Reversibility of chilling injury to corn seedlings

Seedlings of corn (Zea mays) were tested for recovery from chilling injury incurred at 0.3 ± 0.3 C. At 0.3 C visual leaf injury appeared in 36 hours, whereas stem and root injuries appeared later. Appearance of leaf injury was preceded by a rise in O₂ uptake and a lessened effect of 2,4-dinitrophenol on O₂ uptake by leaf segments and was accompanied by increased ion leakage from the leaves. These effects were reversible, in that transfer of seedlings to 21 C after 36 hours at 0.3 C produced a return of O₂ uptake, 2,4-dinitrophenol stimulation, and ion leakage to the levels of unchilled leaves, as well as a disappearance of leaf symptoms, within 72 hours. For most seedlings, transfer to 21 C after 48 to 60 hours at 0.3 C reversed the chilling effects on O₂ uptake, 2,4-dinitrophenol stimulation, and injury symptoms but not on ion leakage within 108 hours. However, some seedlings collapsed during 48 to 60 hours of chilling, and these never recovered. Transfer to 21 C after 72 hours at 0.3 C did not produce recovery from any symptom of chilling injury examined, and these seedlings soon died. No growth occurred at 0.3 C, but growth began soon after transfer to 21 C. Seedlings chilled 24 or 36 hours grew at reduced rates during the first 72 hours at 21 C, but within 96 hours at 21 C were growing at the same rate as nonchilled seedlings. These results demonstrate considerable capacity of growing plants to recover from short chilling treatments even though significant physiological changes occurred at low temperatures.     

Alternative Respiratory Path Capacity in Plant Mitochondria: Effect of Growth Temperature, the Electrochemical Gradient, and Assay pH

Influence of growth temperature on the capacity of the mitochondrial alternative pathway of electron transport was investigated using etiolated corn (Zea mays L.) seedlings. These seedlings were grown to comparable size in either a warm (30°C) or a cold (13°C) temperature regime, and then their respiration rates were measured as O₂ uptake at 25°C. The capacity of the alternative pathway (KCN-insensitive O₂ uptake) was found essentially to double in shoots of cold-grown seedlings. This increased capacity slowly developed over several days growth in the cold, but was lost within 1 day when the seedlings were exposed to a warm regime. When mitochondria were isolated from the shoots of these seedlings, a greater potential for flow through the alternative path was observed in mitochondria from the cold-grown seedlings with all substrates used (an average increase of 84%). Using exogenous NADH as the substrate, the effect of the electrochemical gradient on measurable capacities of the cytochrome and alternative pathways was investigated in mitochondria from both etiolated seedlings and thermogenic spadices. The uncoupler FCCP (p-trifluoromethoxycarbonylcyanide phenylhydrazone) was used to diminish the electrochemical gradient when desired. In corn (Zea mays L.) shoot and mung bean (Vigna radiata L.) hypocotyl mitochondria, which have relatively low capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the electrochemical gradient was found not to influence the potential for flow through the alternative path. However, in mitochondria from skunk cabbage (Symplocarpus foetidus L.) and voodoo lily (Sauromatum guttatum Schott) spadices, which have high capacities of the alternative pathway, increased flow through the cytochrome chain in the absence of the gradient occurred at the expense of flow through the alternative pathway. These results suggest that in mitochondria of thermogenic spadices, the combined capacities of the cytochrome and alternative paths exceed the capacity of the exogenous NADH dehydrogenase. The effect of assay pH on measurable capacities of the cytochrome and alternative paths was determined over a pH range of 5.6 to 8.8 using exogenous NADH as the mitochondrial substrate. When the electrochemical gradient was present, it limited the electron transport rate and little effect of assay pH was observed. However, when formation of the gradient was prevented through inclusion of FCCP, measurable capacities of the cytochrome and alternative paths were found to be greatly influenced by pH. This experiment also revealed that the potential for respiratory control is largely dependent upon the assay pH.     

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.

