Antioxidant function of alternative oxidase in mitochondria of winter wheat during cold hardening

The activity of alternative oxidase (AOX) and generation of reactive oxygen species (ROS) in mitochondria of winter wheat Triticum aestivum L. isolated from seedlings subjected to one (7-day exposure to 2–3°C) and two (7-day exposure to 2–3°C and 2-day exposure to −2°C) phases of a cold hardening has been studied. The antioxidant role of AOX in the first phase of the cold hardening has been determined using inhibitors of respiratory chain. Exposure to low temperature was shown to lead to inhibition of cytochrome pathway in mitochondria, increase of ROS production, and switching of the electron transport to the alternative pathway. Decrease in succinate- and antimycin A-induced ROS generation was found during two phases of cold hardening. This fact may point out to functioning of uncoupling proteins under these conditions. Thus, antioxidant function of AOX during the first phase of cold hardening may be an important component of the cold adaptation mechanism in winter crops. The data suggest that ROS and free fatty acids may be signal molecules regulating the activity of two energy-dissipation systems (AOX and uncoupling proteins).     

Possible plant mitochondria involvement in cell adaptation to drought stress. A case study: durum wheat mitochondria

Although plant cell bioenergetics is strongly affected by abiotic stresses, mitochondrial metabolism under stress is still largely unknown. Interestingly, plant mitochondria may control reactive oxygen species (ROS) generation by means of energy-dissipating systems. Therefore, mitochondria may play a central role in cell adaptation to abiotic stresses, which are known to induce oxidative stress at cellular level. With this in mind, in recent years, studies have been focused on mitochondria from durum wheat, a species well adapted to drought stress. Durum wheat mitochondria possess three energy-dissipating systems: the ATP-sensitive plant mitochondrial potassium channel (PmitoK(ATP)); the plant uncoupling protein (PUCP); and the alternative oxidase (AOX). It has been shown that these systems are able to dampen mitochondrial ROS production; surprisingly, PmitoK(ATP) and PUCP (but not AOX) are activated by ROS. This was found to occur in mitochondria from both control and hyperosmotic-stressed seedlings. Therefore, the hypothesis of a 'feed-back' mechanism operating under hyperosmotic/oxidative stress conditions was validated: stress conditions induce an increase in mitochondrial ROS production; ROS activate PmitoK(ATP) and PUCP that, in turn, dissipate the mitochondrial membrane potential, thus inhibiting further large-scale ROS production. Another important aspect is the chloroplast/cytosol/mitochondrion co-operation in green tissues under stress conditions aimed at modulating cell redox homeostasis. Durum wheat mitochondria may act against chloroplast/cytosol over-reduction: the malate/oxaloacetate antiporter and the rotenone-insensitive external NAD(P)H dehydrogenases allow cytosolic NAD(P)H oxidation; under stress this may occur without high ROS production due to co-operation with AOX, which is activated by intermediates of the photorespiratory cycle.     

Influence of simulated snow cover on the cold tolerance and freezing injury of yellow-cedar seedlings

It has been hypothesized that yellow‐cedar [Chamaecyparis nootkatensis (D. Don) Spach] decline may result from root freezing injury following climate change‐induced reductions in protective snow cover. To test this hypothesis, we measured the freezing tolerance and injury expression of yellow‐cedar seedlings in three treatments that differed in the insulative protection they provided to soils during winter and spring: (1) full exposure to ambient temperatures (exposed treatment), (2) continuous protection from ambient temperatures via addition of perlite over pots (full protection), and (3) perlite protection only during winter and exposure to ambient temperatures during spring (partial protection). Foliage from all treatments was cold tolerant enough to prevent foliar freezing injury throughout the study period. However, on all sample dates, roots of seedlings from all treatments were only tolerant to about ?5 °C – a level considerably warmer than the reported maximum cold tolerance for the species and well above the soil temperature recorded in the exposed treatment. As a result of this limited root cold tolerance, visibly uninjured roots of seedlings from the exposed treatment had significantly higher relative electrolyte leakage (REL) throughout the winter and early spring than seedlings in soil protection treatments. Seedlings from the exposed treatment also had significantly higher foliar REL values and greater visual foliar injury than seedlings from the other treatments starting in early spring. For both roots and foliage, REL measurements consistently detected tissue damage before visual injury was evident. Patterns of injury from both REL and visual injury assessments showed the same pattern: damage began with freezing injury to roots and subsequently became evident as foliar browning after spring temperatures increased. All seedlings in the exposed treatment eventually had 100% fine root damage and died. This progression of initial root damage followed by foliar browning and mortality after the onset of warming conditions is consistent with reports of yellow‐cedar decline symptom development in the field.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

