Sulfide-Resistant Respiration in Leaves of Elodea canadensis Michx: Comparison with Cyanide-Resistant Respiration

The rate of dark O₂ uptake of Elodea canadensis leaves was titrated with either cyanide or sulfide in the presence and in the absence of 5 millimolar salicylhydroxamic acid (SHAM), an inhibitor of the alternative oxidase. The inhibition of O₂ uptake by SHAM alone was very small (3-6%), suggesting that actual respiration mainly occurred through the cytochrome pathway. O₂ uptake was slightly stimulated by cyanide at concentrations of 50 micromolar or higher, but in the presence of SHAM respiration was strongly suppressed. The effects of sulfide on O₂ uptake were similar to those of cyanide, except that the percent stimulation of O₂ uptake by sulfide alone was somewhat higher than that of cyanide. However, the estimates of the capacity of the alternative pathway were similar with both inhibitors. Another difference is that maximal inhibition of respiration in the presence of SHAM was observed with lower concentrations of sulfide (50 micromolar) than cyanide (250 micromolar). The results suggest that sulfide can be used as a suitable inhibitor of cytochrome c oxidase in studies with intact plant tissues, and that sulfide does not apparently inhibit the alternative oxidase.     

Cyanide-resistant respiration in light- and dark-grown soybean cotyledons

Measurements of respiration were made on intact tissue and mitochondria isolated from soybean (Glycine max [L.] Merr. cv `Corsoy') cotyledons from seedlings of different ages grown in light and darkness. Effects of cyanide (KCN) and salicylhydroxamic acid (SHAM) on O₂ uptake rates were determined. O₂ uptake was faster in light-grown tissue and was inhibited by both KCN and SHAM in all except light-grown tissue older than 9 days. Both inhibitors stimulated O₂ uptake in tissues more than 9 days old. Mitochondria in which O₂ uptake was coupled to ATP synthesis were isolated from all tissues. O₂ uptake by mitochondrial preparations from light- and dark-grown cotyledons was equally sensitive to KCN. Similarly, age did not affect KCN sensitivity, but sensitivity to SHAM declined with age both in the presence and absence of KCN. Estimated capacities of the cytochrome and alternative pathways of the mitochondrial preparations indicated considerably larger cytochrome than alternative pathway capacities. The cytochrome pathway capacities paralleled the state 3 mitochondrial respiration rates, which increased from day 5 to day 7 then declined thereafter. The alternative pathway capacities were not affected by light. The uncoupler, p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), increased the flow of electrons through the cytochrome pathway at the expense of flow through the alternative pathway in isolated mitochondria. However, the combined capacities did not exceed the rate in the presence of FCCP. The results are interpreted to indicate that the stimulation of respiration by KCN and SHAM observed in the 12-day-old green cotyledons and previously observed in older soybean leaves is not explained by characteristics of the mitochondria.     

Effects of KCN and Salicylhydroxamic Acid on the Root Respiration of Pea Seedlings

Polarography, using cylindrical platinum electrodes, proved suitable for measuring changes in the internal apical O₂ concentration of the primary root of pea (Pisum sativum L. cv Meteor) effected by KCN and/or salicylhydroxamic acid (SHAM) in the bathing medium. An electrical rootaeration analog was used to help evaluate some of the results. Concentrations of KCN ≤0.05 millimolar had no significant effect. In response to 0.1 millimolar KCN, the O₂ concentration rose substantially for approximately 2 hours, then declined, and after 10 hours had frequently fallen below the pretreatment level. Such changes suggest an initial inhibition of cytochrome oxidase-mediated O₂ uptake followed by an induction of the alternative, cyanide-resistant respiratory pathway. These treatments proved nonlethal. Changes in O₂ concentration similar to those described for 0.1 millimolar KCN were observed in response to 1 and 10 millimolar KCN but these treatments were lethal and the root apex became soft and often appeared flooded. Roots survived and showed no significant responses when treated with SHAM at concentrations ≤5 millimolar. However, when the alternative pathway had been (apparently) induced by 0.1 millimolar KCN, the addition of 5 millimolar SHAM to the bathing medium caused a substantial and persistent rise in the root apical O₂ concentration, suggesting that this (nonlethal) concentration of SHAM could indeed inhibit O₂ uptake via the cyanide-resistant pathway.

