Site of Oxygen Absorption for Downward Transport in Seedlings of Rice and Other Plants

Our earlier reports have shown that appreciable portions (ranging from 20% to 70%) of the total amount of oxygen absorbed by the aerial part can be transported downwards to roots in water cultured intact seedlings of rice, barnyard grass, wheat, pea, etc. By interrupting the alternative paths of transport, it has been demonstrated that oxygen moves downwards mainly through gaseous diffusion along the intercellularspaces in the cortex. The aim of the present investigation is to ascertain the site of oxygen absorption for downward transport in the aerial part and to show that such a transport does not necessarily involve active participation of the absorbing organ. The results are summarized below: 1. Provided that a small upper portion of the leaf is left exposed in air, flooding of the aerial part of the rice seedling does not reduce the amount of total oxygen absorption to any appreciable extent (Fig. 1). In agreement with field observation, the unflooded tip is capable of furnishing the submerged part with enough oxygen to keep it alive. 2. Nor does the complete or partial removal of leaves by cutting in seedlings of rice and pea affect downward oxygen transport appreciably, provided that the stem segment or a leaf sheath is left exposed in air. 3. The following common notion has been confirmed by actual measurement: The abnormal excessive elongation of the coleoptile in rice seedling germinated under water, which may easily extend itself above the water surface, is an adaptive device to furnish the seedling with the oxygen required for root development. 4. The "floating" roots developed at the later stage in rice culture have been demonstrated to be a possible site of oxygen absorption for downward transport. 5. When a rice seedling is held up side down, with its roots exposed in air and the shoot submerged, downward oxygen transport still takes place, although to a lesser extent than in its normal position.     

Path of Downward Oxygen Transport from Aerial to Subterranean Parts in Intact Seedlings of Rice and Other Plants

There are two opposite opinions as regards the mechanism and the path of downward oxygen transport in rice and other higher plants. Van Raalte (1940), Yamada (1952), and others maintain that an oxygen pressure gradient of decreasing magnitude from the stem base down to the root tip exists in the intercellular air spaces, which are interconnected throughout the cortex, and the oxygen transported therein is in free molecular form and moves about by diffusion along its own gradient. Recent diffusion experiments in plants by Barber (1962), employing radioactive O15 as indicator, gave direct confirmation of this hypothesis. The opposite view is held by Brown (1947), Soldatenkov (1963) and others. They consider that the passive diffusion of oxygen along its own gradient is inadequate to account for the actual amount transported downwards. The fact that downward oxygen transport in roots comes almost to a standstill, once the aerial part is removed while the cut end of the short stump is still left in air, casts doubts as to the validity of the diffusion hypothesis; and is in favour of their claim that in addition to, or in placement of, diffusion, active participation of living tissues in shoot is necessary to drive enough oxygen to meet the demand of roots. The oxygen in active transport is no longer in free gaseous state but is in dissolved or combined form (as in peroxides) and moves presumably along the vascular bundles in a way which is hitherto unrevealed but is apparently dependent upon the physiological activity of the conducting tissue. In our previous report (Lou et al 1964), we gave data based on quantitative measurement of the amount of oxygen transported downwards from aerial to submerged parts in intact seedlings with the respiratory hydrometer specially designed for the purpose. In seedlings of marshy plants (e.g. rice), it amounts to about 50% of the total oxygen absorbed by the aerial part; in water cultured seedlings of ordinary land plants (e.g. pea), 20%–30%. By deliberately blocking the alternative paths of oxygen transport in seedlings, one at a time, and measuring the downward oxygen transport accordingly in the same way as before, we should be able to decide which one of the two paths is mainly responsible for the transport. The blocking can be conveniently carried out at the upper end of the radical in a pea (or broadbean) seedling by surgical treatment (see Fig.1); either by ringing off the peripheral cortex where most of the air spaces reside; or by piercing through the central cylinder, within which the vascular bundles are confined. The treated radical is then submerged in water and ready for measurement. Without recourse to surgical treatment and mechanical injury, the air space in the cortex can also be blocked by displacing its air content with water through vacuum infiltration. The present investigation has shown that when the intercellular spaces in the cortex of the radical are blocked either by ringing or by infiltration, the aerial part of the treated seedling absorbs much less oxygen than the control as though its radical were completely severed (Table 2); or, in other words, the downward oxygen transport is effectively stopped by such a means. On the other hand, interruption of vascular bundles in the central cylinder only reduces the amount of oxygen in transport to less than one half, which can be accounted for by the combined effect of the reduced root activity due to shortage of food supply and the unavoidable partial disruption of the peripheral cortex. Besides taking actual measurement, downward oxygen transport in intact pea (or broadbean) seedlings can also be detected by simply noticing the growth rate of its radical. As is shown in this investigation, the radical ceases growing in still water, if the oxygen supply from its aerial part is interrupted. As a result of oxygen deficiency, the radical tip deteriorates in a few days. These effects can be easily realized by ringing off the cortex or by infiltrating its air spaces with water. That the peripheral ringing of the radical does no harm to its growth process is revealed by the fact that if air is bubbled through the water culture steadily, normal growth ensues. The above results leave no doubt that in seedlings of rice, pea, and broadbean, downward oxygen transport mainly takes place in the intercellular spaces in the radical cortex, and seems to have no concern with the activities of vascular bundle and cortex. Although there are evidence that rice roots may actively secrete oxygen in the form of peroxides to its immediate neighborhood (the rhizosphere), the actual amount and the distance traversed in such an active transport however, is very much limited and is insignificant as compared with that taking place in the intercellular spaces.     