     

Effect of photosynthesis and carbohydrate status on respiratory rates and the involvement of the alternative pathway in leaf respiration

In spinach (Spinacia oleracea Hybrid 102 [New World seeds]) and wheat (Triticum aestivum L. cv Gabo) leaves, O₂ uptake rates in the dark were faster after the plants had been allowed to photosynthesize for a period of several hours. Alternative path activity also increased following a period of photosynthesis in these leaves. No such effects were observed with isolated mitochondria. In spinach and wheat leaves, the level of fructose plus glucose decreased during a period of darkness. In pea (Pisum sativum cv Alaska) leaves, the level of these sugars did not vary significantly during the day, and respiratory rates were also constant. In slices cut from wheat leaves harvested at the end of the night, addition of sugars increased the rate of respiration and engaged the previously latent alternative oxidase. In pea leaves, O₂ uptake in the first few minutes following illumination was faster than that observed before illumination, but declined during the next 15 to 20 minutes. Adding the alternative oxidase inhibitor salicylhydroxamic acid, or imposing high bicarbonate concentrations during the period of photosynthesis, prevented the rise in O₂ uptake rate during the immediate post illumination period.

We conclude that the level of respiratory substrate in leaves determines their rate of O₂ uptake, and the degree to which the alternative path contributes to that O₂ uptake.

     

Response to chilling of tomato seedlings and cells in suspension cultures

Tomato cell suspensions and seedlings (Lycopersicon esculentum) responded comparably when exposed to chilling temperatures (10 C or below). Seedling growth and cellular activities related to cell viability and culture growth (triphenyltetrazolium chloride reduction, fluoroscein diacetate uptake, and hydrolysis) were sharply diminished below 10 C. Arrhenius plots of the respiratory O₂ consumption by both seedlings and cell suspensions had a break at 10 C, as is characteristic for chilling-sensitive species. The acyl chains that were found in the phospholipids of both cell cultures and seedlings were similar. These results indicate the potential usefulness of plant suspension cultures for studies of chilling injury.     

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.     

limitation of Alternative Respiratory Pathway Activity in Grapefruit Flavedo Tissue by Oxygen Availability

The capacity of the alternative respiratory pathway increased in the flavedo tissue of `Marsh' grapefruit (Citrus paradisi Macf.) stored at 5°C for 2 weeks or longer. Elevated O₂ levels during respiratory assays enhanced respiration by the tissue at 20°C but not at 5°C. At 20°C, salicylhydroxamic acid alone was inhibitory to O₂ uptake only in elevated O₂. In conventional Warburg studies, alternative pathway respiration may be limited by the low solubility and/or slow rate of O₂ 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.     

Changes in electron transport,superoxide dismutase and ascorbate peroxidase isoenzymes in chloroplasts and mitochondria of cucumber leaves as influenced by chilling

In order to clarify the relationship between chill-induced disturbance in photosynthetic, respiratory electron transport and the metabolism of reactive oxygen species (ROS), leaf gas exchange, chlorophyll fluorescence quenching, respiration, and activities of superoxide dismutase (SOD) and ascorbate peroxidase (APX) were investigated in chloroplasts and mitochondria of cucumber (Cucumis sativus) leaves subjected to a chill (8 °C) for 4 d. Chilling decreased net photosynthetic rate (P _N) and quantum efficiency of photosystem 2 (Φ_PS2), but increased the ratio of Φ_PS2 to the quantum efficiency of CO₂ fixation (ΦCO₂) and non-photochemical quenching (NPQ) in cucumber leaves. While chilling inhibited the activity of cytochrome respiration pathway, it induced an increase of alternative respiration pathway activity and the reduction level of Q-pool. Chilling also significantly increased O₂ ^• production rate, H₂O₂ content, and SOD and APX activities in chloroplasts and mitochondria. There was a more significant increase in SOD and APX activities in chloroplasts than in mitochondria with the increase of membrane-bound Fe-SOD and tAPX in chloroplasts being more significant than other isoenzymes. Taken together, chilling inhibited P _N and cytochrome respiratory pathway but enhanced the photosynthetic electron flux to O₂ and over-reduction of respiratory electron transport chain, resulting in ROS accumulation in cucumber leaves. Meanwhile, chilling resulted in an enhancement of the protective mechanisms such as thermal dissipation, alternative respiratory pathway, and ROS-scavenging mechanisms (SODs and APXs) in chloroplasts and mitochondria.     