It is concluded that while O₂ uptake normally occurs by the cytochrome pathway in the primary pea root, the alternative, cyanide-resistant pathway can be induced by 0.1 millimolar KCN.

     

Cyanide-resistant respiration in photosynthetic organs of freshwater aquatic plants

The rate and sensitivity to inhibitors (KCN and salicylhydroxamic acid[SHAM]) of respiratory oxygen uptake has been investigated in photosynthetic organs of several freshwater aquatic plant species: six angiosperms, two bryophytes, and an alga. The oxygen uptake rates on a dry weight basis of angiosperm leaves were generally higher than those of the corresponding stems. Leaves also had a higher chlorophyll content than stems. Respiration of leaves and stems of aquatic angiosperms was generally cyanide-resistant, the percentage of resistance being higher than 50% with very few exceptions. The cyanide resistance of respiration of whole shoots of two aquatic bryophytes and an alga was lower and ranged between 25 and 50%. These results suggested that the photosynthetic tissues of aquatic plants have a considerable alternative pathway capacity. The angiosperm leaves generally showed the largest alternative path capacity. In all cases, the respiration rate of the aquatic plants studied was inhibited by SHAM alone by about 13 to 31%. These results were used for calculating the actual activities of the cytochrome and alternative pathways. These activities were generally higher in the leaves of angiosperms. The basal oxygen uptake rate of Myriophyllum spicatum leaves was not stimulated by sucrose, malate or glycine in the absence of the uncoupler carbonylcyanide-m-chlorophenylhydrazone (CCCP), but was greatly increased by CCCP, either in the presence or in the absence of substrates. These results suggest that respiration was limited by the adenylate system, and not by substrate availability. The increase in the respiratory rate by CCCP was due to a large increase in the activities of both the cytochrome and alternative pathways. The respiration rate of M. spicatum leaves in the presence of substrates was little inhibited by SHAM alone, but the SHAM-resistant rate (that is, the cytochrome path) was greatly stimulated by the further addition of CCCP. Similarly, the cyanide-resistant rate of O₂ uptake was also increased by the uncoupler.     

Regulation of Respiration in the Leaves and Roots of Two Lolium perenne Populations with Contrasting Mature Leaf Respiration Rates and Crop Yields

Measurements of O₂ uptake were made on leaves and roots of two populations of Lolium perenne L. cv S23 (GL66 and GL72), previously shown to have contrasting rates of CO₂ evolution and yields of dry matter. O₂ uptake was faster in the mature leaves of GL66 than those of GL72, but no difference was observed in the respiratory rates of meristematic leaf bases or mature roots. The growth rate of GL72 was faster than that of GL66. Cyanide resistance was substantial in mature leaves but the alternative path did not contribute to O₂ uptake in the dark. In both populations, adding malate and glycine stimulated O₂ uptake, but exogenous sucrose only stimulated when uncoupler was also present. The difference between the respiratory rates of the two populations was maintained under all investigated conditions. We conclude that the rate of mature leaf respiration in the dark in L. perenne is limited by adenylate control of glycolysis. The difference between the fast (GL66) and slow (GL72) respiring populations reflected a greater respiratory capacity and higher turnover of ATP in GL66. Alternative path capacity was also high in the roots of both and contributed substantially to O₂ uptake, as indicated by inhibition by salicylhydroxamic acid in the absence of KCN. The alternative path capacity of meristematic leaf bases was considerably less than that in mature leaves.

Transverse and cross-sections were made of mature leaves of both populations to study anatomical features which might explain the differences in ATP turnover, suggested by the biochemical experiments. Leaves of GL72 were thicker but did not show a different anatomy when compared with GL66. The increased thickness was not due to more or larger cells but entirely to a larger intercellular volume.

