The influence of externally supplied sucrose on phloem transport in the maize leaf strip

Sucrose (2,5–1000 mmol l^–1), labeled with [¹⁴C]sucrose, was taken up by the xylem when supplied to one end of a 30-cm-long leaf strip of Zea mays L. cv. Prior. The sugar was loaded into the phloem and transported to the opposite end, which was immersed in diluted Hoagland's nutrient solution. When the Hoagland's solution at the opposite end was replaced by unlabeled sucrose solution of the same molarity as the labeled one, the two solutions met near the middle of the leaf strip, as indicated by radioautographs. In the dark, translocation of ¹⁴C-labeled assimilates was always directed away from the site of sucrose application, its distance depending on sugar concentration and translocation time. When sucrose was applied to both ends of the leaf strip, translocation of ¹⁴C-labeled assimilates was directed toward the lower sugar concentration. In the light, transport of ¹⁴-C-labeled assimilates can be directed (1) toward the morphological base of the leaf strip only (light effect), (2) toward the base and away from the site of sucrose application (light and sucrose effect), or (3) away from the site of sucrose application independent of the (basipetal or acropetal) direction (sucrose effect). The strength of a sink, represented by the darkened half of a leaf strip, can be reduced by applying sucrose (at least 25 mmol l^–1) to the darkened end of the leaf strip. However, equimolar sucrose solutions applied to both ends do not affect the strength of the dark sink. Only above 75 mmol l^–1 sucrose was the sink effect of the darnened part of the leaf strip reduced. Presumably, increasing the sucrose concentration replenishes the leaf tissue more rapidly, and photosynthates from the illuminated part of the leaf strip are imported to a lesser extent by the dark sink.Supported by Deutsche Forschungsgemeinschaft     

Effect of Temperature, Photoperiod and Several Growth Substances on the Cold Hardiness of Chrysanthemum morifolium Rhizomes

The effect of 14 combinations of photoperiod, soil and air temperature, and growth substance applications on the cold hardiness of Chrysanthemum morifolium‘Astrid’ rhizomes was evaluated. Both triphenyl tetrazolium chloride and regrowth tests were used to determine the viability of the cold-stressed rhizome tissues. The rhizomes exhibited different degrees of cold hardiness under these environmental conditions. A combination of short photoperiod and low air and soil temperatures induced maximum cold hardiness. Low soil temperature accompanied by long photoperiods and warm aerial temperatures did not induce rhizome hardening, while some hardening in cool soils was evident under either short photoperiods or low aerial temperatures. Warm soils reduced rhizome hardening under the normally inductive short photoperiod-cool aerial conditions. Since the induction of rhizome hardening was dependent on the induction of the aerial organs, the involvement of translocatable hardiness promoters is indicated. Foliar applications of low levels of gibberllic acid (GA₃) or abscisic acid only slightly influenced rhizome hardiness.     

Basipetally polarised transport of [3H]gibberellin A1 and [14C]gibberellin A3, and acropetal polarity of [14C]indole-3-acetic acid transport,in stelar tissues of Phaseolus coccineus roots

Summary Movement of both [³H]GA₁ and [¹⁴C]GA₃ through root segments from P. coccineus seedlings was basipetally polarised. The basipetal/acropetal ratio of radioactivity from [³H]GA₁ in agar receiver blocks was 9.2 for apical, elongating segments, and 4.0 for more basal, non-elongating segments. Polarity of gibberellin transport was restricted to the stele, and absent from cortical tissues. Transport of [¹⁴C]IAA through root segments to agar receivers was preferentially acropetal, particularly so in the stele. Despite the existence of basipetal polarity of gibberellin transport in the root, [³H]GA₁ injected into cotyledons moved into and acropetally along the seedling root.     

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.     

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.     

Action of Abscisic Acid on Auxin Transport and its Relation to Phototropism

The action of abscisic acid on the kinetics of auxin transport through Zea mays L. (cv. Goudster) coleoptiles has been investigated. Abscisic acid applied simultaneously with indoleacetic acid-2-¹⁴C in the donor block reduced the transport intensity without materially affecting the basipetal velocity or the uptake. No effect on acropetal transport was observed. The data have been used to discuss the similarities in effects of abscisic acid and visible radiation and a hypothesis is proposed to explain the phenomena of phototropism.     

