Independence of Lateral and Differential Longitudinal Movement of Indoleactic Acid in Geotropically Stimulated Coleoptiles of Zea mays

The possible interdependence of the differential longitudinal, and the lateral movement of indoleacetic acid (IAA) in horizontal Zea coleoptile segments has been examined. The coleoptiles have been opened out into flat pieces of tissue and supplied apically with IAA-1-¹⁴C.     

Maintenance of polar auxin transport in Zea coleoptiles by anaerobic metabolism

Summary The basipetal movement of IAA in 5-mm Zea coleoptile segments is drastically reduced under anaerobic conditions, but it remains greater than acropetal movement which is closely similar in the presence and absence of oxygen. The polarity of IAA movement has thus been confirmed in Zea coleoptile segments which have been deprived of oxygen. This net polar flux is dependent upon anaerobic metabolism since it is abolished in the presence of the metabolic inhibitiors sodium fluoride and iodoacetic acid.Acropetal movement of IAA is unaffected by the presence of sodium fluoride in air or anaerobic conditions. Uptake of IAA from a basal donor is not affected by sodium fluoride in air, but under anaerobic conditions the inhibitor decreased uptake by approximately 13%.Under anaerobic conditions both inhibitors reduce basipetal movement of IAA to the level of acropetal movement, and both decrease the total uptake of IAA from an apical donor by up to 30–45%. Under aerobic conditions sodium fluoride has no marked effect upon either the uptake of IAA from an apical donor or the basipetal movement of IAA by the segments. On the other hand, iodoacetic acid greatly decreased the uptake of IAA by the segments in air, but the same fraction of the total IAA taken up was recovered in the receiving block in the presence and absence of the inhibitor.This research was supported by Grant Number 83/6 to Professor M. B. Wilkins from the U. K. Agricultural Research Council.     

Dependence of Basipetal Polar Transport of Auxin upon Aerobic Metabolism

The movement of IAA-¹⁴C through coleoptile segments of Avena and Zea has been investigated under aerobic and anaerobic conditions. The results are as follows: Zea. Using a 5-mm segment and a 2-hour transport period anaerobic conditions reduced the total uptake of ¹⁴C from an apical donor by 74% and the proportion of the total found in the receiving block by at least 45%. Anaerobic conditions reduced total uptake from a basal donor by 58% but no ¹⁴C reached the apical receiving block in either air or N₂. Uptake from apical and basal donor blocks in N₂ is closely similar.

The presence of ¹⁴C in the basal receiving blocks, and its absence in the apical receiving blocks, in N₂ suggests that even in anaerobic conditions movement of IAA is polarized basipetally, although the movement occurs at only a fraction of the rate found in air.

Anaerobic conditions induced a similar reduction in basipetal movement of IAA in upper and lower 5-mm segments taken from the apical 10 mm of a Zea coleoptile.

Using 10-mm Zea segments no ¹⁴C was recovered in the receiving blocks at the basal end of the segment after 2 and 4 hours in N₂ whereas large amounts were recovered in air.

Avena: Using 5-mm segments and a 2-hour transport period the total uptake of ¹⁴C from an apical donor is reduced by 83%. Movement of ¹⁴C into the basal donor is totally inhibited in N₂. Total uptake of ¹⁴C from a basal donor is reduced by 61% in nitrogen and no ¹⁴C reached the apical receiving blocks regardless of the atmospheric conditions.

A time course for the movement of ¹⁴C into the basal and apical receiving blocks through 5-mm segments showed that in air the amount in the basal receivers increased for 4 hours and then remained approximately uniform. In N₂ no significant ¹⁴C reached the receivers until 6 to 8 hours after the application of donors but even then the amounts were about 12 to 14% of that in aerobic receivers. Movement of ¹⁴C into apical receivers was similar in air and in nitrogen and even after 6 to 8 hours the amount of radioactivity barely reached significant levels.

