Salicylhydroxamic Acid Mimics Esterase-like Action

Chilling effects on respiration during the recovery period were studied in two maize (Zea mays L.) cultivars differing in their tolerance to chilling: Penjalinan, a chilling-sensitive cultivar, and Z7, a chilling-tolerant cultivar. Both cultivars were exposed to 5 degrees C for 5 d, after which measurements were taken at 25 degrees C. Chlorophyll fluorescence analysis in dark-adapted leaves showed less damage in cv Z7 than in cv Penjalinan during recovery from the chilling treatment. Studies of the electron partitioning between the cytochrome and the alternative respiratory pathways during chilling recovery using the oxygen isotope fractionation technique showed that, although total leaf respiration was not affected by the chilling treatment in either of the two cultivars, electron partitioning to the alternative pathway was significantly increased in the more stressed chilling-sensitive cv Penjalinan, suggesting that increased activity of the alternative pathway is not related to the plant tolerance to chilling. These results suggest a possible role of the alternative pathway in plants under stress rather than specifically contributing to plant resistance to chilling.

Effect of osmotic stress on the respiratory properties of shoots and roots of <i>Arnica alpina</i>

The capacity of the alternative respiratory pathway increased in the flavedo tissue of ;Marsh' grapefruit (Citrus paradisi Macf.) stored at 5 degrees C for 2 weeks or longer. Elevated O(2) levels during respiratory assays enhanced respiration by the tissue at 20 degrees C but not at 5 degrees C. At 20 degrees C, salicylhydroxamic acid alone was inhibitory to O(2) uptake only in elevated O(2). In conventional Warburg studies, alternative pathway respiration may be limited by the low solubility and/or slow rate of O(2) diffusion into plant tissues, such as grapefruit flavedo, and may be responsible for the apparent low utilization of the alternative pathway potential observed in other studies.

In this study the question whether the alternative respiratory pathway acts as an electron bypass for the cytochrome pathway under conditions of growth on limited phosphorus in leaves of bean (Phaseolus vulgaris L.), tobacco (Nicotiana tabacum L.) and Gliricidia sepium Walp was investigated. The oxygen isotope fractionation technique was used to assess the in vivo activities of the cytochrome and alternative respiratory pathways in the absence of added inhibitors. The response of respiration to low phosphorus supply varied among species. Growth at low phosphorus reduced cytochrome pathway activity in bean and tobacco. Alternative pathway activity increased only in bean leaves in response to low phosphorus and not in tobacco. In the case of G. sepium, cytochrome pathway activity remained unchanged whereas the alternative pathway activity increased with low nutritional phosphorus. At low phosphorus, alternative oxidase protein levels increased in the leaves of bean and G. sepium but not in tobacco, suggesting a dependence of alternative pathway activity on protein level. Alternative pathway activity was also not correlated with soluble carbohydrate concentration in bean or tobacco at any phosphorus level. These results show that the alternative pathway does not always act as an electron bypass in response to the downstream restriction of the cytochrome pathway imposed by low phosphorus supply. These results suggest that factors in addition to cellular carbohydrate level and adenylate control can act to regulate alternative pathway activity.

In a survey of several plant species found at Churchill, Manitoba, in the transition zone between the low and subarctic regions, we measured leaf respiration in terms of total respiration and alternative pathway respiration rates. Leaves of arctic plants exhibit higher rates of total respiration and alternative (cyanide insensitive) respiration than temperate species. There is a negative correlation between plant height and alternative pathway activity. Shorter plants have higher rates of alternative pathway respiration. More alternative pathway activity may mean that there is less energy in the form of ATP available for growth. A shorter growth habit keeps these plants in the still air close to the ground. This prevents cooling, water loss and physical damage due to wind abrasion. Thus plants with high rates of alternative pathway respiration may be better adapted to the arctic environment. The alternative pathway respiration of Orchis rotundifolia was shown to be under the influence of the biological clock.Key words: Subarctic, plant respiration, alternative pathway, cyanide insensitiv