Relationship Between Downward Oxygen Transport and Intercellular Spaces in Root Cortex in Water Cultured and Moist Cultured Seedlings

In the present investigation, seedlings of rice, pea, sorghum, and maize are raised both in water culture and moist culture. The former culture is to provide the roots with an oxygen deficient condition; while the latter, a direct access to air. The amount of oxygen transported downwards in the seedlings varies not only with the nature of plants but also with the way how they are raised: More oxygen is transported downwards in marsh plant (rice) than in land plants (pea, sorghum, maize); and, in case the same plant is concerned, more in water cultured seedlings than in moist cultured ones. Downward oxygen transport in the various seedlings is intimately correlated with the relative volume of the intercellular spaces in the root: the more the downward transport, the larger the air spaces in the cortex. The fractional volume of the intercellular spaces in a small plant segment can be conveniently estimated by determining the specific gravities of the fresh turgescent segment before and after it is filled with water by vaccum infiltration. The difference between the two consecutive measurements in specific gravity times 100 gives directly the percentage of the volume occupied by air spaces. When large root segments are used, the relative volume can also be determined by weighing before and after vaccum infiltration. To test whether oxygen diffusion in the intercellular spaces of roots could actually account for its downward transport, a model is built of capillary tubings with dimensions and oxygen pressure gradients similar to those found in roots. The amount of oxygen diffused in such a model is measured with a respiratory hydrometer (see Fig. 1) and fits closely that measured in roots. By comparing the amount of oxygen transported downwards in a seedling with that consumed by its excised roots in air, it can be shown that, in case of rice, it could meet (and at times may even exceed) 100% of that consumed by roots in water cultured seedlings, but is less in moist cultured ones. In land plants (pea, sorghum, and maize), however, the downward oxygen supply is far below its requirement, being 80%–100% in water cultured seedlings and 30%–60% in moist cultured ones. The above results, together with those obtained in previous communications, support the view that adaptation of a plant to flooded condition is primarily achieved by its capacity of providing adequate intercellular spaces for downward oxygen diffusion. The capacity depends not only upon the phylogeny of the plant concerned but also upon its ontogenic development.     

