Effect of Salicylhydroxamic Acid on Endosperm Strength and Embryo Growth of Lactuca sativa L. cv Waldmann''s Green Seeds

Salicylhydroxamic acid (SHAM) stimulated germination of photosensitive lettuce (Lactuca sativa L. cv Waldmann's Green) seeds in darkness. To determine whether SHAM acts on the embryo or the endosperm, we investigated separately effects of SHAM on growth potential of isolated embryos as well as on endosperm strength. Embryo growth potential was quantified by incubating decoated embryos in various concentrations of osmoticum and measuring subsequent radicle elongation. Growth potential of embryos isolated from seeds pretreated with 4 millimolar SHAM was equal to that of untreated controls. Rupture strength of endosperm tissue excised from seeds pretreated with SHAM was 33% less than that of controls in the micropylar region. To determine if the embryo must be in contact with the endosperm for SHAM to weaken the endosperm, some endosperms were incubated with SHAM only after dissection from seeds. Rupture strength of SHAM-treated, isolated endosperms in the micropylar region was 25% less than that of untreated controls. There was no difference in rupture strength in the cotyledonary region of endosperm isolated from seeds treated with SHAM in buffer or buffer alone. SHAM therefore stimulates germination not by enhancing embryo growth potential, but by weakening the micropylar region of the endosperm enclosing the embryo.     

The Effect of pH on the Action of Respiratory Inhibitors in Avena fatua Seeds

The effects of pH on the action of sodium azide, a cytochome-oxidaseinhibitor, and salicylhydroxamate (SHAM), an alternative respirationinhibitor, on the respiration of dormant seeds of wild oat (Avenafatua L.; line AN-51) were studied. While pH had little effecton seed respiration in controls, it strongly affected the activityof azide. One mM azide inhibited seed respiration at pH5, butstimulated it at pH 7. SHAM (10 mM) completely inhibited thestimulation of respiration by 1 mM azide in an unbuffered medium,but failed to do so when the medium was buffered at pH 7. Inunbuffered medium, 10 mM SHAM actually augmented the stimulationof respiration by 0.25 mM azide to the same degree as when theazide solution was acidified to mimic the pH change incurredwith dissolution of 10 mM SHAM. These results suggest that theinhibitory effect of SHAM on the action of azide in an unbufferedsystem may in part be due to its acidification of the incubationmedium rather than by the inhibition of alternative oxidase.Lower pH favours the formation of the undissociated hydrazoatemolecules causing greater inhibition of cytochrome-oxidase andother azide-sensitive enzymes. Avena fatua L, wild oat, seed dormancy, azide, salicylhydroxamate     

Effects of KCN and Salicylhydroxamic Acid on Respiration of Soybean Leaves at Different Ages

Measurements of respiration were made on leaf discs from glasshouse-grown soybean (Glycine max [L.] Merr. cv `Corsoy') plants in the presence and absence of cyanide (KCN) and salicylhydroxamic acid (SHAM). O₂ uptake by mature leaves measured at 25°C was stimulated by 1 millimolar KCN (63%) and also by 5 millimolar azide (79%). SHAM, an inhibitor of the alternative oxidase and a selection of other enzymes, also stimulated O₂ uptake by itself at concentration of 10 millimolar. However, in combination, KCN and SHAM were inhibitory. The rate of O₂ uptake declined consistently with leaf age. The stimulation of O₂ uptake by KCN and by SHAM occurred only after a certain stage of leaf development had been reached and was more pronounced in fully expanded leaves. In young leaves, O₂ uptake was inhibited by both KCN and SHAM individually. The uncoupler, p-trifluoromethoxy carbonylcyanide phenylhydrazone, stimulated leaf respiration at all ages studied, the stimulation being more pronounced in fully expanded leaves. The uncoupled rate was inhibited by KCN and SHAM individually. The capacity of the cytochrome path declined with leaf age, paralleling the decline in total respiration. However, the capacity of the alternative path peaked at about full leaf expansion, exceeding the cytochrome capacity and remaining relatively constant. These results are consistent with the presence in soybean leaves of an alternative path capacity that seems to increase with age, and they suggest that the stimulation of O₂ uptake by KCN and NaN₃ in mature leaves was mainly by the SHAM-sensitive alternative path. The stimulation of O₂ uptake by SHAM was not expected, and the reason for it is not clear.     

