Effect of a short-term hypoxic treatment followed by re-aeration on free radicals level and antioxidative enzymes in lupine roots.

To investigate whether re-aeration after a short-term hypoxic pre-treatment (for 2, 12 or 24 h) induces oxidative stress, the temporal sequence of physiological reactions, including the level of free radicals, hydrogen peroxide production, and changes in antioxidative enzymes, was characterized in roots of hydroponically grown lupine (Lupinus luteus L., cv. Juno) seedlings. By using electron paramagnetic resonance (EPR), we found that the exposure of hypoxically grown roots (hypoxic pre-treatment for 12 and 24 h) to air caused an increase in the level of free radicals. The amount of hydrogen peroxide also tended to increase when hypoxically pre-treated roots were re-aerated, which attests to a higher production of reactive oxygen species. Re-aeration caused a higher activity of superoxide dismutase (SOD, EC 1.15.1.1) and catalase (CAT, EC 1.11.1.6), whereas the activity of peroxidase (POX, EC 1.11.1.7) was only slightly influenced. The roots were less tolerant to longer hypoxic pre-treatments, with a significant decrease in viability, associated with death of root tips immediately after hypoxic stress. Roots exposed to hypoxia for 2 h showed less pronounced responses and their viability was not affected by hypoxic stress and re-aeration. These results indicate that re-aeration following short-term hypoxia imposes a mild oxidative stress. This led us to conclude that re-oxygenation stress per se was not the key factor for cell death in root tips.     

Response of the ascorbate-glutathione cycle to re-aeration following hypoxia in lupine roots.

The response of the enzymes and metabolites of the ascorbate-glutathione pathway to oxidative stress caused by re-aeration following hypoxia was studied in roots of hydroponically grown lupine (Lupinus luteus L. cv. Juno) seedlings. Lupine roots were deprived of oxygen by subjecting them to hypoxia for 48 and 72 h and then re-aerated for up to 4 h. An increased content of total ascorbate was observed in lupine roots immediately after hypoxia, whereas total glutathione level decreased. However, a significant increase in the reduced forms of both metabolites was found directly after hypoxia. Re-admission of oxygen caused the decrease of the ratios of reduced to oxidized forms of ascorbate and glutathione, indicating oxidative stress. While monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) activity remained unaltered during re-aeration the increase in activities of ascorbate peroxidase (APX, EC 1.11.1.11) and glutathione reductase (GR, EC 1.6.4.2) was observed 30 min after transfer from hypoxic condition. Dehydroascorbate reductase (DHAR, EC 1.8.5.1) activity approached the control level during a whole re-aeration period. Native gel electrophoresis combined with specific activity staining revealed seven isoforms of APX, five isoforms of GR and three different proteins with DHA reductase activity in roots extracts. However, immediately after hypoxic treatment APX-5 isoform and GR-1 isoform were not observed in roots. This experimental system was also used to investigate superoxide anion level in roots utilizing the superoxide anion-specific indicator dihydroethidium (DHE). Intense DHE-derived fluorescence was found in re-aerated root tips as compared to control roots, indicating that re-aeration induced superoxide anion production in hypoxically pretreated roots.     

Free radical formation and activity of antioxidant enzymes in lupin roots exposed to lead

Inhibition of root growth and modification of root morphology are the most sensitive responses of Lupinus luteus cv. Ventus L. to lead ions - Pb(NO₃)₂. Using electron paramagnetic resonance (EPR), we found that at the lead concentration of 150 mg.L^–1, the level of free radicals remained at control level, whereas at the higher, sublethal concentration of 350 mg.L^–1, they markedly increased. The EPR signal with the g-value at the maximum absorption of 2.0053 implied that the paramagnetic radical is derived from a quinone. The response of antioxidant enzymes, such as superoxide dismutase (SOD, EC 1.15.1.1), catalase (CAT, EC 1.11.1.6), peroxidase (POX, EC 1.11.1.7) and ascorbate peroxidase (APOX, EC 1.11.1.11), to exogenously applied lead ions was also examined. Enzyme activity was estimated as a function of time and concentration. Native polyacrylamide gel electrophoresis followed by specific staining revealed an increase in the activity of SOD, CAT, POX and APOX coinciding with the time of cultivation. A lead-dependent increase in activities of SOD and POX from root tip extracts was observed, whereas CAT and APOX activities decreased at the higher lead concentrations. These results suggest that at higher lead concentrations, the formation of both free radicals and reactive oxygen species is beyond the capacity of the antioxidant system, which in turn may contribute to the reduced root growth.     