     

Gravitropic Responses of Partially Decapitated Corn Coleoptiles with and without Applied [C]Indoleacetic Acid

The curvature of corn seedling (Zea mays L. Mo17 × B73) coleoptiles which had been half-decapitated and supplied with [¹⁴C]indoleacetic acid (IAA) (3.2 micromolar, 51 milliCuries per millimole) was determined during a 3-hour period of gravitational stimulation. Curvature of such half-decapitated coleoptiles was found to be similar in rate and extent to that of intact coleoptiles responding to gravity. Gravitational stimulation was accomplished by reorienting seedlings to a horizontal position, either up or down with respect to the removed half of the coleoptile tips.

The first set of experiments involved placing aluminum foil barriers along one of the two cut surfaces to restrict the movement of IAA into tissues. The initiation and extent of curvature of these half-decapitated coleoptiles was dependent upon the orientation of the removed half-tip and the accompanying barrier. The distribution of radioactivity from [¹⁴C] IAA after 3 hours indicated that the specific lateral movement of label was also dependent upon orientation of the removed half-tip of the coleoptile. A specific movement to the lower side of approximately 14% of the total recovered radioactivity was found in coleoptiles in which the [¹⁴C]IAA was supplied across a transverse cut surface. In contrast, specific movement of only 4% was found for application across a longitudinal cut surface.

A second series of experiments was conducted using 1.0 and 3.2 micromolar [¹⁴C]IAA (51 milliCuries per millimole) supplied to half-decapitated coleoptiles without inserted barriers. The 3.2 micromolar concentration adequately replaced the removed coleoptile half-tips in terms of straight growth, but it did not result in as much curvature as shown by coleoptiles of intact seedlings. The 1 micromolar concentration was not adequate to replace the removed half-tip in straight growth, but resulted in gravitropic curvature nearly as great as that produced by the higher concentration.

The data presented here suggest that strong auxin gradients are not produced in response to gravity stimulation based on the recovered radioactivity from [¹⁴C]IAA. However, it is evident that auxin is required for the development of normal gravitropic responses. It is possible, therefore, that an important early role of this movement is not to cause a large stimulation of growth on the lower side but to decrease growth on the upper side of a gravitropically responding coleoptile.

     

Effect of Inversion on Growth and Movement of Indole-3-acetic Acid in Coleoptiles

The effect of a 180° displacement from the normal vertical orientation on longitudinal growth and on the acropetal and basipetal movement of ¹⁴C-IAA was investigated in Avena sativa L. and Zea mays L. coleoptile sections. Inversion inhibits growth in intact sections (apex not removed) and in decapitated sections supplied apically with donor blocks containing auxin. Under aerobic conditions, inversion inhibits basipetal auxin movement and promotes acropetal auxin movement, whereas under anaerobic conditions, it does not influence the movement of auxin in either direction. Inversion retards the basipetal movement of the peak of a 30-minute pulse of auxin in corn.

The inversion-induced inhibition of basipetal auxin movement is not explained by an effect of gravity on production, uptake, destruction, exit from sections, retention in tissue, or purely physical movement of auxin. It is concluded that inversion (a) inhibits basipetal transport, the component of auxin movement that is metabolically dependent, and as a result (b) inhibits growth and (c) promotes acropetal auxin movement.

     

Polyamines in Rice Seedlings under Oxygen-Deficit Stress

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

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.     

Plant Growth and Survival under Strict Anaerobiosis

Anaerobic incubation of seedlings and rhizomes reveals interspecificdifferences in the ability of seedlings and rhizomes of higherplants to survive under prolonged and strict anaerobiosis. Rhizomesof several species were killed by an anaerobic incubation at22 °C for 7 d while others survived and showed normal shootextension on return to aerobic conditions. A third group ofspecies showed healthy and geotropically normal bud and shootextension while their rhizomes were in the oxygen free environment.A detailed comparison of bud and shoot growth rates was madewith Scirpus maritimus under aerobic and anaerobic conditionsover a 14 d period. Under similar conditions four species of grass seedlings weresubjected to anaerobic conditions for 2–8 d at 5°C,15 °C and 25 °C. Seedling mortality was highest at 25°C in all four grasses. Interspecific differences were evident.The wetland species were more tolerant of anoxia than the grassesfrom drier habitats.     