     

Cyanide-insensitive, Salicylhydroxamic Acid-sensitive Processes in Potentiation of Light-requiring Lettuce Seeds

Phytochrome-mediated germination of Lactuca sativa L. cv. Waldmann's Green seeds was inhibited strongly by 10 millimolar salicylhydroxamic acid (SHAM), but only slightly delayed by the same level of KCN. SHAM was most effective if applied within the 8-hour potentiation period (release from dormancy) following red light treatment, but much less effective with completely potentiated seeds. SHAM at 3 millimolar actually hastened completion of potentiation, whereas concentrations of 6.6 millimolar or higher retarded the process. A temporary upsurge of O₂ consumption was particularly evident during the period of most rapid potentiation (3 hours after red light), especially in the seed sections containing the embryonic axis. The embryonic axis obtained from dormant seeds also contained most of the SHAM-sensitive O₂ uptake. However, 8 hours of potentiation caused loss of SHAM sensitivity from axes and a simultaneous gain of SHAM sensitivity by cotyledons. Concomitant with this increased sensitivity to SHAM, O₂ uptake by cotyledonary tissues lost some sensitivity to KCN. Red light-stimulated metabolic processes leading to germination were blocked more effectively by SHAM than by KCN, but O₂ consumption by both dormant and nondormant seeds was much less sensitive to 10 millimolar SHAM than to the same concentration of KCN. This apparent contradiction between effects of SHAM on potentiation and O₂ uptake may be a result of: (a) compensatory electron flow through the cytochrome pathway at the expense of the alternate pathway; (b) a functional site of action of SHAM that differs from the organized, energy-coupled respiratory system; or (c) a combination of these possibilities.     

Evidence for a significant contribution by peroxidase-mediated O2 uptake to root respiration of Brachypodium pinnatum

This study describes the O₂ uptake characteristics of intact roots of Brachypodium pinnatum. In the presence of 25 mM salicylhydroxamic acid (SHAM), concentrations of KCN below 3.5 νM had no effect on the rate of root respiration, whereas in the absence of 25 mM SHAM a significant inhibition of approx. 18% was observed. This indicates that an O₂-consuming reaction, not associated with the cytochrome pathway, the alternative pathway or the “residual component”, operates in the absence of any inhibitors in roots of B. pinnatum. We demonstrate here that this fourth O₂-consuming reaction is mediated by a peroxidase. A peroxidase which catalyzed O₂ reduction in the presence of NADH was readily washed from the roots of B. pinnatum. This peroxidase was stimulated by 5 mM SHAM, whereas ascorbic acid, catalase, catechol, gentisic acid, low concentrations potassium cyanide (3.5 μM), sodium azide, sodium sulfide, superoxide dismutase and high concentrations SHAM (25 mM) inhibited this reaction. Except for high concentrations of SHAM and concentrations of KCN higher than approx. 3.5 μM, these effectors could not be used to inhibit the peroxidase-mediated O₂ uptake in intact roots of B. pinnatum. Concentrations of SHAM below 10 mM stimulated O₂ uptake up to 15% of the control rate, depending on concentration, whereas 25 mM SHAM inhibited O₂ uptake by 35%. The stimulation at low concentrations resulted from a SHAM-stimulated peroxidase activity, whereas 25 mM SHAM completely inhibited both the peroxidase-mediated O₂ uptake and the activity of the alternative pathway. A method is presented for determining the relative contributions of each of the four O₂-consuming reactions, i.e. the cytochrome pathway, the alternative pathway, the “residual component” and the peroxidase-mediated O₂ uptake. The peroxidase-mediated O₂ uptake contributed 21% to the total rate of oxygen uptake in roots of B. pinnatum, the cytochrome pathway contributed 41%, the alternative pathway 14% and the “residual component” 24%.     

Inhibition and stimulation of root respiration in pisum and plantago by hydroxamate : its consequences for the assessment of alternative path activity

The contribution of the alternative pathway in root respiration of Pisum sativum L. cv Rondo, Plantago lanceolata L., and Plantago major L. ssp major was determined by titration with salicylhydroxamate (SHAM) in the absence and presence of cyanide. SHAM completely inhibited the cyanide-resistant component of root respiration at 5 to 10 millimolar with an apparent K_i of 600 micromolar. In contrast, SHAM enhanced pea root respiration by 30% at most, at concentrations below 15 millimolar. An unknown oxidase appeared to be responsible for this stimulation. Its maximum activity in the presence of low SHAM concentrations (1-5 millimolar) was 40% of control respiration rate in pea roots, since 25 millimolar SHAM resulted in 10% inhibition. In plantain roots, the maximum activity was found to be 15%. This hydroxamate-activated oxidase was distinct from the cytochrome path by its resistance to antimycin. The results of titrations with cyanide and antimycin indicated that high SHAM concentrations (up to 25 millimolar) block the hydroxamate-activated oxidase, but do not affect the cytochrome path and, therefore, are a reliable tool for estimating the activity of the alternative path in vivo. A considerable fraction of root respiration was mediated by the alternative path in plantain (45%) and pea (15%), in the latter because of the saturation of the cytochrome path.     