Phloem Unloading in Developing Leaves of Sugar Beet : I. Evidence for Pathway through the Symplast

Physiological and transport data are presented in support of a symplastic pathway of phloem unloading in importing leaves of Beta vulgaris L. (`Klein E multigerm'). The sulfhydryl reagent p-chloromercuribenzene sulfonic acid (PCMBS) at concentration of 10 millimolar inhibited uptake of exogenous [¹⁴C]sucrose by sink leaf tissue over sucrose concentrations of 0.1 to 5.0 millimolar. Inhibited uptake was 24% of controls. The same PCMBS treatment did not affect import of ¹⁴C-label into sink leaves during steady state labeling of a source leaf with ¹⁴CO₂. Lack of inhibition of import implies that sucrose did not pass through the free space during unloading. A passively transported xenobiotic sugar, l-[¹⁴C]glucose, imported by a sink leaf through the phloem, was evenly distributed throughout the leaf as seen by whole-leaf autoradiography. In contrast, l-[¹⁴C]glucose supplied to the apoplast through the cut petiole or into a vein of a sink leaf collected mainly in the vicinity of the major veins with little entering the mesophyll. These patterns are best explained by transport through the symplast from phloem to mesophyll.     

Decarboxylation and transport of auxin in segments of sunflower and cabbage roots

Summary The movement of ¹⁴C from indole-3-acetic acid (IAA) ¹⁴C has been examined in 5 mm root segments of dark-grown seedlings of Helianthus annuus and Brassica oleracea. Contaminants from distilled water, phosphate buffer and the razor-blade cutter increase the decarboxylation of IAA-¹⁴C, and cutting of root segments results in an activation of IAA-destroying enzymes at the cut surfaces. When these sources of errors were eliminated the following was shown: a) Both in sunflower and cabbage there is a slight acropetal flux of ¹⁴C through the root segments into the agar receiver blocks. The amount of ¹⁴C found in the receiver blocks increases with the lenght of the transport period. b) When the root segments, after the transport period, are cut in two equal parts and these assayed separately, the amounts of ¹⁴C in the two parts indicate a greater acropetal than basipetal transport. c) The total radioactivity of the receiver blocks is in part due to IAA-¹⁴C and in part to ¹⁴CO₂, the latter being a result of enzymatic destruction of auxin. d) Addition of ferulic acid, an inhibitor of IAA oxidases, to the receiver blocks markedly inhibits the decarboxylation of IAA-¹⁴C and thus increases the amount transported. This effect is more pronounced after a 20 hr than after a 6 hr transport period.     

Regulation of phosphoenolpyruvate carboxylase activity in maize leaves

The aim of this work was to investigate how light regulates the activity of phosphoenolpyruvate carboxylase in vivo in C₄ plants. The properties of phosphoenolpyruvate carboxylase were investigated in extracts which were rapidly prepared (in less than 30 seconds) from darkened and illuminated leaves of Zea mays. Illumination resulted in a significant decrease in the S_0.5(phosphoenolpyruvate) but there was no change in V_max. The form of the enzyme from illuminated leaves was less sensitive to malate inhibition than was the form from darkened leaves. At low concentrations of phosphoenolpyruvate, the activity of the enzyme was strongly stimulated by glucose-6-phosphate, fructose-6-phosphate, triose-phosphate, alanine, serine, and glycine and was inhibited by organic acids. The enzyme was assayed in mixtures of metabolites at concentrations believed to be present in the mesophyll cytosol in the light and in the dark. It displayed low activity in a simulated `dark' cytosol and high activity in a simulated `light' cytosol, but activities were different for the enzyme from darkened compared to illuminated leaves.     

THE MOVEMENT OF IAA-C14 IN THE HYPOCOTYL OF PHASEOLUS VULGARIS

The amount of IAA-C¹⁴ transported basipetally through excised hypocotyl sections was strongly affected by the pH of the donor blocks, less so by the pH of the receivers. The effect of donor pH was mostly on uptake. The small amount of acropetal movement was not noticeably affected by pH. Sucrose added to the donor resulted in increased basipetal transport. The time-course of C¹⁴ movement into basal receivers followed a linear course from 1.5 to 3 hr as expected, but there was no net loss from the donors until after 30-45 min. The usual type of velocity calculation, which assumes uptake starting from zero time, would therefore be lower than the true value. Basipetal transport through segments cut from various positions in the hypocotyl and from seedlings of various ages was maximal in 6-8-day-old hypocotyl segments cut 25-30 mm below the cotyledons. Acropetal movement was minimal at all positions of all ages tested.     