Rice seedlings release momilactone B into the environment

Since the growth inhibitor momilactone B was found recently in root exudates of rice (Oryza sativa L.), 3-day-old rice seedlings were transferred to hydroponic culture and the level of momilactone B released into the environment from the seedlings was measured. At day 15 after transfer, the level of momilactone B in the culture solution was 1.8 nmol per seedling compared with endogenous levels of 0.32 and 0.63 nmol per root and shoot, respectively, suggesting that rice seedlings actively releases momilactone B into the culture solution. This release must occur from the roots because only rice roots were immersed in the culture solution. Momilactone B inhibited the growth of ten cress (Lepidium sativum L.) seedlings at concentrations greater than 3 microM. Ten rice seedlings were incubated with ten cress seeds in a Petri dish containing 1 ml of medium, the medium contained 18 nmol of momilactone B, which came to 18 microM. This level of momilactone B was enough to reveal growth inhibition of the cress seedlings. Release level of momilactone B and its effectiveness as a growth inhibitor suggest that it may play an important role in rice allelopathy.     

Effects of Capsaicin on Plant Growth

Rice (Oryza sativa L.) seedlings inhibited the growth of hypocotyls and roots of cress (Lepidium sativum L.) seedlings when both seedlings were grown together. Two growth inhibiting substances were found in the culture solution in which rice seedlings were hydroponically grown for 14 d. One growth inhibitor was further purified. This suggests that the rice seedlings may produce growth inhibiting substances, acting as allelochemicals to other plants, and release them from their roots into the environment.     

Barbiturate-induced Disorientation of Root and Shoot Geotropism in Germinating Cruciferous Seedlings

Cruciferous seedlings germinated on glass-distilled water andon barbital manifested normal root and shoot geotropisms whereastwisted roots and shoots were evident in seedlings germinatedon amobarbital and secobarbital, the roots often being completelysuspended in the air. The marked difference in germination anddevelopment behaviour of cress and white mustard seedlings germinatedon barbital compared with that on amobarbital and secobarbitalindicate that the Meyer-Overton principle of drug potency andlipid solubility was operative in both species of germinatingseedlings. Lepidium sativum L., Sinapsis alba L., seed germination, geotropism, barbiturates     

Acidification of Deionized Water by Roots of Intact Rice Seedlings

This investigation was designed to examine whether or not deionizedwater could be acidified by roots of intact rice seedlings.Roots of intact rice seedlings caused significant acidificationof the deionized water in which they were immersed and thisacidification could be repeated after replacement of acidifiedwater with fresh deionized water. The addition of K⁺, Na⁺, andMg²⁺ to the deionized water significantly increased the rateand extent of acidification. However, no such increase was foundwhen Ca²⁺ was present in the water. The inhibition of acidificationby vanadate and its promotion by fusicoccin indicated that theacidification of water by roots of intact rice seedlings originatedfrom an ATP-driven proton pump located in the plasmalemma. Ferricyanide was effectively reduced by the roots of intactrice seedlings. This reduction was associated with the acidificationof the bathing solution. 8-Hydroxyquinoline and p-nitrophenyl-acetateinhibited both the reduction of ferricyanide and ferricyanide-inducedacidification. Vanadate, although it slightly inhibited thereduction of ferricyanide, did not inhibit the ferricyanide-stimulateddecrease in pH. It seems that the involvement of redox activityassociated with the plasmalemma in the acidification of deionizedwater cannot be excluded. (Received August 30, 1989; Accepted April 5, 1990)     

Assessment of Allelopathic Potential of Root Exudate of Rice Seedlings

To determine the allelopathic potential of root exudate from early developmental stage of rice (Oryza sativa L), 6-d-old seedlings of eight cultivars were grown with 3-d-old alfalfa (Medicago sativa L.), cress (Lepidium sativum L.) or lettuce (Lactuca sativa L.) seedlings in Petri dishes under controlled condition. All rice cultivars (cv. Norin 8, Kamenoo, Nipponbare, Kinuhikari, Koshihikari, Sasanishiki, Yukihikari and Hinohikari) inhibited growth of roots, shoots and fresh mass of alfalfa, cress and lettuce seedlings. Effectiveness of cv. Koshihikari was the greatest and more than 60% inhibition was recorded in all bioassays, followed by that of cv. Norin 8 of which effectiveness was more than 40%.     

