Differential molecular responses of rice and wheat coleoptiles to anoxia reveal novel metabolic adaptations in amino acid metabolism for tissue tolerance

Rice (Oryza sativa) and wheat (Triticum aestivum) are the most important starch crops in world agriculture. While both germinate with an anatomically similar coleoptile, this tissue defines the early anoxia tolerance of rice and the anoxia intolerance of wheat seedlings. We combined protein and metabolite profiling analysis to compare the differences in response to anoxia between the rice and wheat coleoptiles. Rice coleoptiles responded to anoxia dramatically, not only at the level of protein synthesis but also at the level of altered metabolite pools, while the wheat response to anoxia was slight in comparison. We found significant increases in the abundance of proteins in rice coleoptiles related to protein translation and antioxidant defense and an accumulation of a set of enzymes involved in serine, glycine, and alanine biosynthesis from glyceraldehyde-3-phosphate or pyruvate, which correlates with an observed accumulation of these amino acids in anoxic rice. We show a positive effect on wheat root anoxia tolerance by exogenous addition of these amino acids, indicating that their synthesis could be linked to rice anoxia tolerance. The potential role of amino acid biosynthesis contributing to anoxia tolerance in cells is discussed.     

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.     

Ethanol sensitivity of rice and oat coleoptiles

The ability to avoid the ethanol-induced injury was evaluated in rice ( Oryza sativa L.) and oat ( Avena sativa L.) coleoptiles. The growth of the rice and oat coleoptiles was inhibited by ethanol exogenously applied at concentrations greater than 200 and 30 m M , respectively. At 300 m M ethanol, oat coleoptiles were brown and flaccid but rice coleoptiles did not show any visible symptoms of toxicity. The acetaldehyde level in rice and oat coleoptiles was increased by exogenously applied ethanol and the increases were greater in oat than in rice coleoptiles under aerobic and anaerobic conditions. At 300 m M ethanol, the acetaldehyde concentrations in the rice and oat coleoptiles were 46 and 87 nmol g⁻¹ FW under aerobic conditions, respectively, and 52 and 124 nmol g⁻¹ FW under anaerobic conditions, respectively. The activity of alcohol dehydrogenase (ADH; EC 1.1.1.1) in the direction of ethanol to acetaldehyde was greater in oat than in rice coleoptiles and ADH protein in oat coleoptiles was more induced by exogenously applied ethanol than that in rice coleoptiles. These results suggest that in vivo conversion rate of ethanol to acetaldehyde by ADH is lower in rice than oat coleoptiles, which may be one of the reasons that ethanol sensitivity of rice is much lower than that of oat coleoptiles. The great ability of rice to avoid the ethanol-induced injuries may contribute its anoxia tolerance when glycolysis and ethanolic fermentation replace the Krebs cycle as the main source of energy under anaerobic conditions.     

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.     

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.     

Effect of oxygen concentration on intracellular pH, glucose-6-phosphate and NTP content in rice (Oryza sativa) and wheat (Triticum aestivum) root tips: in vivo 31P-NMR study

Hypoxic pretreatment is known to induce anoxia tolerance in plant species sensitive to oxygen deprivation. However, we still do not have detailed information on changes in cytoplasmic and vacuolar pH (pH_cyt and pH_vac) in plants under low-oxygen availability (hypoxia) and under anoxia. To investigate this, we have studied the influence of hypoxia and anoxia on pH_cyt and pH_vac, glucose-6-phosphate (Glc-6-P) and nucleotide triphosphate (NTP) contents in rice ( Oryza sativa L.) root tips in comparison with those of wheat ( Triticum aestivum L.) with in vivo ³¹P-nuclear magnetic resonance. Both cereals responded to hypoxia similarly, by rapid cytoplasmic acidification (from pH 7.6–7.7 to 7.1), which was followed by slow partial recovery (0.3 units after 6 h). Anoxia led to a dramatic pH_cyt drop in tissues of both species (from pH 7.6–7.7 to less than 7.0) and partial recovery took place in rice only. In wheat, the acidification continued to pH 6.8 after 6 h of exposure. In both plants, NTP content followed the dynamics of pH_cyt. There was a strong correlation between NTP content and cytoplasmic H⁺ activity ([H⁺]_cyt= 10^−pHcyt) for both hypoxic and anoxic conditions. Glc-6-P content increased in rice under anoxia and hypoxia. In wheat, Glc-6-P was not detectable under anoxia but increased under hypoxia. In this study, rice root tips were shown to behave as anoxia tolerant tissues. Our results suggest that the initial cytoplasmic acidification and subsequent pH_cyt are differently regulated in anoxia tolerant and intolerant plants and depend on the external oxygen concentration.     