Protection against activated oxygen following re-aeration of hypoxically pretreated wheat roots. The response of the glutathione system

This paper shows the effects of re-aeration on the glutathionepool following a prolonged period of root hypoxia. An increased content of total glutathione has been measuredin roots of wheat seedlings (Triticum aestivum L. cv. Alcedo),grown in a nitrogen-flushed nutrient solution (HI) with theirshoots in air compared with roots of aerobically grown plants(C). Re-aeration of hypoxically pretreated roots causes oxidativeinjury indicated by the oxidation of reduced glutathione (GSH),decrease of total thiol-groups and increased formation of TBAreactive material (lipid peroxidation). Re-admission of oxygen results in a 50% rise in oxygen uptakeover the whole 16 h re-aeration period compared with the control.During this time the overall glutathione pool of HI treatmentincreases to almost double that of the control, essentiallyreflected in the amount of oxidized glutathione (GSSG). Hypoxically pretreated roots showed lower glutathione reductaseactivity (GR) than the control. Immediately following re-aerationthe activity was further decreased to a limiting value whichseems to prevent full reduction of the newly formed glutathione.Therefore, the capacity to reduce the GSSG pool is below thecapacity for net glutathione synthesis. This results in a declineof the GSH/GSSG ratio which reflects oxidative stress. The enzymeactivity recovers slowly after re-aeration exceeding the valuesof aerobically grown roots only after 16 h correlating witha high reduction state of the glutathione pool. Copper, known to induce the formation of reactive oxygen species,strengthened the effect of re-aeration and enhanced the post-anoxicinjury irreversibly. The importance of the glutathione system in roots to cope withvarying oxygen tension is discussed. Key words: Hypoxia, re-aeration, glutathione system, glutathione reductase, wheat     

Hypoxic induction of alcohol and lactate dehydrogenases in lupine seedlings

Seedlings of lupine (Lupinus luteus L. cv. Juno) were exposed for up to 96 hours to 1 to 2 kPa partial pressure oxygen (hypoxic treatment) and activities of alcohol dehydrogenase (ADH), lactate dehydrogenase (LDH) and their isoform profiles were determined. Roots of lupine seedlings were grown in a nitrogen flushed nutrient solution while their shoots were in air. Prolonged hypoxia led to a reduction of root elongation. This was accompanied by reduced increase in dry weight suggesting that insufficient carbohydrate supply was the cause of retarded growth of lupine roots. Hypoxically treated roots showed induction of ADH and LDH acivities. The maximum increase in LDH activity was low (2-fold) in contrast to ADH activity, which increased up to 7-fold. Hypoxic treatment of roots did not affect the activities of ADH and LDH in hypocotyls and cotyledons. Analysis of ADH and LDH activity gels indicated in roots 1 and 2 isoforms, respectively. The level of isozymes of both enzymes increased in roots upon exposure to hypoxic stress. Differences in isoenzymatic spectrum of ADH and LDH between roots, hypocotyls and cotyledons indicate organ specificity of isozymes of both enzymes. The importance of alcohol and lactate fermentation in roots to cope with hypoxic stress is discussed.     

Hypoxia induces anoxia tolerance in roots and shoots of lupine seedlings

Lupine seedlings were exposed to 4 kPa partial pressure oxygen (hypoxically pretreated) for 18 hours before treatment with strictly anaerobic conditions (anoxia). Seedlings previously exposed to hypoxia were more tolerant than the controls (not hypoxically pretreated) to anoxic stress in both roots and shoots. Hypoxic pretreatment induced roots and shoots survival in anoxia. Improved viability of roots, following hypoxic pretreatment, was associated with increased activity of ADH. In nonacclimated roots and shots significant increase in LDH activity occurd during the first hours under anoxia but the in vitro activity of LDH was two orders of magnitude lower than that of ADH. The results are discussed in relation to the ability of lupine seedlings to survive anoxia.     

