosphofructokinase (E.C. 2.7.1.11) - PK pyruvate kinase (E.C. 2.7.1.40) - PMSF phenylmethylsulfonyl fluoride     

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.     

Salinity-induced subcellular accumulation of H2O2 in leaves of rice

The localization of salt-induced H₂O₂ accumulation in the leaves of rice was examined using 3,3-diaminobenzidine and CeCl₃ staining at ultrastructure level. When the 3-week-old rice plants were affected by 100 mM NaCl for 14 days, the swelling of thylakoids and the destruction of thylakoid membranes were observed. H₂O₂ accumulation was also observed in the chloroplast of the leaf treated with NaCl. The electron dense products of 3,3-diaminobenzidine and CeCl₃ were mainly observed especially around the swelling of thylakoids. H₂O₂ accumulation and any ultrastructural changes were not observed in the chloroplasts under dark condition. Furthermore, treatment with ascorbic acid suppressed both H₂O₂ accumulation and the changes in chloroplast ultrastructure. These results suggest that light-induced production of excess H₂O₂ under salinity is responsible for the changes in chloroplast ultrastructure. H₂O₂ accumulation was also observed in the mitochondria, peroxisomes, plasma membrane, and cell walls under light but not dark, suggesting that these organelles are also the source of H₂O₂ and the production is light dependent under salinity.     

The presence of glycollate oxidase and dehydrogenase in Dunaliella primolecta

Homogenates of Dunaliella primolecta, D. salina and D. tertiolecta were assayed for glycollate oxidase and glycollate dehydrogenase. Both D. primolecta and D. salina but not D. tertiolecta showed substantial glycollate-dependent O₂-uptake which is characteristic of glycollate oxidase. L-Lactate was an alternative substrate and both glycollate- and L-lactate-dependent O₂ uptake were insensitive to 2 mM cyanide. Glycollate dehydrogenase, measured by following the glycollate-dependent reduction of 2,6-dichlorophenolindophenol under aerobic conditions, was present in D. primolecta, D. salina and D. tertiolecta. In the presence of glycollate and D-lactate, rates were additive so both glycollate and D-lactate dehydrogenases are present in the homogenates. Glycollate and D-lactate oxidation were both inhibited by 2 mM cyanide. Organelles released from phototrophically grown cells of D. primolecta were separated by isopycnic centrifugation on sucrose gradients. Glycollate oxidase was present in the peroxisome fraction at an equilibrium density of 1.25 g/cm³, while the major peak of glycollate dehydrogenase activity was in the mitochondrial fraction at an equilibirium density of 1.22 g/cm³.     

Untersuchungen zur Funktionsänderung der Microbodies in den Keimblättern von Helianthus annuus L.

Summary The enzyme patterns in sunflower cotyledons indicate that the glyoxysomal function of microbodies is replaced by the peroxisomal function of these organelles during the transition from fat degradation to photosynthesis. The separation of the microbody population into glyoxysomes and peroxisomes during this transition period is reported. The mean difference in density between the activity peaks of glyoxysomal and peroxisomal marker enzymes on a sucrose gradient was calculated to be 0.007±0.004 g/cm³ and turned out to be significant (t=7.8>4.04=t _5;0.01). The activity peak of catalase coincides with that of isocitrate lyase in early stages of development, but shifts to the activity peak of peroxisomal marker enzymes during the transition period. No isozymes of the catalase could be detected by gel electrophoresis in the microbodies with the two different functions.During the rise of the peroxisomal marker enzymes no synthesis of the common microbody marker, catalase, could be demonstrated using the inhibitor allylisopropylacetamide. Using D₂) for density labeling of newly-formed catalase, no difference is observed between the density of catalase from cotyledons grown on 99.8% D₂O during the transition period and the density of enzyme from cotyledons grown on H₂O. The activity of particulate glycolate oxidase is reduced 30–50% by allylisopropylacetamide, but is not affected by D₂O. The chlorophyll formation in the cotyledons is strongly inhibited by both substances.     

Respiration of Artemia franciscana embryos after continuous anoxia over 1-year period

Encysted embryos of the brine shrimp, Artemia franciscana, exhibit extraordinary longevity when exposed to continuous anoxia. To explore the metabolic basis of this ability, the post-anoxic respiration of embryos exposed to anoxia for periods exceeding 1 year was measured. Since anoxic metabolism might result in the accumulation of metabolic end products, an O₂ debt would be expected. Contrary to that expectation, post-anoxic embryos exhibited a marked depression in respiration rate whether embryos were hydrated under anoxic conditions or were exposed to a previous aerobic incubation and then placed under anoxia. These results, and those of previous studies, suggest that extended anoxia may bring the metabolism of these embryos to a reversible standstill.