Respiratory Properties of Mitochondria from Rice Seedlings Germinated under Water and Their Changes during Air Adaptation

Respiratory activities were compared among rice seedlings germinated in air for 6 days (aerobic seedlings), those germinated under water for 5 days (submerged seedlings), and those grown in air for 1 day after 5 days' submerged germination (air-adapted seedlings). The respiratory activity of the submerged seedlings increased rapidly on transfer to air and reached a plateau at 16 hours in air. Respiration of the submerged seedlings was as sensitive to cyanide as those of aerobic and air-adapted seedlings. 2,4-Dinitrophenol had no effect on the respiration of the submerged seedlings, but stimulated those of the other two types of seedlings. Mitochondria from three types of seedlings did not differ in the ADP/O ratio and the respiratory control ratio (RCR) when succinate was oxidized. However, mitochondria from submerged seedlings (submerged mitochondria) showed poor RCR of about unity when malate was oxidized. Both the rate of succinate oxidation and succinate dehydrogenase activity were low in submerged mitochondria, but increased during air adaptation. Although submerged mitochondria oxidized malate very slowly, this activity increased after exposure to air without any increase in malate dehydrogenase activity. When NAD⁺ was added to submerged mitochondria, oxidation of malate was restored to the level of the aerobic controls. Addition of NAD⁺ enhanced the state 3 rate in submerged mitochondria, and RCR recovered to nearly the same value as that of the aerobic controls. Similar effects of NAD⁺ on 2-oxoglutarate oxidation were observed. All these defects in submerged mitochondria were repaired during air adaptation. These results suggest that NAD⁺-linked substrate oxidation was low in submerged mitochondria because of NAD⁺ deficiency, and that the oxidation increased with an increasing level of NAD⁺ during air adaptation.     

Isolation and identification of a potent allelopathic substance in rice root exudates

A search for growth inhibitors in rice root exudates was undertaken in order to clarify the allelopathic system in rice ( Oryza sativa L.). Rice seedlings inhibited the growth of cress ( Lepidium sativum L.) and lettuce ( Lactuca sativa L.) seedlings when the cress and lettuce were grown with rice seedlings. The putative compound causing the inhibitory effect of rice seedlings was isolated from their culture solution, and the chemical structure of the inhibitor was determined by spectral data as momilactone B. Momilactone B inhibited the growth of cress and lettuce seedlings at concentrations greater than 3 and 30 µ M , respectively. The concentration of momilactone B was 3.4 and 1.1 nmol per seedling in the culture solutions of husked and non-husked rice seedlings, respectively. These results suggest that rice seedlings may release momilactone B into the environment and the stress caused by the husk-treatment may increase the amount of momilactone B released. Thus, momilactone B may play an important role in rice allelopathy.     

The Geotropic Curvature in Roots of Norway Spruce (Picea abies) Containing Anthocyanins

Seedlings of Norway spruce (Picea abies L.) have been found to synthesize anthocyanins in the root tips as well as in the hypocotyls upon irradiation with white light when kept at 4°C for 6–8 days. In addition, it has also been found that the elongation and the geotropic curvature of spruce roots are dependent on the light conditions. The course of the geotropic curvature in spruce roots containing anthocyanins has been followed during a period of 5 h, in which the seedlings were geotropically stimulated continuously in the horizontal position. When the stimulation was performed in white light and in darkness at 21°C, significantly larger curvatures were observed in the roots pretreated at 4°C in darkness than in the roots containing anthocyanins. The specific curvature (curvature in degrees per mm elongation), however, was approximately the same in both types of roots stimulated in white light. This was due to a retarded elongation of the roots pretreated with light at 4°C and containing anthocyanins. A smaller difference in elongation rate between roots with and without anthocyanins was observed in the dark than in the light, but even in the dark the anthocyanin-containing roots grew more slowly than roots without anthocyanins. In order to find out if it is the anthocyanin content or the illumination which affects the elongation and geotropic curvature in the roots, a series of similar experiments was performed using cress seedlings grown at 4°C in light or darkness. Roots of cress seedlings cultivated under conditions which would induce anthocyanin formation in spruce roots exhibited the highest geotropic responses both in light and darkness as compared to cress seedlings grown at 4°C in darkness. As in the case of spruce roots an increase in elongation was observed in cress roots illuminated during the geotropic stimulation. These similarities in the behaviour made it relevant to compare the development of the geotropic curvature in cress and spruce roots.     

