Organ specific analysis of the anaerobic primary metabolism in rice and wheat seedlings II: Light exposure reduces needs for fermentation and extends survival during anaerobiosis

Low oxygen stress in plants can occur during flooding and compromise the availability and utilization of carbohydrates in root and shoot tissues. Low-oxygen-tolerant rice and -sensitive wheat plants were analyzed under anaerobiosis in light to evaluate main factors of the primary metabolism that affect sensitivity against oxygen deprivation: activity of glycolysis and the rate of photosynthesis. Relatively stable ATP contents (93 and 58% of aerated control levels after 24 h anaerobiosis) in illuminated shoot tissues account for enhanced tolerance of rice and wheat seedlings to anaerobiosis upon light exposure in comparison to anoxia in darkness. Although the photosynthetic process was inhibited during low oxygen stress, which was partly due to CO₂ deficiency, more light-exposed than dark-incubated seedlings survived. Illuminated plants could tolerate a 70% lower anaerobic ethanol production in shoots in comparison to darkness, although still an 18-times higher ethanol production rate was determined in rice than in wheat leaves. In conclusion, light-exposed plants grown under anaerobiosis may recycle low amounts of generated oxygen between photosynthesis and dissimilation and generate additional energy not only from substrate phosphorylation during glycolysis but also from other sources like cyclic electron transport.     

Metabolic adaptation to flooding stress in upland and lowland rice seedlings

Ability of metabolic adaptation in upland and lowland rice (Oryza sativa L.) seedlings to flooding stress was compared. Flooding stress increased alcohol dehydrogenase (ADH) activity and ethanol concentration in shoots and roots of the upland and lowland rice seedlings. The difference in ADH activity and ethanol concentration in shoots between the upland and lowland rice was not apparent. However, both ADH activity and ethanol concentration in roots of the lowland rice were 2-fold greater than those in roots of the upland rice, suggesting that flooding-induction of ethanolic fermentation in lowland rice roots may be significantly greater than that in the upland rice roots. Since flooding often causes the anaerobic conditions in rooting zone than aerial part of plants and ethanolic fermentation is essential to survive in the anaerobic conditions, the ability of metabolic adaptation in lowland rice seedlings to flooding stress may be greater than that in upland rice seedlings.     

Effect of low temperature on ethanolic fermentation in rice seedlings

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.     

Kinetics of ethanol and acetaldehyde release suggest a role for acetaldehyde production in tolerance of rice seedlings to micro-aerobic conditions

BACKGROUND AND AIMS: This paper examines the basis of the greater tolerance of an indica rice cultivar FR13A to complete submergence compared with relatively intolerant japonica rice CT6241. We study whether this superior tolerance is related to its greater tolerance to O2 shortage and to an ability to run a more favourable rate of alcoholic fermentation during and after O2 deprivation. METHODS Fermentation products were analysed using sensitive laser-based photoacoustics at high time resolution to establish patterns and rates of ethanol and acetaldehyde emission by intact rice seedlings exposed to micro-aerobic (0.05-0.5 % O2) or zero O2 supply, and also during their return to air. Oxygen and CO2 emission or uptake was also quantified. KEY RESULTS: In the dark, no acetaldehyde and ethanol emission was observed until external O2 concentration in a gas phase decreased to 相似文献   

Der Einfluß der Wurzelanaerobiose auf Äthanol-, Laktat- und Glukosegehalte sowie auf die Aktivitäten von ADH und LDH in Weizenkeimpflanzen

The effect of anaerobiosis of wheat seedling roots during 6 consecutive days on contents of ethanol, lactate and glucose in roots and shoots and on the exudation of ethanol from roots to the medium was examined. Activities of alcohol dehydrogenase (ADH) and lactate dehydrogenase (LDH) were determined. After 36 h of anaerobiosis the concentration of ethanol in roots increased temporarily about 6 times and after 6 days it decreased to the level of control plants. The exudation of ethanol from roots to the medium showed similar pattern. The content of lactate was unaffected by anaerobiosis. In contrast, the content of glucose in roots of seedlings increased already after 1 day of anaerobiosis about 2 times and this higher level of glucose was noticed during consecutive 5 days. Anaerobiosis of roots caused an increase in the activity of ADH in both roots and shoots but the increase was not related to the content of ethanol in tissues, or exudated to the medium. The activity of LDH was unaffected by this factor. The results are discussed in relation to the limitation of energy supply of plants grown under root anaerobiosis.     