Movement of Indoleacetic Acid in Coleoptiles of Avena sativa L. II. Suspension of Polarity by Total Inhibition of the Basipetal Transport

Acropetal and basipetal movement of indole-3-acetic acid through coleoptiles of Avena sativa L. was studied. Sections 10-mm long were supplied with either apical or basal sources containing C(14) carboxyl-labeled indoleacetic acid (10(-5)m). Anaerobic conditions inhibit metabolically dependent movement (transport) thus reducing basipetal but not acropetal movement. Total inhibition of basipetal transport abolishes the polarity of auxin uptake and movement. The nonpolar movement that remains in anaerobic sections is free diffusion with an average diffusion coefficient of approximately 1 x 10(-4) mm(2) per second. During an 8-hour diffusion, at least the first millimeter of the section comes to equilibrium at approximately the same concentration as the donor.Acropetal movement is probably by diffusion and is accompanied by an aerobic immobilization of indoleacetic acid that increases more than proportionally to concentration. Anaerobic conditions totally prevent this immobilization and reduce acropetal uptake but not the amount of indoleacetic acid moving into the upper parts of the section; there is, therefore, no evidence for acropetal transport.Polarity of auxin movement in aerobic coleoptile sections is achieved by strict basipetal transport of auxin. The basipetal transport may intensify the polarity by recycling auxin that is moving acropetally.     

Der basipetale [2-14C] Abscisinsäuretransport in Wurzeln intakter Bohnenkeimlinge und seine Bedeutung für den Wurzelgeotropismus

Summary One h and 3 h after point source application of [2-¹⁴C] ABA to the upper side of geotropically stimulated root tips of intact seedlings of Phaseolus coccineus L. a weak downward lateral transport of radioactivity was observed. A clear upward lateral translocation, however, occurred when ABA was applied to the lower side of the root tip. Thus a uniform distribution of radioactivity was established in horizontal roots regardless of the site of the application point, while in upright roots the distribution was asymmetric. Six h after ABA injection radioactivity was uniformly distributed in both the vertical and horizontal root tips.

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Abkürzungen ABA Abscisinsäure - dpm disintegrations perminute - FG Frischgewicht - GA Gibberellinsäure     

Glucose metabolism in anaerobic rice seedlings

More than 80% of the radioactivity from [U-¹⁴C]glucose metabolised by anaerobic rice seedlings or by excised roots or coleoptiles was recovered as ethanol plus CO₂; less than 5% was recovered as water-soluble acidic components. Rates of ¹⁴CO₂ formation from [U-¹⁴C]glucose were similar in roots and coleoptiles in both N₂ and air atmospheres. More ¹⁴CO₂ was formed from [U-¹⁴C]glucose than could be accounted for by ethanolic fermentation, and the specific yields of ¹⁴CO₂ from [6-¹⁴C]glucose and [1-¹⁴C]glucose gave unusually high C-6/C-1 ratios (1.7) in the anaerobic coleoptile. The results may indicate that appreciable pentan synthesis occurs in the anaerobic coleoptile.     