The plant uncoupling protein homologues: a new family of energy-dissipating proteins in plant mitochondria.

Uncoupling proteins (UCPs) form a subfamily within the mitochondrial carrier protein family, which catalyze a free fatty acid-mediated proton recycling and can modulate the tightness of coupling between mitochondrial respiration and ATP synthesis. As in mammalian tissues, UCPs are rather ubiquitous in the plant kingdom and widespread in plant tissues in which they could have various physiological roles, such as heat production or protection against free oxygen radicals. The simultaneous occurrence in plant mitochondria of two putative energy-dissipating systems, namely UCP which dissipates the proton motive force, and alternative oxidase (AOX) which dissipates the redox potential, raises the question of their functional interactions.     

Involvement of the energy-dissipating systems in modulating the energetic efficiency of respiration in mitochondria from etiolated winter wheat seedlings

Effects of cyanide-resistant alternative oxidase (AOX) and modulators of plant uncoupling mitochondrial proteins (PUMP) on respiration rate and generation of transmembrane electric potential (ΔΨ) were investigated during oxidation of various substrates by isolated mitochondria from etiolated coleoptiles of winter wheat (Triticum aestivum L.). Oxidative phosphorylation in wheat mitochondria during malate and succinate oxidation was quite effective (it was characterized by high respiratory control ratio as defined by Chance, high ADP/O ratio, and rapid ATP synthesis). Nevertheless, the effectiveness of oxidative phosphorylation was substantially modulated by operation of energy-dissipating systems. The application of safranin dye revealed the partial dissipation of ΔΨ during inhibition of cytochrome-mediated malate oxidation by cyanide and antimycin A and demonstrated the operation of AOX-dependent compensatory mechanism for ΔΨ generation. The complex I of mitochondrial electron transport chain was shown to play the dominant role in ΔΨ generation and ATP synthesis during AOX functioning upon inhibition of electron transport through the cytochrome pathway. Effects of linoleic acid (PUMP activator) at physiologically low concentrations (4–10 μM) on respiration and ΔΨ generation in mitochondria were examined. The uncoupling effect of linoleic acid was shown in activation of the State 4 respiration, as well as in ΔΨ dissipation; this effect was eliminated in the presence of BSA but was insensitive to purine nucleotides. The uncoupling effect of linoleic acid was accompanied by reversible inhibition of AOX activity. The results are discussed with regard to possible physiological role of mitochondrial energy-dissipating systems in regulation of energy transduction in plant cells under stress conditions.     

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat was studied by rocket-immunoelectrophoresis and radioactive label. The 1-h low-temperature stress was found to result in an increase in the content of this protein at both above- and below-zero temperatures. It was found that the increase in the relative content of the protein with mol. wt 310 kD, under the effect of short-term low-temperature stress, occurs due to induction of its synthesis. It has been found that during cold hardening of winter wheat the content of this protein decreases up to 64% compared to the control during the first day of hardening but starts to rise by the third day and reaches 179% by the seventh day, which is well correlated with the increase in cold resistance of winter wheat plants during cold hardening.     