Anaerobic induction and tissue-specific expression of maize Adh1 promoter in transgenic rice plants and their progeny.

Summary In order to analyze expression of the maize alcohol dehydrogenase 1 gene (Adh1), its promoter was fused with the gusA reporter gene and introduced into rice by protoplast transformation. Histochemical analysis of transgenic plants and their progeny showed that the maize Adh1 promoter is constitutively expressed in root caps, anthers, anther filaments, pollen, scutellum, endosperm and shoot and root meristem of the embryo. Induction of expression by the Adh1 promoter was examined using seedlings derived from selfed progeny of the transgenic plants. The results showed that expression of the Adh1 promoter was strongly induced (up to 81-fold) in roots of seedlings after 24 h of anaerobic treatment, concomitant with an increase in the level of gusA mRNA. 2,4-D also induced Adh1 promoter-directed expression of gusA to a similar extent. In contrast, little induction by anaerobic treatment was detected in transformed calli, leaves or roots of primary transformants or shoots of seedlings. A detailed examination of seedling roots during anaerobic treatment revealed that the induction started first at the meristem and after 3 h there was strong induction in the elongation zone which is located 1–2 mm above the meristem; the induction then progressed upward from this region. Our results suggest that transgenic rice plants carring the gusA reporter gene fused with promoters are useful for the study of anaerobic regulation of genes derived from graminaceous species.     

Effect of anaerobiosis on alcohol dehydrogenase in oat seedlings

In order to clarify the induction of alcohol dehydrogenase (ADH) by anaerobiosis in oat (Avena sativa L.), the seedlings were exposed to anaerobiosis and activity of ADH and ADH isozyme profiles were determined. The anaerobiosis increased ADH activities in shoots and roots of the seedlings. By day 2, the activity increased 5 and 4 times in the roots and the shoots, respectively, compared with those under aerobic condition. Based on nondenaturing electrophoresis, ADH isozyme composition analysis revealed six bands consisting of a dimmer enzyme with submits encoded by three different Adh genes. Changes in staining intensity of the isozymes indicated that the increase in ADH activity in oat under anaerobiosis resulted from increased enzyme synthesis.     

Sugar utilization and anoxia tolerance in rice roots acclimated by hypoxic pretreatment

Although most cereal roots cannot elongate under anoxic conditions, primary roots of three-day-old rice (Oryza sativa L.) seedlings were able to elongate during a 24-h period of anoxia. Hypoxic pretreatment (H-PT) increased the elongation of their roots. Sucrose synthase (EC 2.4.1.13), glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4), pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) activities were increased by anoxia in both H-PT and non-pretreated (N-PT) roots. However, these activities were greater in the H-PT roots than in the N-PT roots. The average rate of production of ethanol for the initial 6h after the onset of anoxia was 3.7 and 1.4 micromolg(-1) fresh weight h(-1) for the H-PT and N-PT roots, respectively, suggesting that ethanolic fermentation may increase more quickly in the H-PT roots than in the N-PT roots. Roots of the seedlings lost ATP and total adenine nucleotides in anoxia, however, the H-PT roots maintained higher levels of ATP and total adenine nucleotides compared to the N-PT roots. These results show that rice roots are able to utilize the set of enzymes involved in the metabolism of soluble sugars under anoxia. The ability to maintain an active fermentative metabolism for production of ATP by fueling the glycolytic pathway with fermentable carbohydrate is probably greater in H-PT than in N-PT roots.     

Distribution of alcohol dehydrogenase in roots and shoots of rice (Oryza sativa) and Echinochloa seedlings

Abstract Aerobically germinated seedlings of rice and Echinochloa were found to survive when placed in an anaerobic environment for 4 d, whereas pea and maize seedlings did not. Although root and shoot growth were inhibited in rice and Echinochloa under anaerobiosis, growth resumed when the seedlings were returned to aerobic conditions. Alcohol dehydrogenase (ADH) activity increased more, and protein synthesis was greater, in the shoots than in the roots under anaerobic conditions. These results suggest that, in anaerobiosis-tolerant species, ADH activity and protein synthesis in the shoots represents or results from metabolic adaptations to low oxygen. These results are discussed in terms of plant establishment and growth in a low-oxygen environment.     