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.     

Light Actions in the Germination of Cocklebur Seeds: IV. DISAPPEARANCE OF RED LIGHT-REQUIREMENT FOR THE GERMINATION OF UPPER SEEDS SUBJECT TO ANOXIA, CHILLING, CYANIDE OR AZIDEPRETREATMENT

Esashi, Y., Fuwa, Nn Kojima, K. and Hase, S. 1986. Light actionsin the germination of cocklebur seeds. IV. Disappearance ofred light-requirement for the germination of upper seeds subjectto anoxia, chilling, cyanide or azide pretreatmenL—J.exp. Bot. 37: 1652–1662. The effects on the germination of positively photoblastic uppercocklebur (X anthium pennsylvanicum Wallr.) seeds by pretreatingwith anoxia, chilling, cyanide or azide, which stimulates theirdark germination, were examined in relation to light actions.Prior to experiments, seeds were pre-soaked at 23 °C inthe dark for 1 or 2 weeks to remove the pre-existing Pfr. Whenthe prctreatment conditions were suboptimal for germinationinduction, the stimulating effects of the pretreatments on germinationduring a subsequent dark period at 23 °C were manifest onlywhen seeds were irradiated with red light before or after thepretreatment Red light promotion was reversed by blue or far-redlight treatment. However, both prc-chilling for 6 d at 8 °Cand prctreatment with 1· 5 mol m ^{– 3} NaN³ for 2d could induce full germination without red light exposure.On the other hand, both pre-exposure to anoxia for 8 d and pretreatmentwith 30 mol m^–3 KCN could induce the dark germinationonly when germination occurred at 33 °C which is known toaugment the ratio of an alternative respiration flux to a cytochromeone. Moreover, the dark germination in response to these inductionswere strongly inhibited by the inhibitors of alternative respiration,propyl gallate and benzohydroxamic acid, applied during a subsequentdark period. It was thus suggested that Pfr has some relationto the operation of two respiration systems of cocklebur seeds,but it is not indispensable to germination of this positivelyphotoblastic seed. Key words: Anoxia, azide, blue light, chilling cyanide, dark germination, far-red light, red light, seed germination, X anthium pennsylvanicum     

The physiological basis of seed dormancy in Avena fatua VI. Respiration and the stimulation of germination by ethanol

Ethanol induced germination in several partly after-ripened dormant lines of Avena fatua L. The dose-response curves for the stimulation of germination and for oxygen uptake were similar, indicating that ethanol may stimulate germination by promoting oxygen uptake. A time-sequence study showed that ethanol stimulated oxygen uptake by as much as 70% prior to the first visible signs of germination. A similar methanol treatment failed to induce germination or significantly elevate oxygen uptake, indicating that the promotive effects of ethanol are not common to all alcohols. The stimulation of both germination and oxygen uptake by ethanol was not inhibited significantly by salicylhydroxamic acid, an inhibitor of alternative respiration. Thus, stimulation of germination and oxygen uptake by ethanol does not require the operation of the alternative pathway of respiration. Similarly, the stimulation of germination and oxygen uptake by ethanol were not inhibited by sodium azide, an inhibitor of cytochrome-mediated respiration. However, both germination and oxygen uptake were prevented when salicylhydroxamic acid and sodium azide were administered together. Thus, stimulation of these events by ethanol requires only the operation of one or other of these pathways of respiration; a specific requirement for the operation of the alternative pathway of respiration does not exist. The function of ethanol as a promoter of respiration is discussed with reference to dormancy and involvement of the Krebs cycle.     