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%.     

A Comparative Study of CN-Resistant Respiration in Different Cultures of Tobacco Callus

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 O₂ 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.     

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H₂O₂) accumulation. The application of exogenous SA and the chemical scavenger of H₂O₂ reduces the excess H₂O₂ and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H₂O₂ and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H₂O₂ levels.     

Spontaneous Chemiluminescence of Soybean Embryonic Axes during Imbibition

Isolated soybean (Glycine max L. var Hood) embryonic axes have a spontaneous chemiluminescence (about 150 counts per minute per embryo) that increases showing two phases, upon water imbibition. The first photoemission burst was measured between 0 and 7 hours of imbibition with a maximum of about 350 counts per minute per embryo after 2 hours. The second photoemission phase, between 7 and 30 hours, increased from about 220 to 520 counts per minute per embryo. Both chemiluminescence phases were inhibited by infused butylated hydroxyanisole while only the second phase was inhibited by infused salicylhydroxamic acid. On the basis of the sensitivity of the lipoxygenase reaction to both inhibitors (about 90%), the first burst is tentatively assigned to oxy-radicals mobilized upon water uptake by the embryonic axes, and the second phase is tentatively identified as due to lipoxygenase activity. The in vivo lipoxygenase activity of the embryonic axes was estimated by both the fraction of total oxygen uptake that was inhibited by butylated hydroxyanisole and by the fraction of photoemission that was inhibited by butylated hydroxyanisole and by salicylhydroxamic acid. Both approaches indicated marked increases (5-fold and 12-fold, respectively) of lipoxygenase activity between 2 and 30 hours of imbibition. The measured chemiluminescence per O₂ uptake ratio (the experimental quantum yield) for the lipoxygenase reaction (3.3 × 10⁻¹⁴ counts per O₂ molecule) was used to estimate the O₂ uptake due to lipoxygenase activity from the photoemission of the embryonic axes after 30 hours of imbibition. The value (0.54 microliters per minute per axis) was close to the butylated hydroxyanisole-sensitive O₂ uptake (1.2 microliters O₂ per minute per axis) of the same embryonic axes. Chemiluminescence may afford a noninvasive assay for lipoxygenase activity in intact plant tissues.     

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.     

Wounding Stimulates Cyanide-sensitive Respiration in the Highly Cyanide-resistant Leaves of Bryophyllum tubiflorum Harv

The rate of respiration in sectioned leaves of Bryophyllum tubiflorum Harv. increases with decreasing section thickness. The rates of uninhibited respiration in 2- and 8-millimeter-thick sections are 74 and 46 microliters of O₂ per gram fresh weight of unruptured tissue per hour at 20 C, whereas the rate in the presence of cyanide is 31 microliters of O₂ in each case. The rates are unaffected by salicylhydroxamic acid, but cyanide and salicylhydroxamic acid together completely eliminate O₂ uptake. The capacity of the alternative respiratory pathway is thus initially high (estimated at 84% of the uninhibited respiratory rate in whole leaves) and remains constant but probably unexpressed subsequent to the rapid induction of wound respiration.     