Salicylic Acid induces cyanide-resistant respiration in tobacco cell-suspension cultures

Cyanide-resistant, alternative respiration in Nicotiana tabacum L. cv Xanthi-nc was analyzed in liquid suspension cultures using O₂ uptake and calorimetric measurements. In young cultures (4-8 d after transfer), cyanide inhibited O₂ uptake by up to 40% as compared to controls. Application of 20 μm salicylic acid (SA) to young cells increased cyanide-resistant O₂ uptake within 2 h. Development of KCN resistance did not affect total O₂ uptake, but was accompanied by a 60% increase in the rate of heat evolution from cells as measured by calorimetry. This stimulation of heat evolution by SA was not significantly affected by 1 mm cyanide, but was reduced by 10 mm salicylhydroxamic acid (SHAM), an inhibitor of cyanide-resistant respiration. Treatment of SA-induced or uninduced cells with a combination of cyanide and SHAM blocked most of the O₂ consumption and heat evolution. Fifty percent of the applied SA was taken up within 10 min, with most of the intracellular SA metabolized in 2 h. 2,6-Dihydroxybenzoic and 4-hydroxybenzoic acids also induced cyanide-resistant respiration. These data indicate that in tobacco cell-suspension culture, SA induces the activity and the capacity of cyanide-resistant respiration without affecting the capacity of the cytochrome c respiration pathway.     

Cyanide-resistant respiration in roots and leaves. Measurements with intact tissues and isolated mitochondria

A comparison was made between the oxygen uptake of roots and leaves and of mitochondria isolated from the same tissues. Ten species were included in this study: three legumes, one C3-monocotyledon, one C4-monocotyledon, the rest non-leguminous C3-dicotyledons. Root and leaf respiration in all species examined displayed substantial resistance to KCN (0.1–1.0 mM) and the cyanide-resistant respiration was completely inhibited by salicylhydroxamic acid (SHAM; 10–20 mM). SHAM alone inhibited oxygen uptake to varying degrees, depending on the species. Mitochondria were isolated from roots and leaves of many of the species examined and also displayed cyanide-resistant oxygen uptake, which was sensitive to both SHAM and tetraethylthiuram disulfide (disulfiram). Concentrations of SHAM greater than 2 mM caused inhibition of the cytochrome path as well as of the alternative path in isolated mitochondria. Respiration rates of intact roots and leaves in the presence of varying concentrations of SHAM alone were plotted against those obtained in the presence of both SHAM and KCN. This plot showed that in vivo the cytochrome pathway was not affected by 10 or 20 mM SHAM in the external solution. We conclude that the activity of the alternative pathway in intact roots and leaves can be reliably estimated by comparing SHAM-sensitivity and cyanide-resistance of respiration.     

Reversion from light-induced inhibition of seed germination by respiratory inhibitors

Treating of the dark-imbibed lettuce (Lactuca sativa L.) seeds prior to light irradiation with 1 millimolar KCN or NaN₃ in the dark for 3 hours prevented blue light and far-red light-induced inhibitions of phytochrome-mediated germination. Similarly, salicylhydroxamic acid (SHAM) at 10 millimolar counteracted the blue and far-red light inhibitions, the combined application of KCN and SHAM being more effective than KCN or SHAM alone in some experiments. These respiratory inhibitors slightly inhibited phytochrome-mediated lettuce seed germination. These results indicate that both CN-sensitive, conventional cytochrome oxidase and CN-resistant (SHAM-sensitive), alternative respiration may be involved in the light inhibition or that an appropriate balance of both may be necessary for the light inhibition.     