Energy conservation and dissipation in mitochondria isolated from developing tomato fruit of ethylene-defective mutants failing normal ripening: the effect of ethephon,a chemical precursor of ethylene

Alternative oxidase (AOX) and uncoupling protein (UCP) are present simultaneously in tomato fruit mitochondria. In a previous work, it has been shown that protein expression and activity of these two energy-dissipating systems exhibit large variations during tomato fruit development and ripening on the vine. It has been suggested that AOX and UCP could be responsible for the respiration increase at the end of ripening and that the cytochrome pathway could be implicated in the climacteric respiratory burst before the onset of ripening. In this study, the use of tomato mutants that fail normal ripening because of deficiencies in ethylene perception or production as well as the treatment of one selected mutant with a chemical precursor of ethylene have revealed that the bioenergetics of tomato fruit development and ripening is under the control of this plant hormone. Indeed, the evolution pattern of bioenergetic features changes with the type of mutation and with the introduction of ethylene into an ethylene-synthesis-deficient tomato fruit mutant during its induced ripening.     

Cold tolerance and cold hardening strategy of the Japanese pine sawyer Monochamus alternatus (Coleoptera: Cerambycidae)

The Japanese pine sawyer, Monochamus alternatus , is an important pine forest pest and vector transmitting the pine wilt nematode that causes pine wilt disease. Low temperatures in autumn, winter and spring often differentially affect mortality of M. alternatus larvae. In this paper, we mainly compared the differences of mortality and cold hardening of larvae from different seasons, based on supercooling point (SCP) and cumulative probability of individuals freezing (CPIF). The cold hardening of the larvae from autumn, winter and spring seasons were largely different. Correlations between mortality and CPIF of autumn and spring larvae were highest on day 1/4, and gradually decreased with prolonged exposure duration. This beetle's death mainly resulted from freezing in short exposure duration. However, the correlation between mortality and CPIF of winter larvae increased gradually with the prolonged exposure duration. Death did not mainly result from freezing in long exposure duration. Autumn larvae are more susceptible and adaptable than winter and spring larvae. Winter larvae have a slight freeze-tolerance trend. Our research showed that M. alternatus came into complex cold-hardening strategies under natural selection. Freeze avoidance is the primary strategy; with prolonged exposure duration to above SCP or < 0 °C, chill tolerance is more important; this is followed by freeze tolerance during harsh winters.     

Free and Bound Gibberellin Activities and Ent-kaurene Synthesis during Induction of Cold Hardiness in Black Locust Seedlings

The role of gibberellin in the development of cold hardiness in black locust (Robinia pseudoacacia L.) seedlings was investigated. Free and bound gibberellin activities were followed during induction of cold hardiness using ethyl acetate partitioning and pH variation, with subsequent paper chromatographic fractionation and gibberellin bioassay. While total gibberellin activity decreased during the induction of cold hardiness in black locust seedlings, no convincing evidence was found to support conversion of free gibberellin to a bound form. However, bound gibberellin activity did appear to be more stable than did free gibberellin activity during the final stages of cold hardening at freezing temperatures. Gibberellin synthesis was followed using ¹⁴C-mevalonate conversion to ent-kaurene in a cell-free extract of the tissue. Ent-kaurene synthesis decreased during cold hardening with no detectable synthesis in fully hardened seedlings. However, since growth cessation precedes development of cold hardiness, decreased gibberellin synthesis and corresponding trends in free and bound fractions might have been expected, and a cause and effect relationship is difficult to establish. Even so, a decline in one step in gibberellin synthesis and a greater stability of bound than free gibberelin activity are associated with induction of cold hardiness in black locust seedlings.     

Adaptation of membrane fluidity of rye wheat seedlings according to temperature

Order parameters of chloroplast membrane lipids of rye wheat seedlings differing in cold hardiness were compared before after hardening. Seedlings grown at 25° exhibited similar membrane microviscosities. When hardened, the cultivars most resistant to freezing temperatures possessed the most fluid membranes, while those sensitive to cold were unable to alter them. Changes in linolenic acid levels alone cannot be responsible for the observed phenomena.     