Hypoxically inducible barley alanine aminotransferase: cDNA cloning and expression analysis

A 1.75 kb cDNA containing the entire coding sequence of the hypoxically inducible alanine aminotransferase (AlaAT) from barley roots was isolated and sequenced. This clone has an open reading frame of 1446 bp, and a deduced amino acid sequence of 482 residues, giving an estimated protein molecular mass of 52 885 Da. RNA blot analysis of barley root tissue showed a 4-fold increase of a single AlaAT-2 mRNA band after 12–24 hours of hypoxic stress, followed by a decrease in message levels after 48 h of hypoxic conditions. AlaAT-2 protein concentration increased in a similar pattern to AlaAT activity in root tissue, to almost 6-fold the aerobic level after 96 h of hypoxic stress. AlaAT-2 activity increased more than 2-fold in roots of Panicum miliaceum exposed to hypoxia, and is the same isoform as the light inducible AlaAT in P. miliaceum leaves. The unique expression patterns of AlaAT-2 in root and leaf tissue upon exposure to different environmental stimuli is also discussed.     

Ability of lupine seeds to germinate and to tolerate desiccation as related to changes in free radical level and antioxidants in freshly harvested seeds

Seeds of yellow lupine (Lupinus luteus L. cv. Juno) were collected throughout their development on the mother plant to determine whether the ability to germinate and to tolerate desiccation is related to the level of free radicals and the changes in the redox state of ascorbate and glutathione as well as the activities of antioxidative enzymes. Electron paramagnetic resonance (EPR)-based analyses showed that development of lupine seed was accompanied by generation of free radicals with g₁ and g₂ values of 2.0049 ± 0.0004 and 2.0029 ± 0.0003, respectively. Free radical level increased significantly 25 DAF and decreased thereafter. The amount of hydrogen peroxide was high in fresh immature seeds and decreased during maturation drying. Ascorbate accumulated in lupine embryos during early seed filling stage whereas glutathione content increased during late seed filling phase. During maturation drying the redox state of both ascorbate and glutathione pools shifted towards the oxidized forms. While superoxide dismutase (SOD, EC 1.15.1.1), and ascorbate peroxidase (APX, EC 1.11.1.11) activities remained high at the early seed filling stage the activities of both dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) and that of catalase (CAT, EC 1.11.1.6) increased before seeds reached physiological maturity and decreased thereafter. The changes of isoform patterns of antioxidative enzymes were observed during seed maturation. Immature lupine seeds tested immediately after harvest acquired the ability to germinate when less than half-filled and reached high tolerance to desiccation just after physiological maturity. The physiological implications of the changes in antioxidative machinery for the acquisition of desiccation tolerance and seeds germinability are discussed.     

Prolonged root hypoxia effects on enzymes involved in nitrogen assimilation pathway in tomato plants

In order to investigate the effects of root hypoxia (1–2% oxygen) on the nitrogen (N) metabolism of tomato plants (Solanum lycopersicum L. cv. Micro-Tom), a range of N compounds and N-assimilating enzymes were performed on roots and leaves of plants submitted to root hypoxia at the second leaf stage for three weeks. Obtained results showed that root hypoxia led to a significant decrease in dry weight (DW) production and nitrate content in roots and leaves. Conversely, shoot to root DW ratio and nitrite content were significantly increased. Contrary to that in leaves, glutamine synthetase activity was significantly enhanced in roots. The activities of nitrate and nitrite reductase were enhanced in roots as well as leaves. The higher increase in the NH₄⁺ content and in the protease activities in roots and leaves of hypoxically treated plants coincide with a greater decrease in soluble protein contents. Taken together, these results suggest that root hypoxia leaded to higher protein degradation. The hypoxia-induced increase in the aminating glutamate dehydrogenase activity may be considered as an alternative N assimilation pathway involved in detoxifying the NH₄⁺, accumulated under hypoxic conditions. With respect to hypoxic stress, the distinct sensitivity of the enzymes involved in N assimilation is discussed.Key words: tomato, hypoxia, nitrogen, glutamine synthetase, protease, glutamate dehydrogenase     