Auxin-Promoted Transport of Metabolites in Stems of Phaseolus vulgaris L.: EFFECTS REMOTE FROM THE SITE OF HORMONE APPLICATION

Phloem transport in stems of Phaseolus vulgaris was found tobe sensitive to treatment with the auxin transport inhibitor,2,3,5-triidobenzoic acid (TIBA). The response was dependenton the concentration of TIBA applied. A concentration of TIBA(0?5% in lanolin) which did not interfere with normal phloemtransport proved inhibitory to both basipetal transport of IAAand the acropetal component of IAA-promoted metabolite transport.In contrast, both acropetal IAA transport and basipetal IAA-promotedmetabolite transport were unaffected by TIBA treatment. Theinhibitory effect of TIBA on acropetal IAA-promoted transportwas overcome by providing IAA below the point of TIBA application.Both acropetal and basipetal IAA-promoted transport in stemsegments were unaccompanied by any corresponding changes inthe accumulation of [¹⁴C]sucrose by the segments.     

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.

     

Response of Phloem Loading and Export to Rapid Changes in Sink Demand

Sink demand was abruptly changed for an illuminated sugar beet source leaf by shading the six to ten other source leaves. Export of recently assimilated, labeled material underwent a transient increase and then returned to a steady rate approximately equal to the pretreatment rate. Uncovering the darkened leaves caused a transient decrease in export of ¹⁴C; following recovery there was a gradual decline. It remains to be established whether export of unlabeled reserves occurs in response to increased sink demand. The possibility that phloem loading increases in response to decreased sieve tube turgor was tested. Phloem loading of exogenous ¹⁴C-sucrose increased when turgor in leaf cells was decreased by floating leaf discs on solutions with up to 1 M mannitol osmoticum. However, the increase appeared to be the result of plasmolysis of mesophyll cells possibly resulting from easier access to minor veins via the free space. Phloem loading in leaf discs continued undiminished even though sieve tube-companion cell sucrose concentration exceeded a calculated value of 1 M. Regulation of export to meet sink demand by a direct response of phloem loading to a turgor or concentration set point does not appear to occur. Phloem loading may be promoted by the influx of water which drives mass flow, increasing phloem loading in response to increased velocity of transport.     

Response of Respiration of Tobacco Leaves in Light and Darkness and the CO(2) Compensation Concentration to Prior Illumination and Oxygen

The course of respiration in control leaves of tobacco (Nicotiana tabacum L.) that were illuminated 4 to 5 hours and then darkened 0.25 to 10 hours and in tobacco leaves starved of carbohydrate by 14 hours or more of darkness was measured as CO₂ efflux in light and darkness into CO₂-free atmospheres containing 0.04, 2.23, 21, 40, and 100% O₂.     

Sugar Transport in Immature Internodal Tissue of Sugarcane: II. Mechanism of Sucrose Transport

The mechanism by which sucrose is transported into the inner spaces of immature internodal parenchyma tissue of sugarcane (Saccharum officinarum L. var. H 49-5) was studied in short term experiments (15 to 300 seconds). Transport of sucrose, glucose, and fructose was each characterized by a V_max of 1.3 μmoles/gram fresh weight·2 hours, and each of these three sugars mutually and competitively inhibited transport of the other two. When ¹⁴C-glucose was supplied exogenously, ¹⁴C-glucose 6-phosphate and ¹⁴C-glucose were the first labeled compounds to appear in the tissue; no ¹⁴C-sucrose was detected until after 60-second incubation. After 15-second incubation in ¹⁴C-sucrose, all intracellular radioactivity was in glucose, fructose, glucose 6-phosphate, and fructose 6-phosphate; trace amounts of ¹⁴C-sucrose were found after 30 seconds and after 5 minutes, 71% of the intracellular radioactivity was in sucrose. Although it was possible that sucrose was transported intact into the inner space and then immediately hydrolyzed, it was shown that the rate of hydrolysis under these conditions was too low to account for the rate of hexose accumulation. Pretreatment of the tissue with rabbit anti-invertase antiserum eliminated sucrose transport, but had no effect on glucose transport. Since the antibodies did not penetrate the plasmalemma, it was concluded that sucrose was hydrolyzed by an invertase in the free space prior to transport. The glucose and fructose moieties, or their phosphorylated derivatives, were then transported into the inner space and sucrose was resynthesized. No evidence for the involvement of sucrose phosphate in transport was found in these experiments.     