Changes in plant mitochondrial electron transport alter cellular levels of reactive oxygen species and susceptibility to cell death signaling molecules

Transgenic tobacco (Nicotiana tabacum) lacking mitochondrial alternative oxidase (AOX) have been compared with wild-type (Wt) tobacco using two different systems, either suspension cell cultures or leaves. In both systems, a lack of AOX was accompanied by an increase in some anti-oxidant defenses, consistent with the hypothesis that a lack of AOX increases the mitochondrial generation of reactive oxygen species (ROS). In most cases, this increase in anti-oxidant defenses could more than offset the presumed increased rate of ROS generation, resulting paradoxically in a lower steady-state level of ROS than was found in Wt leaves or suspension cells. We also found that the amount of cell death induced by salicylic acid or nitric oxide correlated strongly with the level of ROS (irrespective of the level of AOX), while death induced by azide was dependent upon the presence or absence of AOX. These results suggest that susceptibility to cell death by signaling molecules (salicylic acid and nitric oxide) is dependent upon the steady-state cellular level of ROS and that AOX levels clearly contribute to this steady state, perhaps by influencing the rate of mitochondrial-generated ROS and hence the cellular level of anti-oxidant defenses.     

Conversion of Phosphatidyl Choline to Phosphatidic Acid in Freeze Injured Rye and Wheat Cultivars

The effect of exposure to freezing temperature (?15°C) on leaf phospholipid composition of hardened rye (Secale cereale L.) and hardened wheat cultivars (‘Miranovskaja 808’, ‘Bezostaja 1’, ‘Short Mexican’ and ‘Penjamo 62’), which differ in their resistance to frost, was investigated. Hardening took place under natural conditions. All the seedlings attained an equal level of linolenic acid in their leaves during hardening. Exposure to freezing temperatures resulted in a loss of phosphatidyl choline and accumulation of phosphatidic acid in the leaves. The ratio of phosphatidic acid to phosphatidyl choline, but not the level of poly-unsaturated fatty acids in the leaves, was related to their ability to survive at low temperatures. As freezing injury is caused by the formation of ice crystals in both extra- and intracellular space, it is probable that the plasma membranes of the investigated cultivars differed with respect to their water permeability. It is concluded that the plants, depending on the degree of their resistance to cold, produce an unknown substance of lipidic nature upon exposure to cold, with the aid of which they adjust the transitional state of their membranes to the prevailing temperature and, at the same time, facilitate the efflux of water from the cells.     

Responses of the bed bug,Cimex lectularius,to temperature extremes and dehydration: levels of tolerance,rapid cold hardening and expression of heat shock proteins

This study of the bed bug, Cimex lectularius, examines tolerance of adult females to extremes in temperature and loss of body water. Although the supercooling point (SCP) of the bed bugs was approximately −20°C, all were killed by a direct 1 h exposure to −16°C. Thus, this species cannot tolerate freezing and is killed at temperatures well above its SCP. Neither cold acclimation at 4°C for 2 weeks nor dehydration (15% loss of water content) enhanced cold tolerance. However, bed bugs have the capacity for rapid cold hardening, i.e. a 1‐h exposure to 0°C improved their subsequent tolerance of −14 and −16°C. In response to heat stress, fewer than 20% of the bugs survived a 1‐h exposure to 46°C, and nearly all were killed at 48°C. Dehydration, heat acclimation at 30°C for 2 weeks and rapid heat hardening at 37°C for 1 h all failed to improve heat tolerance. Expression of the mRNAs encoding two heat shock proteins (Hsps), Hsp70 and Hsp90, was elevated in response to heat stress, cold stress and during dehydration and rehydration. The response of Hsp90 was more pronounced than that of Hsp70 during dehydration and rehydration. Our results define the tolerance limits for bed bugs to these commonly encountered stresses of temperature and low humidity and indicate a role for Hsps in responding to these stresses.     