The effect of post-hypoxia on roots inSenecio andMyosotis species related to the glutathione system

This paper shows the effect of re-aeration following hypoxic pretreatment on the glutathione system in plants with different flooding tolerance. Re-aeration of hypoxically pretreated roots led to an increase of TBA-rm content indicating an accelerated lipid peroxidation (post-anoxic injury). Re-admission of oxygen resulted in a clear increase in the content of total glutathione in both flooding-intolerant speciesMyosotis arvensis andSenecio jacobaea. Simultaneously, the high ratio between reduced (GSH) and oxidized (GSSG) glutathione decreased in these species upon the onset of re-aeration, while the tolerantMyosotis palustris andSenecio aquaticus showed only little changes in contents of GSH and GSSG. An imbalance in GSH/GSSG ratio reflects oxidative stress. The glutathione reductase (GR) reacted very differently in the investigated genera. The metabolic response to varying oxygen pressure is much stronger in the flooding-intolerant species compared to species naturally growing in wetlands. The present results suggest that glutathione system is an important component in overcoming oxidative stress.     

Root hypoxia reduces leaf growth : role of factors in the transpiration stream

This study examined the potential role of restricted phloem export, or import of substances from the roots in the leaf growth response to root hypoxia. In addition, the effects of root hypoxia on abscisic acid (ABA) and zeatin riboside (ZR) levels were measured and their effects on in vitro growth determined. Imposition of root hypoxia in the dark when transpirational water flux was minimal delayed the reduction in leaf growth until the following light period. Restriction of phloem transport by stem girdling did not eliminate the hypoxia-induced reduction in leaf growth. In vitro growth of leaf discs was inhibited in the presence of xylem sap collected from hypoxic roots, and also by millimolar ABA. Disc growth was promoted by sap from aerated roots and by 0.1 micromolar ZR. The flux of both ABA and ZR was reduced in xylem sap from hypoxic roots. Leaf ABA transiently increased twofold after 24 hours of hypoxia exposure but there were no changes in leaf cytokinin levels.     

Comparative lipid peroxidation,antioxidant defense systems and proline content in roots of two rice cultivars differing in salt tolerance

The changes in the activity of antioxidant enzymes such as superoxide dismutase (SOD: EC 1.15.1.1), catalase (CAT: EC 1.11.1.6), peroxidase (POX: EC 1.11.1.7), ascorbate peroxidase (APOX: EC 1.11.1.11) and glutathione reductase (GR: EC 1.6.4.2), free proline content, and the rate of lipid peroxidation level in terms of malondialdehyde (MDA) in roots of two rice cultivars (cvs.) differing in salt tolerance were investigated. Plants were subjected to three salt treatments, 0, 60, and 120 mol m⁻³ NaCl for 7 days. The results showed that activated oxygen species may play a role in cellular toxicity of NaCl and indicated differences in activation of antioxidant defense systems between the two cvs. The roots of both cultivars showed a decrease in GR activity with increase in salinity. CAT and APOX activities increased with increasing salt stress in roots of salt-tolerant cultivar Pokkali but decreased and showed no change, respectively, in roots of IR-28 cultivar. POX activity decreased with increasing NaCl concentrations in salt-tolerant Pokkali but increased in IR-28. SOD activity showed no change in roots of both cultivars under increasing salinity. MDA level in the roots increased under salt stress in sensitive IR-28 but showed no change in Pokkali. IR-28 produced higher amount of proline under salt stress than in Pokkali. Increasing NaCl concentration caused a reduction in root fresh weight of Pokkali and root dry weight of IR-28. The results indicate that improved tolerance to salt stress in root tissues of rice plants may be accomplished by increased capacity of antioxidative system.     

