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.     

Tissue Respiration and Mitochondrial Oxidative Phosphorylation of NaCl-Treated Pea Seedlings

The effect of sodium chloride added to root medium of pea seedlings on respiratory activity of tissue segments and on isolated mitochondria was studied. Salinization enhances the respiration of leaves about one-third on a fresh weight, dry weight or protein basis. Roots and stems show only 10 to 15% respiratory stimulation. The onset of respiratory increase in leaves roughly parallels the increase in NaCl content and the decrease in growth rate. At a later stage the elevated respiration is apparent in treated plants even though the concentration of NaCl reaches a plateau and osmotic adjustment is being reached. Stimulation of respiration was found in both etiolated and green plants. Experiments with DNP show that simple uncoupling by salt is not involved; the respiratory increase in control and treated tissue is proportionally the same.

In accordance with increased respiration rates observed in vivo, mitochondria from salt-treated plants show higher rates of oxygen uptake on several substrates. The effect of NaCl added during growth is long term and is distinct from the effect of NaCl added to mitochondria isolated from control plants. Since P/O ratios are not affected by NaCl, the potential for oxidative phosphorylation in salt-affected tissue appears to increase. It is postulated that this increase may lead to changes in ADP and ATP content, and in turn, affect regulation of metabolic pathways.

     

Effects of Hydroxyproline and other Amino Acid Analogues on the Growth of Pea Root Segments

Changes in the lengths and growth rates of isolated 2–4 mm pea root segments, cultured in sucrose media under aseptic conditions, were paralleled by changes in invertase development and in chloride and leucine uptakes. The amino acid analogues o-, m- and p-fluorophenylalanine, azetidine-2-carboxylic acid and ethionine inhibited growth with corresponding changes in invertase activity and in chloride and leucine uptakes. In contrast hydroxyproline, which under the conditions used may be regarded as an analogue of proline, enhanced both the growth rate and duration of growth but had little effect on the several parameters of protein synthesis which were measured. No amino acid tested affected changes in growth, invertase activity or the uptake of chloride and leucine, but they prevented the effects of the corresponding analogues. The results show that although extension growth is dependent on continuous protein synthesis, only specific proteins, probably in the cell wall, play a key role in this process.     

Effects of Gibberellic Acid on the Transport of Nitrogen from the Cotyledons of Young Pea Seedlings

Gibberellic acid increased shoot length in tall and dwarf peaseedlings, but whilst in the former it had no effect on therate of breakdown of reserve proteins nor on the transport ofnitrogen to the axis and its final distribution between theshoot and the root, it promoted these processes in the dwarfpea. The results are discussed in relation to the overall controlof the mobilization of reserves in the cotyledons during germination.     

Isoxazolin-5-ones and Amino Acids in Root Exudates of Pea and Sweet Pea Seedlings

Seeds of Pisum sativum L. cv Finale and Lathyrus odoratus L. cv Spencer were germinated aseptically in moistened sand in the dark. At several stages, the amino acid composition of the exudate and of the corresponding roots was analyzed. A number of common amino acids, including homoserine, were exuded by the growing seedling root in an early stage and were partly reabsorbed later. A number of uncommon amino acids, including several isoxazolin-5-one derivatives, uracil alanines, l-γ-glutamyl-d-alanine, and α-aminoadipic acid were exuded at different rates.     

Effects of KCN and NaN3 Pretreatment on the Cyanide-Resistant Respiration in Tobacco Callus

The effects of KCN (0.5mmol/L) and NaN3 (0.01 mmol/L) pretreatment on the operation of the alternative pathway in subcultured tobacco (Nicotiana rustica L. cv. Gansu yellow flower) callus were analyzed. After treatment with KCN and NaN3 for 12 h, the total respiration rate (Vt) decreased by 12% and 17%, whereas oxygen consuption by the cytochrome pathway decreased by 22% and 28% respectively. The capacity of the alternative pathway (Valt) remained constant, while the activity of the alternative pathway (ρ· Valt ) inreased slightly. This changing pattern led to a declined contribution of the cytochrome pathway to the total respiration rate and an increased activity of the alternative pathway. Treatment with KCN for 24 h brought about a slight rise of oxygen consumption by the cytochrome pathway as compared with that in callus treated for 12 h, but the oxygen consumption was still lower than that in the untreated callus. Treatment with NaN3 for 24 h resulted in a profound decrease of the cytochrome pathway operation and a continuing increase of the alternative pathway operation. These data indicated that the enhanced operation of the alternative pathway played a compensatory role to the total respiration when the cytochrome pathway was partially inhibited in tobacco callus.     