The resistance to anoxia and the mitochondrial fine structure of rice seedlings

Summary Under anoxia, rice coleoptiles have a remarkable capacity to grow and to preserve undamaged mitochondrial structure and functions. The transfer of aerobically grown intact seedlings to anaerobic conditions resulted in the appearance of unusual mitochondria in coleoptiles as well as in leaves and roots. These mitochondria become filled with stacks of extended cristae, but, obviously, are not affected structurally (only in the root cortex the cells are damaged after a longer period without oxygen). On the contrary, the mitochondria and other organelles of excised coleoptiles, roots and leaves disintegrate after a relatively short exposure to an oxygen-free environment. The degeneration can be avoided if the excised organs are supplied with glucose. Then the mitochondrial fine structure resembles that of intact plants kept under anaerobic conditions.The observations suggest that the capacity of rice coleoptiles to grow under anoxia and to preserve undamaged mitochondria and other organelles is not caused by the resistance of the cell organelles to oxygen deficiency, but rather by the ability of the seedling to transport organic compounds easily, even under the exclusion of oxygen, from the grain to the coleoptile where they can be utilized by glycolysis.     

Growth of submerged mycelia of Aspergillus kawachii in solid-state culture

Submerged mycelial growth of Aspergillus kawachii IFO4308 in solid-state culture (SSC) was studied. From the result of Northern blot analysis, acid-stable α-amylase was found to be produced mainly by the submerged mycelia rather than the aerial mycelia. The submerged mycelia showed better growth in SSC using rice as the solid substrate (koji) than in agar plate culture in spite of low concentrations of dissolved oxygen in koji. Good growth in SSC suggested the existence of an effective oxygen transfer mechanism in koji which governed the mycelial growth. When koji was submerged in water, small bubbles were generated. This phenomenon indicated the formation of vacant spaces in koji during SSC. The submerged mycelia showed better growth in the koji having a larger number of vacant spaces. Considering these facts it was concluded that the vacant spaces participate in effecting an oxygen transfer mechanism in koji as air vents because the diffusivity of oxygen in an air is larger than in koji itself.     

Committee for the study of the Scottish Flora

Summary

Three different oxygen-containing germination environments demonstrate the profound influence exerted by environmental oxygen (0₂) on growth and plastogenesis in coleoptiles of light-germinated rice seedlings. Coleoptile greening is extensive in low numbers of seedlings germinated in a sealed, initially air-saturated, static water environment and in large numbers of seedlings germinated under unagitated water underambient gaseous exchange conditions. In seedlings germinated in air (?21% 0₂), coleoptile greening is sparse and extension growth is much reduced compared with coleoptile extension growth of the submerged seedlings. Coleoptile greening and shoot and root growth are completely inhibited under hypoxia resulting from large numbers of germinating seedlings competing for the limited 0₂ supply in the sealed, initially air-saturated, static water environment. Coleoptile extension growth is highest under hypoxia and lowest under ?21% 0₂. The observations presented here demonstrate that 0₂ stress and non- stress conditions serve as environmental signals which influence growth behaviour and plastogenesis in coleoptiles of light-germinated rice seedlings.     