The lateral transport of IAA in intact coleoptiles of Avena sativa L. and Zea mays L. during geotropic stimulation

Summary Movement of IAA was studied in excised coleoptile apices and whole seedlings of Zea mays L. and Avena sativa L. during geotropic stimulation. A micropipette technique permitted the application of [5-³H]IAA at predetermined points on the coleoptiles with minimal tissue damage.When [5-³H]IAA was applied to the upper side of a horizontal excised Zea coleoptile, about 60% of the recoverable radioactivity had moved into the lower half after 2 h. In contrast, when application was made to the lower side of a horizontal excised coleoptile, only 4% of the radioactivity migrated to the upper half. There was, thus, a net downward movement of 56%. Similar patterns of distribution were found for radioactivity in both the tissue and the basal receiver blocks. In horizontal shoot tissues of intact Zea seedlings a net downward movement of about 30% of the recoverable radioactivity occurred after 1 h of geotropic stimulation. Comparable experiments with Avena indicated a net downward movement of 6–12% in excised apices of coleoptiles and in the intact shoot. In both Zea and Avena chromatographic analyses of tissue and receiver blocks indicated that the movement of radioactivity reflected that of IAA.In Zea coleoptiles, the lateral migration of radioactivity after 2 h was 3 to 4 times greater in the apical tissues than in the basal tissues. A significant net downward movement of radioactivity was detected after 10 min of geotropic stimulation in the extreme apex of Zea coleoptiles but not in the more basal regions.These experiments show that downward lateral transport of IAA occurs in intact shoots of Zea and Avena seedlings upon geotropic stimulation. Lateral transport of IAA had previously been demonstrated only in sub-apical segments of Zea coleoptiles.     

Indoleacetic Acid biosynthesis in Avena coleoptile tips and excised bean shoots

Avena coleoptiles did not elongate when incubated with tryptophan under sterile conditions. Indole, anthranilic acid, and tryptamine promoted elongation. Under the same conditions, the tissue converted tryptophan-¹⁴C to IAA-¹⁴C. More IAA-¹⁴C was produced from indole-¹⁴C than from tryptophan-¹⁴C; however, the free tryptophan content of the tissue was also greatly increased by the indole treatment. Tryptophan-¹⁴C was readily taken up by the tissue but was mainly incorporated into protein and did not increase the free tryptophan level. When bean shoots were labeled with tryptophan-¹⁴C or indole-¹⁴C, the label incorporation into IAA-¹⁴C was very nearly the same. In this tissue the free tryptophan level in the tryptophan-¹⁴C and indole-¹⁴C treatments was also about equal. These results suggest that failure of exogenously supplied tryptophan to promote the elongation of Avena coleoptiles is a result of its predominant incorporation into protein and consequent unavailability for conversion to IAA.     

Effect of Oxygen on the Light-enhanced Dark Carbon Dioxide Fixation in Chlorella Cells

With Chlorella ellipsoidea cells, the effect of oxygen was investigated on the products of enhanced dark ¹⁴CO₂ fixation immediately following preillumination in the absence of CO₂. When the reaction mixture was made aerobic by bubbling air (CO₂-free) throughout preillumination and the following dark ¹⁴CO₂ fixation periods, the initial fixation product was mainly 3-phosphoglyceric acid. When nitrogen gas had been used instead of air, only about one-half of the total radioactivity in the initial fixation products was in 3-phosphoglyceric acid and the rest in aspartic, phosphoenolpyruvic, and malic acids. The percentage distribution of radioactivity incorporated in these initial products rapidly decreased during the rest of the dark period. Concurrent with the decrease in the initial ¹⁴CO₂ fixation products, some increase was observed in the radioactivities of the sugar phosphates. The maximal radioactivity incorporated in sugar mono- and diphosphates accounted for only 10% of total ¹⁴C, under either the aerobic or anaerobic conditions. Under anaerobic conditions most of the ¹⁴C incorporated was eventually transferred to alanine, whereas the main end products under aerobic conditions were aspartate and glutamate. The pattern of ¹⁴CO₂ fixation products was unaffected by the atmospheric condition during the period of preillumination. The preferential flow of the fixed carbon atom to alanine or aspartate depended on the presence or absence of oxygen during the period of dark CO₂ fixation.     