The physiological basis of seed dormancy in Avena fatua IV. Alternative respiration and nitrogenous compounds

Sodium nitrate and nitrite induced germination in a partly after-ripened dormant line of Avena fatua L. The dose-response curves for their stimulation of germination and oxygen uptake were similar, indicating that these compounds may stimulate germination by promoting oxygen uptake. Time-sequence studies showed that nitrate and nitrite stimulated oxygen uptake as much as 70% prior to the first signs of germination. A similar ammonium chloride treatment failed to induce germination or elevate the rate of oxygen uptake, indicating that nitrate and nitrite may promote these events by acting as electron acceptors. The stimulation of germination and oxygen uptake by nitrate and nitrite were not significantly inhibited by salicylhydroxamic acid, an inhibitor of alternative respiration. Thus, stimulation of germination and oxygen uptake by nitrate and nitrite do not require the operation of the alternative pathway of respiration. The stimulation of germination and oxygen uptake by nitrate and nitrite were not inhibited by sodium azide, an inhibitor of cytochrome-mediated respiration; however both germination and oxygen uptake were prevented when salicylhydroxamic acid and sodium azide were applied together. Thus, stimulation of germination and oxygen uptake by nitrate and nitrite require the operation of only one or other of the pathways of respiration; however a specific requirement for the operation of the alternative pathway of respiration does not exist. The function of these compounds as promotors of respiration is discussed with reference to dormancy and the involvement of the Krebs cycle.     

Salicylhydroxamic Acid Potentiates Germination of `Waldmann''s Green'' Lettuce Seed

A combination of salicylhydroxamic acid (SHAM) + cyanide (CN) is known to stimulate dark germination of Lactuca sativa L. seeds. Further studies were done to characterize SHAM and CN action in stimulating dark germination of lettuce seed. Germination was stimulated slightly by either SHAM or CN, whereas when SHAM and CN were combined germination was greatly enhanced. Treatment of seeds with SHAM + CN only during the first 8 hours of hydration stimulated germination as much as did treatment for 72 hours. During the first 8 hours of incubation in SHAM + CN, potentiation (i.e. dormancy-breaking) of germination occurs. SHAM alone stimulated potentiation nearly to the level of SHAM + CN but inhibited subsequent radicle elongation, thereby decreasing germination when present for 72 hours. Oxygen must be present for SHAM or SHAM + CN to potentiate dark germination. The ability of SHAM and SHAM + CN to potentiate germination is influenced by O₂ concentration and the timing of chemical treatment.     

The physiological basis of seed dormancy in Avena fatua L. I. Action of the respiratory inhibitors sodium azide and salicylhydroxamic acid

The dormancy breaking effect of sodium azide was studied in seeds of several genetically pure lines of Avena fatua L. isolated from field populations. Sodium azide (0.8 and 1 m M ) induced germination in several dormant lines (characterized by long term dormancy) after two weeks of treatment. By about five weeks, germination was nearly complete in azide treated seeds as compared to little or no germination in controls. (2-chloroethyl) trimethylammonium chloride (an inhibitor of gibberellin biosynthesis) completely inhibited the azide effect suggesting that stimulation of germination by azide requires gibberellin biosynthesis. Azide was very effective in breaking dormancy in lines AN-51, AN-86, AN-127 and AN-265, but failed to induce germination in Montana 73. In this line there was a synergism between azide and gibberellic acid in promotion of germination. Thus, at least two metabolic blocks are involved in the stimulation of germination in this line. Salicylhydroxamic acid (an inhibitor of alternative respiration) at 3 m M completely inhibited the germination induced by 1 m M azide. At this concentration, salicylhydroxamic acid did not inhibit germination in 1) genetically nondormant seeds (line SH-430), 2) afterripened seeds of a dormant line (AN-51), and 3) gibberellic acid-treated dormant seeds. These findings suggest that salicylhydroxamic acid-sensitive process(es), presumably alternative respiration, is necessary for the stimulation of germination in the presence of azide, but not in the germination of genetically nondormant, gibberellic acid-treated dormant, or afterripened seeds.     