Nippostrongylus brasiliensis and Ascaridia galli: Mitochondrial respiration in free-living and parasitic stages

Respiration in the filarial nematode Brugia pahangi

The role of ethylene and hydrogen peroxide (H₂O₂) in the induction of the alternative respiratory pathway (AP) in calluses from wild-type (WT) Arabidopsis and ethylene-insensitive mutant etr1-3 under salt stress was investigated. The capacity and the contribution of the AP to the total respiration were significantly induced by 100 mM sodium chloride (NaCl) in WT calluses but only slightly induced in etr1-3 calluses. Ethylene emission was enhanced in WT calluses under salt stress. Application of 1-aminocyclopropane-1-carboxylic acid (an ethylene precursor) further increased the AP capacity in WT calluses but not in etr1-3 calluses under salt stress. Reduction of ethylene production by aminooxyacetic acid (AOA, an ethylene biosynthesis inhibitor) in WT calluses eliminated the NaCl-induced increase of ethylene emission and inhibited AP induction under salt stress, suggesting that ethylene is required for AP induction. H₂O₂ enhanced ethylene production while ethylene reduced H₂O₂ generation in WT calluses under salt stress. In addition, ethylene and H₂O₂ modulated NaCl-induced alternative oxidase gene (AOX1a) expression and the increase in pyruvate content in WT calluses. Inhibition of the AP by salicylhydroxamic acid in WT calluses under salt stress resulted in severe cellular damage as indicated by the high content of H₂O₂, malondialdehyde and more electrolyte leakage. Taken together, ethylene and H₂O₂ are involved in the salt-induced increase of the AP, which plays an important role in salt tolerance in WT calluses, and ethylene may be acting downstream of H₂O₂.

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14 degrees C were measured at 25 degrees C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30 degrees C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14 degrees C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14 degrees C after growth at 30 degrees C for 2 days had the same alternative oxidase capacity as seedlings grown at 30 degrees C. Seedlings grown at 14 degrees C for 10 to 12 days, then transferred to 30 degrees C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20 degrees C. At 41 degrees C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41 degrees C was reversible. After incubation at 41 degrees C, the alternative oxidase capacity measured at 25 degrees C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41 degrees C as they did when incubated at 25 degrees C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.

The callus of Nicotiana rustica cv Gansu yellow flower and N. tabacum cv willow leaf were cultured on ordinary subculture medium (M-1) and on regeneration medium (M-2), respectively. No differentiation was observed in Gansu yellow flower tobacco callus cultures grown on both M-1 and M-2 medium. The respiration of both cultures was partially resistant to cyanide and markedly inhibited by m-chlorobenzhydroxamic acid. The relative contributions of alternative and cytochrome pathway were 31% and 47% of the total respiration, respectively, in M-1 callus cultures. The relative O(2) uptake of the two pathways was not changed significantly in M-2 callus cultures. In subcultured M-1 callus cultures of Willow leaf tobacco, the respiration mediated via alternative pathway was about 29 to 38% of the total respiration, and the cytochrome pathway still was the major respiratory pathway. In M-2 callus cultures in which differentiation occurred, the relative contribution of alternative pathway increased to 41 to 47% of the total respiration, and the cytochrome pathway decreased considerably. These results suggested that the change of respiratory electron transport pathway was probably related to the differentiation of tobacco callus cultures.

Wild-type and cytochrome-deficient strains of Schwanniomyces castellii were cultivated aerobically on citrate, and the effects of respiratory inhibitors on respiration, ATP levels and energy charge were measured. The obtained results demonstrate that every respiratory pathway of Schwanniomyces castellii involves phosphorylation site I, and suggest that the functioning of this site allows the alternative pathways to produce ATP.