Rapid elicitation of second messengers by nanomolar doses of triacontanol and octacosanol

Triacontanol (TRIA) increases the dry weight and alters the metabolism of rice (Oryza sativa L.) seedlings within 10 min of application to either the shoots or roots. This activity is prevented if octacosanol (OCTA, C₂₈ primary alcohol) is applied with the TRIA on the roots or shoots. Triacontanol activity is also stopped if the OCTA is applied at least 1 min before the TRIA on the opposite part of the seedling.Triacontanol rapidly elicits a second messenger that moves rapidly throughout the plant resulting in stimulation of growth (dry-weight increase) and water uptake. Octacosanol also produces a second messenger that inhibits TRIA activity. We have named the putative secondary messengers elicited by TRIA and OCTA, TRIM and OCTAM, respectively. The water-soluble TRIM extracted from plants treated with TRIA increases the growth of rice seedlings about 50% more than extracts from untreated plants, within 24 h of application. Both OCTAM and OCTA inhibit the activity of TRIA but not of TRIM.The TRIA messenger was isolated from rice roots within 1 min of a foliar application of TRIA. The TRIM elicited by TRIA will pass through a 4-mm column of water connecting cut rice shoots with their roots and can also be recovered from water in which cut stems of TRIA-treated plants have been immersed. Triacontanol applied to oat (Avena sativa L.) or tomato (Lycopersicon esculentum Mill.) shoots connected to rice roots by a 4-mm water column also results in the appearance of TRIM in rice roots.Abbreviations OCTA octacosanol - OCTAM second messenger elicited by OCTA - TAS tallow alkyl sulfate - TRIA triacontanol - TRIM second messenger elicited by TRIA Michigan Agricultural Experiment Station Journal Article No. 12001     

Fermentation metabolism in roots of wheat seedlings after hypoxic pre-treatment in different anoxic incubation systems

A hypoxic pre-treatment (HPT) can improve the anoxic survival of flooding sensitive plants. Here, we tested whether a 4-d HPT of wheat plants (Triticum aestivum L.) would improve their anoxic resistance, and if so, why. We found that the metabolic adjustment during prolonged HPT involved an increased lactate excretion rate, the up-regulation of glycolytic and fermentative enzymes as well as the accumulation of various sugars. Therefore, HPT wheat roots could sustain a 3 times higher ethanolic fermentation rate during an anoxic period compared to non-pre-treated (NHPT) roots. Nevertheless, the enhanced fermentation rate provided temporary relief to the energy crisis only, and both NHPT and HPT plants died after 5d of anoxia in illumination. Comparison of different low oxygen incubation systems using excised roots or roots of intact plants revealed striking differences. The benefits of intact shoots, oxygen transport as well as additional sugar supply enabled a more stable energy supply of anoxia-treated NHPT and HPT roots. However, the height of the fermentation rate was correlated with a high ATP content during dark anoxic incubation, but not in illumination.     

Levels of endogenous polyamines and NaCl-inhibited growth of rice seedlings

The role of endogenous polyamines in the control of NaCl-inhibited growth of rice seedlings was investigated. Putrescine, spermidine and spermine were all present in shoots and roots of rice seedlings. NaCl treatment did not affect spermine levels in shoots and roots. Spermidine levels in shoots and roots were increased with increasing concentrations of applied NaCl. NaCl at a concentration of 50 mM, which caused only slight growth inhibition, drastically lowered the level of putrescine in shoots and roots. Addition of precursors of putrescine biosynthesis (L-arginine and L-ornithine) resulted in an increase in putrescine levels in NaCl-treated shoots and roots, but did not allow recovery of the growth inhibition of rice seedlings induced by NaCl. Pretreatment of rice seeds with putrescine caused an increase in putrescine level in shoots, but could not alleviate the inhibition effect of NaCl on seedling growth. The current results suggest that endogenous polyamines may not play a significant role in the control of NaCl-inhibited growth of rice seedlings.Abbreviations PUT putrescine - SPD spermidine - SPM spermine     

Phytotoxic, clastogenic and bioaccumulation effects of the environmental endocrine disruptor bisphenol A in various crops grown hydroponically