Decomposition of14C- and15N-labeled organisms in soil under anaerobic conditions

Carbon and nitrogen mineralized from soil under waterlogged conditions may come from the soil microbial biomass pool and potentially could be used for biomass estimations.¹⁴C and¹⁵N labeled cells added to soil were monitored for decomposition under aerobic and anaerobic conditions. Under aerobic conditions 12–42% of the added organism C was mineralized and 1–30% of the N. Under waterlogged conditions 13–33% of the C and 4–13% of the N was mineralized. The mineralized organism C as a percent of the total C evolved was consistent for both aerobic and anaerobic conditions, however the nitrogen showed extreme variations     

Enhancement of Anaerobic Respiration in Root Tips of Zea mays following Low-Oxygen (Hypoxic) Acclimation

Root tips (10-millimeter length) were excised from hypoxically pretreated (HPT, 4% [v/v] oxygen at 25°C for 16 hours) or nonhypoxically pretreated (NHPT, 40% [v/v] oxygen) maize (Zea mays) plants, and their rates of respiration were compared by respirometry under aerobic and anaerobic conditions with exogenous glucose. The respiratory quotient under aerobic conditions with 50 millimolar glucose was approximately 1.0, which is consistent with glucose or other hexose sugars being utilized as the predominant carbon source in glycolysis. Under strictly anaerobic conditions (anoxia), glycolysis was accelerated appreciably in both HPT and NHPT root tips, but the rate of anaerobic respiration quickly declined in NHPT roots. [U-¹⁴C]Glucose supplied under anaerobic conditions was taken up and respired by HPT root tips up to five times more rapidly than by NHPT roots. When anaerobic ethanol production was measured with excised root tips in 50 millimolar glucose, HPT tissues consistently produced ethanol more rapidly than NHPT tissues. These data suggest that a period of low oxygen partial pressure is necessary to permit adequate acclimation of the root tip of maize to subsequent anoxia, resulting in more rapid rates of fermentation and generation of ATP.     

Auxin transport in roots

Summary Light promotes the net acropetal movement of ¹⁴C through 6-mm subapical segments of dark-grown roots of Zea mays supplied at their basal ends with 1 M IAA-1-¹⁴C in agar blocks. This promotion occurs only when the segments are irradiated during the transport period, and both red and blue light appear to be as effective as white light at the radiant flux densities used in this investigation. The promotion is not found if the segments are pretreated with light and then returned to darkness before the trasport of IAA-1-¹⁴C is determined. The very slight basipetal movement of ¹⁴C through the segments supplied with an apical source of IAA-1-¹⁴C is unaffected by light.Only one radioactive substance is found in the apical receiver blocks. This substance has an R_f virtually identical to those of the stock solution of IAA incorporated into the donor block and of unlabelled IAA. The movement of radioactivity into the receiver blocks through, the illuminated segments therefore appears to reflect the movement of IAA. Light thus increases the acropetal movement of IAA through the Zea root segment.The primary roots of Zea mays var. Giant Horse Tooth seedlings grown in total darkness do not exhibit a positive geotropic response. When the seed is orientated with the embryo uppermost the radicle grows out horizontally. On exposure to light, however, the roots bend down. This reaction appears about 3–9 hours after the onset of illumination, and white, red and blue light appear to be equally effective at the flux densities employed in this study. Green light in the spectral band between 510–530 nm did not appear to induce this positive geotropic responsiveness.     

Auxin Transport in Zea mays L. Coleoptiles I. Influence of Gravity on the Transport of Indoleacetic Acid-2-C

¹⁴C-methylene labeled IAA was used to determine the influence of reorientation with respect to gravity on auxin transport in Zea mays L. coleoptile segments. It was observed that inversion of the segments leads to a decrease in the capacity to transport ¹⁴C-IAA basipetally, as well as, in certain instances, the linear velocity of that transport. Segments were also reoriented horizontally, and the transport velocity and capacity of the upper and lower tissue halves compared with vertical halves. There was no significant change in the velocity, but the transport capacity of lower halves was higher than that of the vertical halves, which in turn was higher than the capacity of the horizontal upper halves. It is suggested that the geocurvature of horizontally placed coleoptiles may be caused primarily by the effect of reorientation on auxin transport.     