Hydroxamate-Stimulated O(2) Uptake in Roots of Pisum sativum and Zea mays, Mediated by a Peroxidase : Its Consequences for Respiration Measurements

Low concentrations of salicylhydroxamic acid (<5 millimolar) stimulate O₂ uptake in intact roots of Pisum sativum. We demonstrate that the hydroxamate-stimulated O₂ uptake does not reside in the mitochondria. We also show that the hydroxamate-stimulated O₂ uptake is due to the activation of a peroxidase catalyzing reduction of O₂. This peroxidase, which can use both NADH and NADPH as a substrate, is stimulated by low concentrations of monophenols, e.g. salicylhydroxamic acid and 2-methoxyphenol. It is inhibited by high (20 millimolar) concentrations of salicylhydroxamic acid, cyanide, and scavengers of the superoxide free radical ion, e.g. ascorbate, gentisic acid, and catechol. In the presence of gentisic acid, O₂ uptake by intact pea roots was no longer stimulated by low concentrations of salicylhydroxamic acid. The consequence of the present finding for in vivo respiration measurements is that the use of low concentrations of salicylhydroxamic acid and uncoupler is reliable only in the presence of a suitable superoxide free radical scavenger which prevents activation of the peroxidase. It also confirms that high concentrations of salicylhydroxamic acid (20-25 millimolar) can be safely used in short-term experiments to assess the activity of the alternative path in intact roots.     

Effect of water deficit on respiration of conducting bundles in leaf petioles of sugar beet

Isolated fibrovascular bundles from source leaf petioles of sugar beet (Beta vulgaris L.) and hog-weed (Heracleum sosnovskyi L.) were used to study the influence of long-term drought on the oxygen uptake rate and activities of mitochondrial oxidases, i.e., cytochrome oxidase and salicylhydroxamic acid-sensitive alternative oxidase (AO). Under normal soil moisture content (70% of full water-retaining capacity, WRC), the oxygen uptake by sugar beet conducting bundles was characterized by a high rate (> 700 μl O₂/(g fr wt h)) and by distinct cytochrome oxidase-dependent manner of terminal oxidation (up to 80% inhibition of respiration in the presence of 0.5 mM KCN). After long-term water deficit (40% of WRC), the bundle respiration proceeded at nearly the same rate but featured an elevated resistance to cyanide. At early drought stage (10 days), a decrease in the activity of cytochrome-mediated oxidation pathway was largely counterbalanced by activation of mitochondrial AO, whereas long-term dehydration of plants was accompanied by activation of additional oxidative systems insensitive to both KCN and SHAM. Similar but even more pronounced changes in activities of terminal oxidases were discovered in conducting bundles of wild-grown hogweed plants exposed to long-term natural drought. It is supposed that the suppression of cytochrome-mediated oxidation coupled with ATP synthesis in the cells of sugar beet source leaves impedes the translocation of assimilates and their accumulation in the taproot, which represents an important factor of drastic decrease in the yield of this agricultural crop under conditions of water deficit.     

Dual action of respiratory inhibitors: inhibition of germination and prevention of dormancy induction in lettuce seeds

`Grand Rapids' lettuce Lactuca sativa L. seeds germinate readily at 15°C but poorly at 25°C in darkness. When held in dark at 25°C for an extended period, the ungerminated seeds become dormant as shown by their inability to germinate or transfer to 15°C in darkness. Induction of dormancy at 25°C was prevented by exposure to CN<sup>−</sup>, azide, salicylhydroxamic acid (SHAM), dinitrophenol, and pure N<sub>2</sub> as determined by subsequent germination at 15°C on removal of inhibitors. The effectiveness of inhibitors to break dormancy declined as dormancy intensified. At relatively low levels, CN<sup>−</sup>, SHAM, and azide promoted dark germination at 25°C while at high levels they were inhibitory. Uptake of O<sub>2</sub> by seeds held at 25°C for 4 days in 1.0 millimolar KCN was inhibited by 67% but was promoted 61% when KCN was removed. Correspondingly greater inhibition (79%) and promotion (148%) occurred when 1.0 millimolar SHAM was added to KCN solution. When applied alone, SHAM had little effect on O<sub>2</sub> uptake. These data indicate that Cyt pathway of respiration plays a dominant role in the control of both dormancy induction and germination of lettuce seeds, and `alternative pathway' is effectively engaged in presence of CN⁻. The channeling of respiratory energy use for processes governing germination or dormancy is subject to control by physical and chemical factors.

A scheme is proposed that illustrates compensatory use of energy for processes controlling dormancy induction and germination. A block of germination, e.g. by low water potential polyethylene glycol solution or a supraoptimal temperature spares energy to be utilized for dormancy induction while a block of dormancy induction by low levels of CN⁻ (similar to GA and light effects) drives germination. Blocking both processes by inhibitors (e.g. CN⁻, CN⁻ + SHAM) presumably leads to accumulation of `reducing power' with consequent improvement in O₂ uptake and oxidation rates of processes controlling germination or dormancy induction upon removal of the inhibitors.