The effects of the endocrine disruptor bisphenol A (BPA) at concentrations of 10 and 50 mg l⁻¹ were evaluated on the germination and morphology, micronuclei (MN) content in root tip cells and BPA bioaccumulation of hydroponic seedlings of broad bean (Vicia faba L.), tomato (Lycopersicon esculentum Mill.), durum wheat (Triticum durum Desf.) and lettuce (Lactuca sativa L.) after 6 and 21 days of growth. In general, BPA at any dose used did not inhibit germination and early growth (6 days) of seedlings of the species examined, with the exception of primary root length of tomato which decreased at the higher BPA dose. In contrast, an evident phytotoxicity was induced by BPA in all species after 21 days of growth with evident morphological anomalies and significant reductions of the lengths and fresh and dry weights of shoots and roots of seedlings. With respect to the nutrient medium without seedlings, BPA concentration decreased markedly during the growth period in the presence of broad bean and tomato seedlings, and limitedly in the presence of durum wheat and, especially, lettuce. Further, the presence of BPA measured in roots and shoots of broad bean and tomato after 21-day growth indicated that bioaccumulation of BPA had occurred. The number of MN in broad bean and durum wheat root tip cells increased markedly by treatment with BPA at both concentrations, thus suggesting a potential clastogenic activity of BPA in these species.     

Effects of ethanol on growth of rice seedlings

Effects of ethanol, the end product of ethanolic fermentation, on the growth of rice (Oryza sativa L.) seedlings were determined as a means of evaluating growth responses under anoxia. The ethanol concentrations in roots and coleoptiles of the seedlings subjected to 48 h-anoxia, and in their culture medium were 23 and 32 µmol g^–1 fresh weight, and 19 µmol ml^–1, respectively. The growth of the roots and coleoptiles of the seedlings was restricted by exogenous ethanol at concentrations above 50 mM and 100 mM, respectively, suggesting that the roots are more sensitive to ethanol than the coleoptiles.     

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.     

Influence of NO3 - and NH4 + nutrition on fermentation,nitrate reductase activity and adenylate energy charge of roots of Carex pseudocyperus L. and Carex sylvatica Huds. exposed to anaerobic nutrient solutions

The adenylate energy charge, production of ethanol and lactate, and nitrate reductase activity were determined in order to study the influence of different nitrogen sources on the metabolic responses of roots of Carex pseudocyperus L. and Carex sylvatica HUDS. exposed to anaerobic nutrient solutions. Determination of adenylates was carried out by means of a modified HPLC technique. Total quantity of adenylates was higher in Carex pseudocyperus than in Carex sylvatica under all conditions. In contrast, the adenylate energy charge was only slightly different between the species and decreased more or less in relation to the applied nitrogen source under oxygen deficiency. The adenylate energy charge in roots of plants under nitrate nutrition showed a smaller decrease under anaerobic environmental conditions than plants grown with ammonium or nitrate/ammonium. Roots of nitrate-fed plants showed a lower ethanol and lactate production than ammonium/nitrate- and ammonium-fed plants. Ethanol production was higher in C. pseudocyperus, formation of lactate was lower compared to that in Carex sylvatica. The activity of enzymes involved in fermentation processes (ADH, LDH and PDC) was enhanced significantly after 24 hours of exposure to anaerobic nutrient solutions in roots of both species. The induction of these enzymes was only slightly influenced by different nitrogen supply. In vivo nitrate reductase activity increased almost 3-fold compared to the aerobic treatment in both species and overcompensated loss of NADH reoxidation capacity caused by decrease of ethanol and lactate development. Induction of in vitro nitrate reductase activity was enhanced 313% in C. pseudocyperus and 349% in C. sylvatica under anaerobic environmental conditions and nitrate supply. These results indicate that nitrate may serve as an alternative electron acceptor in anaerobic plant root metabolism and that the nitrate-supported energy charge may be due to an accelerated glycolytic flux resulting from a more effective NADH reoxidation capacity by nitrate reduction plus fermentation than by fermentation alone.Abbreviations ADH alcohol dehydrogenase - AEC adenylate energy charge - DMSO dimethyl sulfoxide - EDTA ethylen diamine tetraacetic acid - HPLC high performance liquid chromatography - LDH lactate dehydrogenase - NRA nitrate reductase activity - PCA perchloric acid - PDC pyruvate decarboxylase - PVP polyvinylpyrrolidone - PVPP polyvinylpolypyrrolidone - TCA trichloroacetic acid, Tris-tris(hydroxymethyl)aminomethane     