Changes in the Root System of Wheat Seedlings Following Root Anaerobiosis: III. Oxygen Concentration in the Roots

The oxygen status in roots of wheat seedlings (Triticum aestivum)was determined by a volumetric micro-absorption method. Plantsgrew in nutrient solution (aerated or nitrogen-flushed) or onflooded sand up to the 10th day. The roots were then exposedto aerated or hypoxic conditions for several hours before gaswas extracted by reducing the pressure within a concentratedsalt solution or by physical crushing. The oxygen content ofthe extracted gas bubbles was measured with pyrogallol. Comparativeexperiments with the helophytes Phalaris arundinacea and Carexacutiformis yielded similar oxygen concentrations to those alreadydescribed in literature. The concentrations of oxygen (13–16%)in young wheat roots were surprisingly high when exposed tonutrient solution flushed with nitrogen gas. Removal of the shoots decreased the oxygen concentration inthe roots, indicating some internal oxygen transport from shootsto roots. Detached, submerged roots of wheat still contained6% oxygen following 20 h of submergence in nitrogen-flushedsolution. A linear relationship was found between the oxygenconcentration in roots of Triticum aestivum, Zea mays and thetwo helophytes and the volume of extractable gas per volumeof root. This ratio corresponded to the extent of aerenchymaformation. Hence, a certain amount of oxygen may have been adsorbedonto the inner surfaces of the lacunae of the roots. However, the large amount of oxygen in the roots of intact wheatplants suggest that some parts of the root system are unlikelyto suffer from the oxygen shortage imposed by oxygen-deficientexternal conditions such as flooded soil. Triticum aestivum L. cv. Hatri, wheat, helophytes, roots, micro-absorption method, oxygen concentration, hypoxia, intercellular space     

Salicylic Acid Enhances Resistance to Fusarium oxysporum f. sp. phaseoli in Common Beans (Phaseolus vulgaris L.)

Fusarium wilt caused by Fusarium oxysporum f. sp. phaseoli (Fop) is one of the most serious diseases of common bean (Phaseolus vulgaris L.) and is especially prevalent in China. In this study, we demonstrated that exogenous application of 2 mM salicylic acid (SA) by leaf spraying could induce resistance against Fop in common beans. Accumulation of free and conjugated SA in roots was detected by HPLC analysis and compared. After 168 h of daily SA treatment, the free SA level in roots was eight times higher than in control plants. However, the conjugated SA level reached a peak at 72 h of SA treatment, which was nine times higher than in control plants, and then sharply declined at 168 h. The activities of phenylalanine ammonia lyase (PAL, EC 4.3.1.5) and peroxidases (POX, EC 1.11.1.7) in roots were 9.4 and 6.3 times higher than in control plants after 168 h of SA treatment, respectively. H₂O₂ and O₂ ^? levels reached 2.6 and 13.6 times higher, respectively, than in the control plants at 168 h after SA treatment. Host reactions of SA-treated plant roots infected by Fop observed in microscopy included the deposition of electron-dense materials along the secondary walls. However, untreated inoculated plants showed marked cell wall degradation and total cytoplasm disorganization of root cells. These results indicated that SA applied to foliar tissue is capable of enhancing the systemic acquired resistance of common bean roots to infection by Fop.     

Cell cycle kinetics of aerated, hypoxic and re-aerated cells in vitro using flow cytometric determination of cellular DNA and incorporated bromodeoxyuridine