Anoxic stress leads to hydrogen peroxide formation in plant cells.

Hydrogen peroxide (H2O2) was detected cytochemically in plant tissues during anoxia and re-oxygenation by transmission electron microscopy using its reaction with cerium chloride to produce electron dense precipitates of cerium perhydroxides. Anoxia-tolerant yellow flag iris (Iris pseudacorus) and rice (Oryza sativa), and anoxia-intolerant wheat (Triticum aestivum) and garden iris (Iris germanica) were used in the experiments. In all plants tested, anoxia and re-oxygenation increased H2O2 in plasma membranes and the apoplast. In the anoxia-tolerant species the response was delayed in time, and in highly tolerant I. pseudacorus plasma membrane associated H2O2 was detected only after 45 d of oxygen deprivation. Quantification of cerium precipitates showed a statistically significant increase in the amount of H2O2 caused by anoxia in wheat root meristematic tissue, but not in the anoxia-tolerant I. pseudacorus rhizome parenchyma. Formation of H2O2 under anoxia is considered mainly an enzymatic process (confirmed by an enzyme inhibition analysis) and is due to the trace amount of dissolved oxygen (below 10(-5) M) present in the experimental system. The data suggest oxidative stress is an integral part of oxygen deprivation stress, and emphasize the importance of the apoplast and plasma membrane in the development of the anoxic stress response.     

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.     

Manipulation of ethanol production in anoxic rice coleoptiles by exogenous glucose determines rates of ion fluxes and provides estimates of energy requirements for cell maintenance during anoxia

Ethanol production by anoxic, excised, 7-10 mm tips of rice coleoptiles was manipulated using a range of exogenous glucose concentrations. Such a dose-response curve enabled good estimates at which level of ethanol production (and hence by inference ATP production), injury commenced and also allowed assessments of energy requirements for maintenance in anoxia. Rates of net uptake or loss of K+ and P by these excised coleoptile tips were related to rates of ethanol production (r2 of 0.59 and 0.68, respectively). At 72 h anoxia, ATP levels in excised tips were similar at 0, 2.5, and 50 mol m(-3) exogenous glucose, despite large differences in the inferred rates of ATP production. At 96 h anoxia, tips without exogenous glucose had low ATP concentrations; these may be the cause or the consequence of cell injury. In tips without glucose, injury was indicated by losses of K+ and Cl- between 72-96 h anoxia, and during the first hour after re-aeration, while later than 1 h after re-aeration, rates of net uptake were substantially lower than for re-aerated tips previously in anoxia with exogenous glucose. Between 96 h and 124 h anoxia, ion losses from tips without exogenous glucose increased while recovery of net uptake after re-aeration was very sluggish and incomplete. The energy requirement for maintenance of health and survival of anoxic coleoptile tips, expressed on a fresh weight basis, was lower than for three other anoxia-tolerant plant tissues/cells, studied previously. However, the energy requirement on a protein basis was assessed at 1.4 micromol ATP mg(-1) protein h(-1) and this value is 2.6-5.4-fold higher than for the other plant tissues/cells. Yet, this requirement was still only 58-88% of the published values for aerated tissues. The reason for this relatively high ATP requirement per unit protein in anoxic rice coleoptiles remains to be elucidated.     

Stabilization of adenine nucleotide ratios at various values by an oxygen limitation of respiration in germinating lettuce (Lactuca sativa) seeds.

The concentrations of adenine nucleotides were determined in germinating lettuce (Lactuca sativa) seeds after transitions from air to hypoxic or anoxic atmospheres. The ratio ATP/ADP and the energy charge were rapidly lowered after the transitions and remained stable at low values for hours. The energy charge in anoxia stabilized at a value close to 0.3. After 24 h in anoxia the energy charge rose rapidly to high values (0.9) when N2 was replaced by air. The metabolic properties of lettuce seeds had then been conversed for hours at low energy charge. In hypoxia the O2 uptake was decreased and the energy charge was stabilized at values intermediate between that in air and that in anoxia. When the O2 partial pressures (pO2) were 5 and 2kPa, the values of O2 uptake were one-third and one-sixth of that in air, and the energy charges were 0.7 and 0.5. These results show that the energy charge is regulated over a wide range of values. The ratio ATP/ADP and the energy charge are indicators of the limitation of metabolic activity by hypoxia.     