     

Differential Carbon Monoxide Sensitivity of Cytochrome-Oxidase in the Leaves of Tall and Dwarf Wheat Cultivars

Differential response in the leaves of tall and dwarf wheat to CO, an inhibitor of cytochrome oxidase and to SHAM, an inhibitor of alternative oxidase appears to be correlated with presence of Rht dwarfing genes. This was detected by in vivo nitrate reductase assay after CO treatment and direct O₂ uptake in presence of SHAM. Pretreatment of the leaves with Triton X-100 at a concentration which specifically inhibits the accessibility of exogenous NAD(P)H to alternative oxidase, Significantly enhanced the CO response as assessed by in vivo NR assay. This supports the hypothesis that the competition for NADH between NR and mitochondrial respiration is regulated by NADH-dehydrogenase located on the outer surface of inner mitochondrial membrane.     

Alternative respiration and heat evolution in plants

The alternative respiratory pathway dissipates most of the chemical energy of respiratory substrates as heat. We have shown that this heat can be quantified by microcalorimetry and is a measure of alternative pathway activity in vivo. The alternative pathway is known to increase in aged potato (Solanum tuberosum) slices and in chill-stressed leaves. Aging of potato slices for 24 hours was accompanied by an almost fourfold increase in the rate of heat evolution. This heat increase was resistant to KCN but could be blocked by an alternative pathway inhibitor, salicylhydroxamic acid (SHAM). In cucumber (Cucumis sativus) leaves subjected to chilling stress (between 4 and 16°C), the rate of heat evolution was inversely related to temperature. As in aged potato slices, the increased rate of heat evolution in cucumber leaves was blocked by SHAM, but not by KCN. Nitrogen or the combination of SHAM and KCN blocked most of the heat evolution in both aged potato slices and chill-stressed cucumber leaves. Calorimetric measurements of the alternative pathway corresponded to respiration measurements performed using an oxygen electrode.     

Effect of Salicylhydroxamic Acid on Respiration, Photosynthesis, and Peroxidase Activity in Various Plant Tissues

Earlier reports from our laboratory described salicylhydroxamicacid (SHAM) stimulation of O₂ uptake by expanded soybean leavesor older green cotyledons. This stimulation could not be interpretedin terms of engagement or capacity of the cytochrome and alternativerespiratory pathways. In this report, we tested the possibilitythat a soluble peroxidase, which can be easily eluted from soybeanleaves and cotyledons, might be responsible for SHAM stimulationin whole tissue. The peroxidase catalyzes oxidation of NADHby O₂, is strongly stimulated by SHAM and benzhydroxamic acid(BHAM) and inhibited by KCN, propyl gallate and gentisic acid.This peroxidase, however, does not seem to be responsible forSHAM-stimulated O₂ uptake in whole, green tissue. In our earlier work reporting SHAM-stimulated respiration ingreen tissue, the samples had not been shielded from room light(10–20 µmol photons m^–2.s^–1). In thisreport, we show that O²-uptake rates of controls measured indarkness were always greater than those measured in room light.SHAM stimulation was not observed in the dark or in tissue withoutchlorophyll. We also found that CO₂ uptake of whole leafletsin saturating light was completely inhibited by SHAM fed throughthe transpiration stream. SHAM, therefore, is a potent inhibitorof photosynthesis. We conclude that the SHAM-stimulated respirationof green tissues we reported earlier likely was due to verylow rates of photosynthesis occurring under room light. ³Present address: SANDOZ Ltd., Agrobiological Research Station,4108 Witterswil, Switzerland ⁴Present address: WTC 1A3, Weyerhaeuser Co., Tacoma, WA 98477,U.S.A. (Received June 23, 1989; Accepted October 20, 1989)     

Dark respiration in the marine macroalga Chondrus crispus (Rhodophyceae)