Effects of salicylhydroxamate on respiration, seed germination and seedling growth in Avena fatua

The effects of inhibitors of alternative respiration [salicylhydroxamate (SHAM) and propyl gallate (PG)] on germination, seedling growth and O₂ uptake in Avena fatua L. (wild oats) were studied. SHAM did not inhibit germination or O₂ uptake prior to germination. SHAM-sensitive (alternative) respiration, therefore, cannot be a pre-requisite for germination. Following germination, both chemicals inhibited seedling growth with the root being more susceptible than the shoot. SHAM concentrations that inhibited root growth by 90 to 95%, inhibited O₂ uptake of 1 cm root apices by less than 15%. While sodium azide (a cytochrome-oxidase inhibitor; 1 m M ) alone inhibited O₂ uptake by only 40 to 50%, in the simultaneous presence of SHAM (or PG), O₂ uptake was inhibited by 90 to 99%. Thus: 1) respiration of wild oat seedling root apices is predominantly cytochrome-mediated and incomplete inhibition of O₂ uptake in the presence of azide alone is due to diversion of electrons to the alternative pathway and 2) even though these roots have little alternative respiration, they maintain the capacity to support a much greater flux of electrons via this path way. SHAM and PG at concentrations (0.05 to 0.4 m M ) which inhibited O₂ uptake significantly in the presence (but not in the absence) of azide had little effect on root growth suggesting that an effect(s) other than that on respiration is involved in the inhibition of root growth at higher concentrations. The effect of SHAM on wild oat root growth is not selective as it also inhibits growth of a number of crop species.     

Azide-stimulated oxygen consumption by the green alga Selenastrum minutum

Dark O₂ consumption by the green alga Selenastrum minutum was sensitive to inhibition by the cytochrome pathway respiration inhibitor cyanide in the absence of an alternative oxidase inhibitor, consistent with previous work that suggested that this alga lacks alternative oxidase capacity. In contrast, addition of low concentrations of the cytochrome pathway inhibitor azide (50–750 μ M ) resulted in a stimulation of dark O₂ consumption, while higher concentrations of azide (1–2 m M ) partially inhibited O₂ consumption. Measurements of changes in cellular levels of pyruvate, malate and pyridine nucleotides upon cyanide addition were consistent with the absence of alternative oxidase capacity, and suggested that cyanide inhibition of O₂ consumption was not due to nonspecific effects of cyanide. Addition of salicylhydroxamic acid (SHAM) also resulted in an increase in the rate of O₂ consumption. Both azide- and SHAM-stimulated O₂ consumption were sensitive to inhibition by 50 m M ascorbate or by cyanide. However, the ubiquinone analogs chloroquine and quinacrine specifically inhibited azide-stimulated O₂ consumption, with only minor effects on SHAM-stimulated O₂ consumption. These results suggest that azide-stimulated O₂ consumption was not mediated by the previously characterized SHAM-stimulated oxidase, and are consistent with the possibility that azide-stimulated O₂ consumption is mediated by a plasma membrane redox system.     

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.     

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%.     

Presence and role of jasmonate in apple embryos

(-)Jasmonic acid (JA) was identified in extracts front embryos of apple (Mulus domestica) by combined gas chromatography-mass spectromelry. Quantification of JA in embryos isolated from seeds at different perexts of stratification by gas chromalography combined with mass spectrometry/selective ion monitoring indicated a sharp peak at day 30. At the same time the maximal ratio of conjugated to free JA was found by enzyme-linked imrnunosorbent assay (ELISA). Germination of embryo.s was stimulated by added JA and inhibited by salieylhydroxamic acid (SHAM, an inhibitor of lipoKygenase). Both stimulation and inhibition disappeared in embryos stratified for more than 30 days. Methyl jasmonate was more effective in stimulation of embryo germination than free JA. while JA-isoleucine inhibited germination. The possible mechanism responsible for changes in JA level as wel! as the role of JA and its conjugates in removal of dormancy in apple seeds are discussed.     