We investigated the differential expression of AOX1 multi-gene family and the regulation of alternative respiratory pathway during initial greening development in leaves of rice (Oryza sativa L.) seedlings. After exposing the dark-grown rice seedlings to continuous irradiation, total respiration (Vt), capacity of alternative pathway (Valt), and their ratio (Valt/Vt) increased with the greening of leaves. In this process, AOX1c transcript increased under constant irradiation, while AOX1a and AOX1b transcripts were hardly detected. Thus AOX1c in rice presents a similar expression pattern as AOX2 does in many dicotyledonous species during greening development. Compared with the rapid increase of cyanideresistant respiration in the presence of photon energy, CO2 fixation was not observed until 8 h after the onset of irradiation. The AOX inhibitor salicylhydroxamic acid (SHAM; 1 mM) inhibited 67.3 % of cyanide-insensitive oxygen uptake in dark-grown leaves and 69.4 % of it in leaves grown under irradiation. Dark-grown plants pre-treated with SHAM were then irradiated for 12 h. SHAM did not obviously modify photosynthetic CO2 fixation rate on a chlorophyll (Chl) content basis in both leaves and simultaneously isolated chloroplasts. Hence during initial greening steps of the plants, the induction of alternative pathway and AOX1 expression by irradiation is not directly linked with carbon assimilation of photosynthesis. The application of SHAM partially limited Chl production in rapidly greening leaves, indicating that Chl synthesis in the process of greening might be medicated to some extent by alternative respiratory pathway.

Plants have a non-energy conserving bypass of the classical mitochondrial cytochrome c pathway, known as the alternative respiratory pathway (AP). This involves type II NAD(P)H dehydrogenases (NDs) on both sides of the mitochondrial inner membrane, ubiquinone, and the alternative oxidase (AOX). The AP components have been widely characterised from Arabidopsis, but little is known for monocot species. We have identified all the genes encoding components of the AP in rice and barley and found the key genes which respond to oxidative stress conditions. In both species, AOX is encoded by four genes; in rice OsAOX1a, 1c, 1d and 1e representing four clades, and in barley, HvAOX1a, 1c, 1d1 and 1d2, but no 1e. All three subfamilies of plant ND genes, NDA, NDB and NDC are present in both rice and barley, but there are fewer NDB genes compared to Arabidopsis. Cyanide treatment of both species, along with salt treatment of rice and drought treatment of barley led to enhanced expression of various AP components; there was a high level of co-expression of AOX1a and AOX1d, along with NDB3 during the stress treatments, reminiscent of the co-expression that has been well characterised in Arabidopsis for AtAOX1a and AtNDB2.

During dark-induced senescence isovaleryl-CoA dehydrogenase (IVDH) and D-2-hydroxyglutarate dehydrogenase (D-2HGDH) act as alternate electron donors to the ubiquinol pool via the electron-transfer flavoprotein/electron-transfer flavoprotein:ubiquinone oxidoreductase (ETF/ETFQO) pathway. However, the role of this pathway in response to other stresses still remains unclear. Here, we demonstrated that this alternative pathway is associated with tolerance to drought in Arabidopsis. In comparison with wild type (WT) and lines overexpressing D-2GHDH, loss-of-function etfqo-1, d2hgdh-2 and ivdh-1 mutants displayed compromised respiration rates and were more sensitive to drought. Our results demonstrated that an operational ETF/ETFQO pathway is associated with plants' ability to withstand drought and to recover growth once water becomes replete. Drought-induced metabolic reprogramming resulted in an increase in tricarboxylic acid (TCA) cycle intermediates and total amino acid levels, as well as decreases in protein, starch and nitrate contents. The enhanced levels of the branched-chain amino acids in loss-of-function mutants appear to be related to their increased utilization as substrates for the TCA cycle under water stress. Our results thus show that mitochondrial metabolism is highly active during drought stress responses and provide support for a role of alternative respiratory pathways within this response.

Potentiation of factor H by heparin: A rate-limiting mechanism for inhibition of the alternative complement pathway