Dark respiration in the red macroalga Chondrus crispus was studied under a variety of conditions. The components of respiration were examined using selective inhibitors in order to characterise pathways of respiration and examine regulation of respiration in marine macroalgae.In comparison to respiration rates generally reported for higher-plant leaves and roots, the steady-state rate of O₂ consumption by this alga, after 30 min dark pretreatment, was found to be quite low (three- to sixfold lower than in higher plants). The addition of uncoupler had only a slight effect on the basal respiration rate, indicating that in these conditions, substrate supply could be limiting respiration. The addition of KCN inhibited respiration by approx. 60%, indicating the presence of alternative oxidase activity. The coefficient of engagement of the alternative pathway (calculated from the data herein) showed that under normal conditions there was little participation of the alternative pathway in O₂ consumption. The response of respiration to O₂ tension was examined with and without inhibitors and the apparent K _m was 17 to 21 M. The addition of KCN plus salicylhydroxamic acid almost completely blocked respiration in C. crispus. The hypothesis that respiratory substrate limits respiration in this alga was investigated by measuring respiration rates immediately after periods of photosynthetic activity. It was found that the respiration rate was dependent on the duration of the light period and could increase up to twofold. This stimulated rate of respiration declined in a first-order fashion during the next 20 to 30 min, finally reaching the basal, zero-order rate measured before illumination. These results strongly indicate a change in the nature of the respiratory substrates during this period. No change in the contribution of the alternative pathway of respiration could be detected following light pretreatment.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - SHAM salicyl hydroxamic acid     

Participation of the cyanide-resistant pathway in respiration of winter rape leaves as affected by plant cold acclimation

Leaf slices sampled from winter rape plants ( Brassica napus L., var. oleifera L., cv. GórczaánAski), grown in cold (5°C), showed an increase in the dark respiration rate (measured at 25°C) as compared to slices cut from control plants (grown at 20/15°C). The effect of low temperature was most pronounced after 4 days of plant growth in the cold. Oxygen uptake by control slices was 60% inhibited by 1 m M KCN and was insensitive to 2.5 m M salicylhydroxamic acid (SHAM). On the contrary, respiration of leaf slices from cold-pretreated plants was more resistant to cyanide (35% inhibition after 4 days of cold treatment) and was 30% inhibited by SHAM. The patterns of cold-induced changes in total respiratory activity and in the estimated activity of alternative pathway were similar. It seems that in leaf slices from plants grown in the cold, the cyanide-resistant, alternative pathway participates in oxygen uptake. Cold treatment of plants also brought about a 4-fold increase in the level of soluble sugars, which reached a maximum on day 4 of exposure to cold. Addition of sucrose to the incubation medium resulted in an immediate increase in oxygen uptake by slices with low endogenous sugar level. The respiration stimulated by sucrose addition was more resistant to cyanide than the basal respiration and it was inhibited by SHAM. It is concluded that the operation of the alternative pathway is responsible for the increased oxygen uptake by the cold-grown winter rape leaves and it may be induced by an increased sugar supply for respiratory processes.     

Alternative Path Mediated ATP Synthesis in Roots of Pisum sativum upon Nitrogen Supply

Changes in the efficiency of root respiration were examined on intact plants of Pisum sativum L. cv Rondo after addition of nitrate or ammonium to the culture solutions. Nitrate was absorbed immediately after addition and elicited a respiratory rise (O₂-uptake as well as CO₂-production) to 160% at most. This occurred both in roots of plants fixing N₂ and in those of non-nodulated plants pregrown for 1 or 2 weeks on a nitrogen-free culture solution. In older plants, used after 2 weeks of N-free growth, the full capacity of the cytochrome path was engaged in root respiration. This was demonstrated by the absence of an effect of the uncoupler carbonylcyanide m-chlorophenylhydrazone in the presence of 25 millimolar salicylhydroxamate, an inhibitor of the alternative path. In these plants more than 90% of the nitrate-induced stimulation of root respiration was salicylhydroxamate-sensitive. In young plants, used after 1 week of N-free growth, the cytochrome path was not saturated. Its activity increased instantaneously at the expense of alternative path activity, which initially dropped to zero and subsequently increased to 160% of the control 7 hours after nitrate supply. The rate of photosynthesis rose to 120% of the control, but not before 1 hour after nitrate supply, suggesting that the stimulation of root respiration was not due to a higher rate of photosynthesis. Experiments with plants grown with a split-root system showed that respiration rate and alternative path activity only increased in the root halves exposed to nitrogen. Ammonium was equally effective as nitrate in stimulating root respiration. These results lead to the conclusion that alternative-path mediated root respiration contributes to synthesis of ATP during at least the first 24 hours following nitrogen supply.