Production and organ distribution of succinate in rice seedlings during anoxia

Anaerobic production of succinate, a common feature in animals able to sustain anoxia, has seldom been reported in plants. By the use of ¹H-nuclear magnetic resonance spectroscopy we show here that succinate is produced by rice seedlings (Oryza sativa L. cv. Arborio) subjected to anoxic conditions. Starting from levels below I μmol (g fresh weight)⁻¹ in air, after 48 h of anoxia the levels of alanine, succinate and lactate had increased to 23.8, 5.2 and 1.0 μmol (g fresh weight) ⁻¹, respectively, in shoot tissues. Succinate was accumulated in shoots, notably in the coleoptiles, but not in roots of the rice seedlings, suggesting its involvement in rice coleoptile elongation under anoxia. Other possible functions of succinate production in rice seedling, an organism highly tolerant to anoxia, are discussed.     

pH regulation in anoxic rice coleoptiles at pH 3.5: biochemical pHstats and net H+ influx in the absence and presence of NOFormula

During anoxia, cytoplasmic pH regulation is crucial. Mechanisms of pH regulation were studied in the coleoptile of rice exposed to anoxia and pH 3.5, resulting in H(+) influx. Germinating rice seedlings survived a combination of anoxia and exposure to pH 3.5 for at least 4 d, although development was retarded and net K(+) efflux was continuous. Further experiments used excised coleoptile tips (7-10 mm) in anoxia at pH 6.5 or 3.5, either without or with 0.2 mM NO(3)(-), which distinguished two processes involved in pH regulation. Net H(+) influx (μmol g(-1) fresh weight h(-1)) for coleoptiles with NO(3)(-) was ～1.55 over the first 24 h, being about twice that in the absence of NO(3)(-), but then decreased to 0.5-0.9 as net NO(3)(-) uptake declined from ～1.3 to 0.5, indicating reduced uptake via H(+)-NO(3)(-) symports. NO(3)(-) reduction presumably functioned as a biochemical pHstat. A second biochemical pHstat consisted of malate and succinate, and their concentrations decreased substantially with time after exposure to pH 3.5. In anoxic coleoptiles, K(+) balancing the organic anions was effluxed to the medium as organic anions declined, and this efflux rate was independent of NO(3)(-) supply. Thus, biochemical pHstats and reduced net H(+) influx across the plasma membrane are important features contributing to pH regulation in anoxia-tolerant rice coleoptiles at pH 3.5.     

草酸氧化酶在水稻胚芽鞘衰老中的作用

草酸氧化酶（OxO）催化草酸氧化产生CO2和H2O2,其在植物发育及防御过程中可能具有重要作用。本文以水稻品种‘湘糯1号’（‘Xiangnuo 1’）为材料,对胚芽鞘中的OxO及其生理功能进行了研究。结果表明,胚芽鞘中的H2O2含量在其衰老时增加;OxO活性在浸种后96h时较低,之后也迅速增加,在240h达到最高;而可溶性蛋白、O2-·和草酸含量以及过氧化氢酶（CAT）活性则随着胚芽鞘的衰老迅速降低。由于H2O2能够诱导细胞死亡,推测OxO可能通过降解草酸产生H2O2参与胚芽鞘的衰老。     

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.     

Tolerance of crop plants to oxygen deficiency stress: fermentative activity and photosynthetic capacity of entire seedlings under hypoxia and anoxia

The study investigates the reactions of rice, wheat and maize to anoxia (plants without access to oxygen) and hypoxia (roots with very limited access to oxygen). We studied the adaptations of these intact crop plants because they are known to differ widely in their tolerance to oxygen deficiency. In hypoxia, there was an accumulation of sugars, especially in wheat and maize, although both flood-sensitive species significantly increased the activities of fermentative and glycolytic enzymes, clearly more than in rice. In rice, avoiding an oxygen limitation due to the effective aeration system (30% of root cross-sectional area) may have accounted for only a minor metabolic reaction to hypoxia. In anoxia, maize and wheat quickly lost viability and nearly all photosynthetic capacity, while most rice leaves stayed turgid and green, losing only 50% of the photosynthetic capacity. A strong metabolic arrest under anoxia was obvious for the sucrolytic, glycolytic and fermentative enzymes in all tested species, but was most pronounced in rice. Of the 14 enzymes studied, rice showed the lowest activity increase in hypoxia for 11 enzymes, and the strongest activity decrease in anoxia for 8 enzymes. However, rice was able even under anoxia to keep a 1/4 of the ATP level of the aerated control, while it was at the detection limit in maize and wheat. It appears that in anoxic rice, the switch to metabolic dormancy and maintenance of basic shoot meristems diminishes the needs for energy and substrate. Additionally, rice already has lower sugar demand under hypoxia, and sugar supply appears to be sustained under anoxia by a functioning anaerobic amylase and by the photosynthetically active shoot.