The effects of extreme hypoxia on cell cycle progression were studied by simultaneous determination of DNA and bromodeoxyuridine (BrdU) contents of individual cells. V79-379A cells were pulse-labelled with BrdU (1 microM, 20 min, 37 degrees C) and then incubated for up to 12 hr in BrdU-free medium under either aerated or extremely hypoxic conditions. After the incubation interval (0-12 hr), the cells were trypsinized and fixed in 50% EtOH. Propidium iodide and a fluorescein-labelled monoclonal antibody to BrdU were then used to quantify DNA content and incorporated BrdU, respectively. Measurements in individual cells were made by simultaneous detection of green and red fluorescence upon excitation at 488 nm using flow cytometry. Bivariate analysis revealed progression of BrdU-labelled cells in aerated cultures out of S phase, into G2 and cell division, with halving of mean fluorescence, and back into S phase by approximately 9 hr after the BrdU pulse. Hypoxia immediately arrested cells in all phases of the cell cycle. Both the DNA distribution and the bivariate profile of cells that were fixed from 2 to 12 hr after induction of hypoxia were identical to the 0 hr controls. The percent of cells with green fluorescence in a mid-S phase window remained 100% and the mean fluorescence of these cells remained at control (0 hr) levels. This indicates that, under hypoxic conditions, cells were moving neither into nor out of S phase. Cultures that had been hypoxic for 12 hr exhibited an increasing rate of BrdU uptake with time after re-aeration. Re-aerated cells were able to complete or initiate DNA synthesis, but their rates of progression through the cell cycle were markedly reduced. A large fraction of cells appeared unable to divide up to 12 hr following release from hypoxia.     

Cloning and expression of a hypoxic and nitrogen inducible maize alanine aminotransferase gene

Alanine aminotransferase (ALT, EC 2.6.1.2) is regulated by hypoxia in the roots of several plant species, and by light and nitrogen stress in the leaves of the C₄ plant, broomcorn millet ( Panicum miliaceum ). In order to more fully characterize the regulation of ALT, we isolated a maize alt genomic clone that has high sequence homology to the coding regions of both the barley ( Hordeum vulgare ) and P. miliaceum alt cDNA clones. This is the first plant alt gene isolated to date. The alt gene consists of 15 exons, and has regions of the promoter that are similar to the maize Anaerobic Responsive Element, and to the maize 27‐kDa zein promoter. ALT activity increased 2.1‐fold in roots after 96 h of hypoxic stress, but did not increase significantly in either root or leaf tissue when the plant was subjected to anaerobic conditions. Northern analysis of hypoxic root tissue showed an 18‐fold increase in a single alt mRNA band after 8 h of hypoxic stress, followed by a continual decline in mRNA levels. ALT activity and mRNA levels were also found to increase in root tissue after recovery from nitrogen stress but not after salt, cold or heat stress conditions. These interesting patterns of ALT expression in maize roots as a result of exposure to hypoxia and nitrogen stress are discussed.     

Sucrose synthase activity does not restrict glycolysis in roots of transgenic potato plants under hypoxic conditions

The effect of hypoxia on root development and carbon metabolism was studied using potato (Solanum tuberosum L.) plants as a model system. Hypoxia led to a cessation of root elongation, and finally to the death of meristematic cells. These changes were accompanied by a 4- to 5-fold accumulation of hexoses, suggesting that insufficient carbohydrate supply was not the cause of cell death. In addition, prolonged hypoxia (96 h) resulted in a 50% increase in activity of most glycolytic enzymes studied and the accumulation of glycerate-3-phosphate and phosphoenolpyruvate. This indicates that endproduct utilisation may restrict metabolic flux through glycolysis. As expected, the activities of alcohol dehydrogenase (EC 1.1.1.1) and pyruvate decarboxylase (EC 4.1.1.17) increased during hypoxia. Apart from the enzymes of ethanolic fermentation the activity of sucrose synthase (SuSy; EC 2.4.1.13) was enhanced. To investigate the in-vivo significance of this increase, transgenic plants with reduced SuSy activity were analysed. Compared to untransformed controls, transgenic plants showed a reduced ability to resume growth after re-aeration, emphasising the crucial role of SuSy in the toleration of hypoxia. Surprisingly, analysis of glycolytic intermediates in root extracts from SuSy antisense plants revealed no change as compared to wildtype plants. Therefore, limitation of glycolysis is most likely not responsible for the observed decreased ability for recovery after prolonged oxygen starvation. We assume that the function of SuSy during hypoxia might be to channel excess carbohydrates into cell wall polymers for later consumption rather than fuelling glycolysis. Received: 17 February 1999 / Accepted: 10 June 1999