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)     

The Stability and Movement of Gibberellic Acid in Pea Seedlings

The stability and movement of gibberellic acid (GA) in intactdwarf pea seedlings growing in the light was studied by meansof both unlabelled GA and GA labelled with isotopic carbon (¹⁴C).After ¹⁴C-GA had been applied to the mature leaves of pea seedlingsmuch remained in association with the treated leaflets, but¹⁴C-GA was also extractable from the young shoots. The yieldwas approximately the same 5 to 96 hours after treatment. GApenetrated leaf surfaces only while the application solventwas moist (about 1 hour), but moved from the treated leafletsinto the shoots for at least 24 hours. Some hours after treatmentthere was an abrupt increase in the growth-rates of the plants,and crude estimates suggest that an effective dose of GA movedto the elongating tissue at about 5 cm/hr. The pattern of distributionof ¹⁴C was examined by autoradiography. The data suggest thatGA which enters the plant is redistributed from maturing leavesto immature leaves, passing through the elongating tissue, foras long as any of the substance is present. The hypothesis remainstenable that GA produces its growth effects by acting only uponexpanding tissue     

Studies on the Residual Respiration not Inhibited by KCN Plus Hydroxamic Acid in Tobacco Callus Cultures

A residual respiration not inhibited by KCN plus hydroxamic acid had been observed in many plant organs and tissues. The relative O2 uptake of it was 20–30% of total respiration in tobacco callus cultures. However, there is no report concerning the nature of the residual respiration and its localization in cell. The object of this study is to elucidate the characteris- tics of this residual respiration and its localization in cell. The experimental results are as follows: 1. The additions of glycolate and glyoxylate cause a marked rise in residual respiration not inhibited by KCN (or NaN3) plus m-CLAM. 2. The O2 uptake induced by glycolate and the residual respiration is inhibited by the addition of α-hydroxy ethanesulfonate. 3. The mitochondrial respiration is completely inhibited by KCN plus m-CLAM, but no effect by adding of glycolate. 4. Oxidation reactions of glycolate and glyoxylate in supernatant are observed after mitochondria are removed. Based on the above results, it is suggested that the residual respiration not inhibited by KCN plus m-CLAM in tobacco callus cultures is primarily catalyzed by glycolic acid oxidase localized within microbodies.     

Effects of Low Irradiance Stress on Gibberellin Levels in Pea Seedlings

Using gas chromatography-selected ion monitoring with internalstandards we analyzed endogenous levels of GA₁, GA₈, GA₁₉, GA₂₀,GA₂₉, GA₄₄ and GA₅₃ in shoots of pea cv. Alaska grown underdifferent levels of irradiance: high irradiance, 386±70µmolm^-2s^-1, control (100%); medium (50%); low (10%); darkness (0%).The average plant heights for medium and low irradiance anddark grown plants were 157%, 275%, and 460% of the control plants,respectively. Plants grown in medium and low irradiance developedthe same numbers of internodes as control plants but plantsin darkness developed fewer internodes and exhibited suppressedleaf expansion. The endogenous levels of GA₁ GA₈ and GA₂₉ werehigher in medium and low irradiance grown plants than thoseof the high irradiance control. In particular, the GA₂₀ levelof low irradiance plants was markedly higher (7.6-fold) thanthat of control plants. In dark-grown plants GA₁, and GA₈ levelsalso slightly increased but GA₂₀ and GA₂₉ levels decreased andthe levels of GA₁₉, GA₄₄ and GA₅₃ did not change. Feeding ofGA₁, and a GA biosynthesis inhibitor (uniconazole) to plantsgrown at reduced irradiance and in darkness suggests that theresponsiveness of plants to GA₁, also increased at low irradianceand in darkness. In conclusion, plants increase both GA₁, andGA₂₀ biosynthesis or altered catabolism and GA₁, responsivenessunder low irradiance stress ¹Present address: Dept. of Plant Physiol., Warsaw AgriculturalUniversity, Rakowiecka 26-30, 02-528 Warsaw, Poland     

Respiration of Gametangia of the Aquatic Phycomycete Allomyces macrogynus: Inhibition by Cyanide or Antimycin and Salicylhydroxamic Acid or Propyl Gallate

Gametangia of the aquatic phycomycete Allomyces macrogynus have a cyanide- and antimycin A-insensitive respiration, which is sensitive to salicylhydroxamic acid (alternative respiration). Propyl gallate is also an inhibitor of this alternative pathway, and propyl gallate is more efficient than hydroxamic acid. Gametangial respiration is insensitive to propyl gallate, but propyl gallate sensitivity is gradually established when the gametangia are titrated with cyanide. Carbonyl cyanide m-chlorophenyl hydrazone stimulates the cyanide-sensitive respiration and engages the alternative sensitive respiration. Sodium azide inhibits both the alternative and the cyanide-sensitive respiration, but the cyanide-sensitive respiration is inhibited 10 times more efficiently than the alternative respiration. Rotenone inhibits the total respiration and the propyl gallate-insensitive respiration by 33% and the cyanide-insensitive respiration by 43%.

The kinetic results reported here are discussed with respect to the models of de Troostembergh and Nyns (1977 Arch Int Physiol Biochem 85:404-406; 1978 Eur J Biochem 53:423-432) and of Bahr and Bonner (1973 J Biol Chem 248:3446-3450) for the partitioning of electrons between cyanide-insensitive and propyl gallate-insensitive respiration. The results reported here do not agree with the model of de Troostembergh and Nyns.