Fusicoccin Counteracts the n-Ethylmaleimide and Silver-Induced Stimulation of Oxygen Uptake in Egeria densa Leaves

It was previously shown that a number of sulfhydryl [SH] group reagents (N-ethylmaleimide [NEM], iodoacetate, Ag⁺, HgCl₂, etc.) can induce a marked, transitory stimulation of O₂ uptake (QO₂) in Egeria densa leaves, insensitive to CN⁻ and salicylhydroxamic acid and inhibited by diphenylene iodonium and quinacrine. The phytotoxin fusicoccin (FC) also induces a marked increase in O₂ consumption in E. densa leaves, apparently independent of the recognized stimulating action on the H⁺-ATPase. In this investigation we compared the FC-induced increase in O₂ consumption with those induced by NEM and Ag⁺, and we tested for a possible interaction between FC and the two SH blockers in the activation of QO₂. The results show (a) the different nature of the FC- and NEM- or Ag⁺-induced increases of QO₂; (b) that FC counteracts the NEM- (and Ag⁺)-induced respiratory burst; and (c) that FC strongly reduces the damaging effects on plasma membrane permeability observed in E. densa leaves treated with the two SH reagents. Two alternative models of interpretation of the action of FC, in activating a CN⁻-sensitive respiratory pathway and in suppressing the SH blocker-induced respiratory burst, are proposed.     

NaCl胁迫下交替呼吸途径对烟草悬浮细胞死亡调节作用的研究

盐分胁迫是植物在自然环境中经常遭遇的环境胁迫因素之一,会引起植物代谢紊乱乃至细胞死亡,这严重限制了植物的生长、繁育和生存。交替呼吸途径是植物较之动物独特的线粒体呼吸途径。该研究在烟草悬浮细胞中调查了交替呼吸途径对Na Cl胁迫引起的植物细胞死亡过程的调节作用及相应的内在机制,以及在200 mmol·L~(-1)Na Cl处理的烟草悬浮细胞中研究了交替呼吸途径和细胞死亡发生及H_2O_2之间的关系。结果表明:(1)随着Na Cl处理浓度的增加,烟草悬浮细胞死亡水平逐渐增加,而交替呼吸途径的容量也逐渐上升。(2)与Na Cl处理相似,外源H_2O_2的处理也能导致烟草悬浮细胞死亡水平的增加。200 mmol·L~(-1)Na Cl的胁迫导致明显的细胞死亡发生和H_2O_2产量的显著性增加;而较之200 mmol·L~(-1)Na Cl胁迫下的细胞,用水杨基氧肟酸(交替呼吸途径的抑制剂)预处理后的细胞再置于200 mmol·L~(-1)Na Cl的胁迫下导致更高水平的细胞死亡和H_2O_2的产生。综上表明,高盐胁迫诱导了烟草悬浮细胞的交替呼吸途径的增加,而交替呼吸途径则可能通过抑制活性氧的产生而起到缓解细胞死亡发生的作用。     

Cyanide-insensitive and Cyanide-sensitive O(2) Uptake in Wheat: I. GRADIENT-PURIFIED MITOCHONDRIA

The mitochondrial fraction isolated from durum wheat seedlings by differential centrifugation demonstrated antimycin A- or cyanide-insensitive O₂ uptake. Further purification of this initial mitochondrial pellet using a linear Percoll (Pharmacia) density gradient separated the mitochondria into two bands of physiologically distinct activity. Based on the usual mitochondrial respiratory criteria of ADP/O and respiratory control values, these fractions were qualitatively similar to the crude pellet. However, we observed no antimycin A-insensitive O₂ uptake in either gradient band. Antimycin A-insensitive O₂ consumption could be restored to the upper gradient band of mitochondria by the addition of linoleic acid. This activity was inhibited either by salicylhydroxamic acid or propyl gallate, a known lipoxygenase inhibitor. Likewise, addition of linoleic acid to the crude mitochondrial pellet elicited a 4- to 5-fold increase in O₂ uptake. This O₂ consumption was insensitive to antimycin A and cyanide but was inhibited by either propyl gallate or salicylhydroxamic acid. Electron microscopic examination revealed that only the lower gradient band contained contamination-free mitochondria, which, in turn, lacked ability to oxidize linoleic acid. Antimycin A-insensitive O₂ consumption in the differential centrifugation fraction from germinating durum wheat seedlings decreased over 64 hours of development.