In Vitro Sugar Transport in Zea mays L. Kernels : I. Characteristics of Sugar Absorption and Metabolism by Developing Maize Endosperm

Short-term transport studies were conducted using excised whole Zea mays kernels incubated in buffered solutions containing radiolabeled sugars. Following incubation, endosperms were removed and rates of net ¹⁴C-sugar uptake were determined. Endogenous sugar gradients of the kernel were estimated by measuring sugar concentrations in cell sap collected from the pedicel and endosperm. A sugar concentration gradient from the pedicel to the endosperm was found. Uptake rates of ¹⁴C-labeled glucose, fructose, and sucrose were linear over the concentration range of 2 to 200 millimolar. At sugar concentrations greater than 50 millimolar, hexose uptake exceeded sucrose uptake. Metabolic inhibitor studies using carbonylcyanide-m-chlorophenylhydrazone, sodium cyanide, and dinitrophenol and estimates of Q₁₀ suggest that the transport of sugars into the developing maize endosperm is a passive process. Sucrose was hydrolyzed to glucose and fructose during uptake and in the endosperm was either reconverted to sucrose or incorporated into insoluble matter. These data suggest that the conversion of sucrose to glucose and fructose may play a role in sugar absorption by endosperm. Our data do not indicate that sugars are absorbed actively. Sugar uptake by the endosperm may be regulated by the capacity for sugar utilization (i.e. starch synthesis).     

Characterization of the active sucrose transport system of immature soybean embryos

Immature soybean embryos were isolated from soybean [Glycine max (L.) Merr.] seeds at various stages of development to study their accumulation of [¹⁴C]sucrose in vitro. Isolated embryos accumulate sucrose at a constant rate over several hours, the label entering large, endogenous pools of sucrose from which starch, protein, and lipid storage products are formed. Accumulation is without extracellular sucrose hydrolysis and occurs predominantly by active transport at physiological sucrose concentrations. A nonsaturable diffusion component, apparently superimposed upon the active saturable component, dominates overall uptake at exogenous concentrations greater than approximately 50 millimolar sucrose. Active transport is sensitive to uncoupling agents and the sulfhydryl-modifying reagent p-chloromecuribenzene sulfonate, is dependent on more than one energy source, and exhibits well-defined requirements for incubation temperature, pH, and oxygen availability. Under optimal incubation conditions of 35°C, saturating illumination (pH 6), and 21% oxygen, the apparent K_m for sucrose is approximately 8 millimolar and V_max is approximately 0.6 micromoles per hour per 100 milligrams fresh weight. Embryos readily accumulate sucrose from dilute exogenous solutions and, when preloaded with large amounts of sucrose, maintain the internal sucrose pool against steep outward gradients. These and other observations indicate that, although perhaps fully saturated in vivo, active sucrose transport is a significant component of photosynthate uptake in developing soybean embryos, enhancing uptake at physiological sucrose concentrations 2- to 5-fold over diffusion alone.     

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.

     

Polar transport of kinetin in tissues of radish

Polar transport of kinetin-8-¹⁴C occurred in segments of petioles, hypocotyls, and roots of radish (Raphanus sativus L.). The polarity was basipetal in petioles and hypocotyls and acropetal in roots. In segments excised from seedlings with fully expanded cotyledons, indole-3-acetic acid was required for polarity to develop. In hypocotyl segments isolated at this stage, basipetal and acropetal movements were equal during the first 12 hours of auxin treatment after which time acropetal movement declined. Pretreatment with auxin eliminated this delay in the appearance of polarity. In hypocotyl segments excised from seedlings with expanding cotyledons, exogenous auxin was unnecessary for polarity. Potassium cyanide abolished polarity at both stages of growth by allowing increased acropetal movement. The rate of accumulation of kinetin in receiver blocks was greater than the in vivo increase in cytokinin content of developing radish roots.     

Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.