Auxin transport in roots

Summary The reasons underlying the initial increase and subsequent decrease in the amount of radioactivity in the receiver block at the apical end of a Zea root segment supplied with a basal donor block containing labelled IAA have been investigated.The phenomenon was observed in segments supplied with IAA-1-¹⁴C, IAA-2-¹⁴C and IAA-5-³H. An acropetal polarity in the movement of radioactivity into the receiver blocks was observed using donor blocks containing IAA-5-³H at concentrations as low as 10^-10M.The decrease in the amount of radioactivity in the receiver block begins after 6–8 h of transport at 25° C, and is unaffected by renewal of the donor block every 2 h, or the presence of 2% sucrose in the donor and receiver blocks.The net export of radioactivity into the receiver block at the apical end of the segment virtually ceases after 6–8 h of transport at 25° C, and is not prolonged by the presence of 2% sucrose in the donor and receiver blocks. At 10° C, net export of radioactivity continues for at least the first 50 h of transport, and the amount of radioactivity in a continuously applied receiver block continues to increase over this period.Receiver blocks removed from the apical end of segments after 8 h of transport and placed on planchettes show little or no decrease in the amount of radioactivity they contain as a function of time, in marked contrast to those left in contact with the segment.There is a marked, and metabolically dependent, resorption of radioactivity from the receiver block at the apical end of the segment after about 8 h of transport at 25° C; most of the resorbed radioactivity remains in the apical 2–4 mm of the segment.There is a loss of radioactive CO₂ from segments supplied with a basal donor block containing 10^-6M IAA-1-¹⁴C at 25° C, the emission beginning after 6–8 h of transport. Segments similarly supplied with 10^-6M IAA-2-¹⁴C did not begin to lose radioactive CO₂ until after about 10–12 h of transport.The ability of the segments to transport radioactivity in a polar manner declines with time after they are excised from the root, regardless of whether their cut ends are kept in the intervening period in contact with plain agar blocks, or ones containing unlabelled IAA at 10^-6M. By the 6th h after excision at 25° C no transport of radioactivity through the segments and into the receiver blocks could be detected in either the aropetal or basipetal direction.The decrease in radioactivity in the receiver block after transport periods of 6–8 h at 25° C is therefore due to (1) a cessation of net export of radioactivity into the block, and (2) the onset of a metabolically-dependent, net resorption of radioactivity. At this time substantial amounts of radioactive CO₂ begin to be evolved from segments supplied with IAA-1-¹⁴C, whereas with IAA-2-¹⁴C radioactive CO₂ is not evolved for a further 4–6 h.     

The effect of experimental conditions on the anaerobic metabolism of bivalve molluscs

Experiments were designed to assess the effect of various experimental conditions on the anaerobic metabolism of three bivalve species inhabiting different types of habitat, Mytilus edulis, Cardium edule and Scrobicularia plana. Animals were either immersed in sea water bubbled with air (aerobic) or nitrogen (anaerobic) or exposed to the atmosphere (exposed) or buried in damp sand (buried) and then injected with Na₂¹⁴CO₃ and returned immediately to their experimental environments. The incorporation of ¹⁴CO₂ into their metabolic pools was measured. The results obtained from these experiments are discussed in relation to the ecological distribution of each species. The results showed that M. edulis was able to utilise the succinate pathway under both anaerobic and exposed conditions. The results from the gill of M. edulis under both anaerobic and exposed conditions are different from those cited in the literature but this can be explained in terms of seasonal variations. Whilst C. edule utilise this pathway only under anaerobic conditions, S. plana was observed to use it under all conditions.