The energy-conserving and energy-dissipating processes in mitochondria isolated from wild type and nonripening tomato fruits during development on the plant

Bioenergetics of tomato (Lycopersicon esculentum) development on the plant was followed from the early growing stage to senescence in wild type (climacteric) and nonripening mutant (nor, nonclimacteric) fruits. Fruit development was expressed in terms of evolution of chlorophyll a content allowing the assessment of a continuous time-course in both cultivars. Measured parameters: the cytochrome pathway-dependent respiration, i.e., the ATP synthesis-sustained respiration (energy-conserving), the uncoupling protein (UCP) activity-sustained respiration (energy-dissipating), the alternative oxidase(AOX)-mediated respiration (energy-dissipating), as well as the protein expression of UCP and AOX, and free fatty acid content exhibited different evolution patterns in the wild type and nor mutant that can be attributed to their climacteric/nonclimacteric properties, respectively. In the wild type, the climacteric respiratory burst observed in vitro depended totally on an increse in the cytochrome pathway activity sustained by ATP synthesis, while the second respiratory rise during the ripening stage was linked to a strong increase in AOX activity accompanied by an overexpression of AOX protein. In wild type mitochondria, the 10-M linoleic acid-stimulated UCP-activity-dependent respiration remained constant during the whole fruit development except in senescence where general respiratory decay was observed.     

Photosynthetic acclimation to low temperature by western red cedar seedlings

Imposition of low, but above freezing, temperatures resulted in a gradual increase in the cold hardiness of western red cedar seedlings. This was associated with a decrease in the maximum rates of photosynthetic CO₂ fixation and O₂ evolution, and changes in chlorophyll a fluorescence transients which indicated that photoinhibition had occurred. Maximum photosynthetic rates declined approximately 40% during cold hardening. The leaves changed colour from green to red-brown during the hardening process. The colour change was due to the synthesis of large amounts of the carotenoid rhodoxanthin. Lutein levels doubled, while chlorophyll declined slightly. Dehardening resulted in the rapid recovery of photosynthesis to control levels, the rapid disappearance of rhodoxanthin, and the return of lutein levels to control. It is suggested that rhodoxanthin accumulation at low temperature functions to decrease the light intensity reaching the photosynthetic apparatus. The combination of photoinhibition and rhodoxanthin synthesis probably serves to protect the photosynthetic capacity of the seedlings at low temperature.     

Acute and chronic cold exposure differentially affects the browning of porcine white adipose tissue

Piglets are characteristically cold intolerant and thus susceptible to high mortality. However, browning of white adipose tissue (WAT) can induce non-shivering thermogenesis as a potential strategy to facilitate the animal’s response to cold. Whether cold exposure can induce browning of subcutaneous WAT (sWAT) in piglets in a similar manner as it can in humans remains largely unknown. In this study, piglets were exposed to acute cold (4°C, 10 h) or chronic cold exposure (8°C, 15 days), and the genes and proteins of uncoupling protein 1 (UCP1)-dependent and independent thermogenesis, mitochondrial biogenesis, lipogenic and lipolytic processes were analysed. Interestingly, acute cold exposure induced browning of porcine sWAT, smaller adipocytes and the upregulated expression of UCP1, PGC1α, PGC1β, C/EBPβ, Cidea, UCP3, CKMT1 and PM20D1. Conversely, chronic cold exposure impaired the browning process, reduced mitochondrial numbers and the expression of browning markers, including UCP1, PGC1α and PRDM16. The present study demonstrated that acute cold exposure (but not chronic cold exposure) induces porcine sWAT browning. Thus, browning of porcine sWAT could be a novel strategy to balance the body temperature of piglets, and thus could be protective against cold exposure.