Ethylene production by cress roots and excised cress root segments and its inhibition by 3,5-diiodo-4-hydroxybenzoic acid

Summary 3,5-Diiodo-4-hydroxybenzoic acid (DIHB) has been shown to exert an inhibitory effect on the formation of ethylene by the roots of intact cress Lepidium sativum seedlings in light, and by excised cress root segments. Adding IAA to the culture solution greatly promoted ethylene production, which was suppressed by DIHB. The findings together with results obtained with dinitrophenol (DNP), L-methionine and L-ethionine and also the horseradish peroxidase/methional system of Yang are discussed.The results indicate that the effect of DIHB in promoting the root growth of cress seedlings in nutrient solution in the light operates, at least in part, by suppressing the formation of the root growth inhibitor ethylene.Abbreviation GLC gas-liquid chromatography - dIHB 3,5-diiodo-4-hydroxybenzoic acid This study forms part of research to be submitted for PhD degree and supported by a grant from Consejo Nacional de Ciencia y Tecnología (México).     

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     

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.     

Suppression of gravitropic response of primary roots by submergence

Primary roots of six plant species were placed horizontally either in humid air or under water, and their growth and gravitropic responses were examined. In air, all the roots showed a normal gravitropic curvature. Under water without aeration, roots of rice (Oryza sativa L.), oat (Avena sativa L.), azuki bean (Vigna angularis Ohwi et Ohashi), and cress (Lepidium sativum L.) curved downward at almost same rate as in air, whereas the curvature of roots of maize (Zea mays L.) and pea (Pisum sativum L.) was strongly suppressed. Submergence did not cause a decrease in growth rate of these roots. When roots of maize and pea were placed horizontally under water without aeration and then rotated in three dimensions on a clinostat in air, they showed a significant curvature, suggesting that the step suppressed by submergence is not graviperception but the subsequent signal transmission or differential growth process. Constant bubbling of air through the water partly restored the gravitropic curvature of maize roots and completely restored that of pea roots. The curvature of pea roots was also partly restored by the addition of an inhibitor of ethylene biosynthesis, aminooxyacetic acid. In air, ethylene suppressed the gravitropic curvature of roots of maize and pea. Furthermore, the level of ethylene in the intercellular space of the roots was increased by submergence. These results suggest that the accumulation of ethylene in the tissue is at least partly involved in suppression of transmission of the gravity signal or of differential growth in maize and pea roots under conditions of submergence.Abbreviations AOA aminooxyacetic acid - 3-D three-dimensional Dedicated to Professor Andreas Sievers on the occasion of his retirementWe thank Professor H. Suge and Drs. H. Takahashi and H. Kataoka, Tohoku University and Dr. T. Suzuki, Yamagata University, for helpful suggestions. The present study was supported in part by a Grant for Basic Research in Space Station Utilization from the Institute of Space and Astronautical Science, Japan.     

Antioxidative enzymes in seedlings of Nelumbo nucifera germinated under water

Dry seeds of anoxia-tolerant lotus ( Nelumbo nucifera Gaertn= Nelumbium speciosum Willd.) have green shoots with plastids containing chlorophyll, so photosynthesis starts even in seedlings germinated under water, namely hypoxia. Here we investigated antioxidative enzyme changes in N. nucifera seedlings responding to oxygen deficiency. The activity of superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (DHAR; EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) were lower in seedlings germinated under water (submerged condition) in darkness (SD seedlings) than those found in seedlings germinated in air and darkness (AD seedlings). In contrast, ascorbate peroxidase (APX; EC 1.11.1.11) activity was higher in SD seedlings and the activity of catalase (EC 1.11.1.6) and monodehydroascorbate reductase (MDAR; EC 1.6.5.4) in SD seedlings was nearly the same as in AD seedlings. When SD seedlings were exposed to air, the activity of SOD, DHAR and GR increased, while the activity of catalase and MDAR decreased. Seven electrophoretically distinct SOD isozymes were detectable in N. nucifera . The levels of plastidic Cu,Zn-SODs and Fe-SOD in SD seedlings were comparable with those found in AD seedlings, which may reflect the maintenance of green plastids in SD seedlings as well as in AD seedlings. These results were substantially different from those previously found in rice seedlings germinated under water.