Growth and development ofRumex roots as affected by hypoxic and anoxic conditions

The growth characteristics of threeRumex species were determined under different solution oxygen concentrations in hydroculture. These species all occur in a river foreland ecosystem and they were found to differ in their flood tolerance. The flood-tolerantR. maritimus undR. crispus developed a large number of new, aerenchymatous roots within a short period under low solution oxygen concentrations. Biomass production was not affected. In the flood-intolerantR. thyrsiflorus, however, only few slow-growing new roots were developed and biomass production was significantly reduced at solution oxygen concentrations below 2% (v:v). These different responses could be partly explained by a differential aerenchyma formation in new roots of the flood-tolerant species. Aerenchyma can relieve the oxygen stress of the root systems via internal aeration.The fast development of new roots of the flood-tolerantR. maritimus andR. crispus after the onset of anaerobiosis coincided with the reduction or cessation of growth of the primary roots. Notwithstanding the cessation of growth, however, primary roots of both species were able to recover following restoration of aerobic conditions after a 13-day anaerobic period. However, the roots ofR. thyrsiflorus ceased growing very soon after the onset of anaerobiosis. All had died within 10 days.The balance between the growth rates of the primary and the newly formed root system are discussed and related to the differential tolerance of theRumex species to transient flooding.     

Nitrite-driven anaerobic ATP synthesis in barley and rice root mitochondria

Mitochondria isolated from the roots of barley (Hordeum vulgare L.) and rice (Oryza sativa L.) seedlings were capable of oxidizing external NADH and NADPH anaerobically in the presence of nitrite. The reaction was linked to ATP synthesis and nitric oxide (NO) was a measurable product. The rates of NADH and NADPH oxidation were in the range of 12–16 nmol min⁻¹ mg⁻¹ protein for both species. The anaerobic ATP synthesis rate was 7–9 nmol min⁻¹ mg⁻¹ protein for barley and 15–17 nmol min⁻¹ mg⁻¹ protein for rice. The rates are of the same order of magnitude as glycolytic ATP production during anoxia and about 3–5% of the aerobic mitochondrial ATP synthesis rate. NADH/NADPH oxidation and ATP synthesis were sensitive to the mitochondrial inhibitors myxothiazol, oligomycin, diphenyleneiodonium and insensitive to rotenone and antimycin A. The uncoupler FCCP completely eliminated ATP production. Succinate was also capable of driving ATP synthesis. We conclude that plant mitochondria, under anaerobic conditions, have a capacity to use nitrite as an electron acceptor to oxidize cytosolic NADH/NADPH and generate ATP.     

Metabolic origin of acetaldehyde emitted by poplar (Populus tremula x (P. alba) trees

The metabolic origin and emission by the leaves of the tropospheric trace gas acetaldehyde were examined in 4-month-old poplar trees (Populus tremula x P. alba) cultivated under controlled environmental conditions in a greenhouse. Treatments which resulted in increased ethanol concentration of the xylem sap caused significantly enhanced rates of acetaldehyde and ethanol emission by the leaves. Leaves fed [¹⁴C]-ethanol via the transpiration stream emitted [^<14C]-acetaldehyde. These findings suggest that acetaldehyde in the leaves is synthesized by a metabolic pathway that operates in the opposite direction of alcoholic fermentation and results in oxidation of ethanol. Enzymatic studies showed that this pathway is mediated either by alcohol dehydrogenase (ADH; EC 1.1.1.1) or catalase (CAT; EC 1.11.1.6), both constitutively present in the leaves of poplar trees. Labelling experiments with [¹⁴C]-glucose indicated that the ethanol delivered to the leaves by the transpiration stream is produced in anaerobic zones of submersed roots by alcoholic fermentation. Anoxic conditions in the rhizosphere caused by flooding of the root system resulted in an activation of alcoholic fermentation and led to significantly increased ethanol concentrations in the xylem sap. These results support the hypothesis that acetaldehyde emitted by the leaves of trees is derived from xylem transported ethanol which is synthesized during alcoholic fermentation in the roots.Keywords: Acetaldehyde, emission, ethanol, anaerobiosis, Populus tremula x P. alba