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.     

Anoxic Seed Germination of Erythrina caffra: Ethanol Fermentation and Response to Metabolic Inhibitors

Erythrina caffra seeds were shown to be true anaerobic germinators.They exhibit a Pasteur effect, high alcohol dehydrogenase activityand produce high levels of ethanol under anoxia, in which situationgermination starts to be suppressed by as little as 0.1% externallyapplied ethanol. Toxic levels of ethanol production appear tobe prevented by a decrease in the rate of ethanol accumulation.Carbon monoxide does not inhibit germination. Cyanide, SHAM,iodoacetate, pyrazole, and 4-methylpyrazole are more inhibitoryto anoxic than aerobic germination whereas azide, arsenate,and fluoride inhibit both. Azide, pyrazole, 4-methylpyrazoleand a low concentration of cyanide and SHAM tend to stimulateethanol production in air. At 10 mol m^–3, 4-methylpyrazolestimulates anaerobic ethanol production. At higher concentrationsthis compound and all other inhibitors used suppress anaerobicethanol production initially. Inhibition of ethanol productionby 10 mol m^–3 cyanide is paralleled by an accumulationof acetaldehyde. Azide and cyanide appear to exert their inhibitoryeffect at different loci. Key words: Erythrina caffra, anoxic germination, fermentation, metabolic inhibitors     

Respiratory pathways in aged soybean seeds

Respiratory activities in soybean seeds (Glvcine max (L.) Merr. cv. Chippewa 64) have been examined in the first minutes after water imbibition and after three hours of imbibition, using either particles or intact axes. Cyanide and azide were utilized as inhibitors of the cytochrome oxidase pathway of respiration, and SHAM inhibitions were interpreted as effects on the alternative pathway, since in unaged axes inhibitions by SHAM were obtained only when respiratory activity had either been restricted with inhibitors of the cytochrome oxidase pathway or expanded by an uncoupler (CCCP). From the experiments with these inhibitors, it is suggested that unaged seeds utilize both respiration pathways in the cotyledon but only the cytochrome pathway in the axis. Accelerated aging causes a marked deterioration of respiration, especially that through the cytochrome pathway, and there is an associated engagement of the alternative pathway in the seed axis. It is suggested that the lowering of respiratory activity and the shift in respiratory pathways may play a major role in the decline of germinability and vigor.     

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.     

Respiration of barley roots: Assessment of activity of the alternative path using SHAM

The activity of the alternative path of O₂ consumption in detached and intact roots of barley [ Hordeum distichum (L.) Lam. cv. Maris Mink] was determined by titration with salicylhydroxamic acid (SHAM) in the presence and absence of cyanide. In the absence of cyanide, only high concentrations were inhibititory (> 5 m M ). whilst in its presence low SHAM concentrations (2.5–5.0 m M ) gave maximum inhibition: the resulting ϱ V_alt plots were non-linear. A SHAM-stimulated peroxidase could readily be washed from these roots, but non-linearity cannot be explained in terms of SHAM-stimulation of this peroxidase as it is not active in the absence of an exogenous supply of NADH. In detached roots the degree of inhibition of respiration with 25 m M SHAM was nearly double the capacity of the alternative path (measured as the degree of inhibition by SHAM in the presence of cyanide), suggesting non-specific inhibition. Effects of SHAM on cytochrome path activity in intact roots were examined by reverse titration with cyanide in the presence and absence of SHAM. At 5 m M SHAM had no effect on the cytochrome path, but at 25 m M it inhibited. We conclude that the only factor causing non-linearity of ϱV_alt plots in barley roots is non-specific inhibition of the cytochrome path by high concentrations of SHAM; consequently only low concentrations of SHAM (2.5–5.0 m M ) are suitable for estimating alternative path activity in barley roots.     

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.