Cold-Shock 310-kD Protein Uncouples Oxidative Phosphorylation in Plant Mitochondria

We studied the effects of cold-shock 310-kD protein (CSP310) isolated from winter rye seedlings on the energetic activity of plant mitochondria. CSP310 was shown to enhance nonphosphorylating respiration and uncoupled oxidative phosphorylation in isolated mitochondria. The uncoupling effect was enhanced with increasing protein concentration. An antibody against CSP310 interfered with the uncoupling effect of CSP310. Free fatty acids were not evidently involved in uncoupling. The physiological role of uncoupling between oxidation and phosphorylation during plant adaptation to low temperatures is discussed.     

Functioning of a CSP310 stress protein is related to the shunting of electron transfer along the respiratory chain of winter wheat mitochondria

Using three-day-old winter-wheat (Triticum aestivum L.) and six-day-old pea (Pisum sativum L.) seedlings as examples, we studied the effects of inhibitors of the electron transfer chain of plant mitochondria on the uncoupling between oxidation and phosphorylation brought about by the CSP310 stress protein. This uncoupling was inhibited by cyanide and by antibodies against CSP310, but not inhibited by antimycin A. It was shown that, in plant mitochondria, the CSP310 stress protein is involved in the electron transfer via shunting the major cytochrome pathway. In this case, the electron transfer bypasses complex II, ubiquinone, and complex III of the mitochondrial respiratory chain and is realized in the following succession: complex I-CSP310-cytochrome c-complex IV. This electron-transfer pathway was found in winter grass mitochondria during the low-temperature stress and resulted in thermogenesis. It was concluded that CSP310 is a thermogenic system, which is activated in winter grass mitochondria during the low-temperature stress.     

Stress-induced protein CSP 310: a third uncoupling system in plants

Addition of the cold-stress-related protein CSP 310 to mitochondria isolated from winter wheat ( Triticum aestivum L. cv. Zalarinka), winter rye ( Secale cereale L. cv. Dymka), maize ( Zea mays L. cv. VIR 36) and pea ( Pisum sativum L. cv. Marat) caused an increase in non-phosphorylative respiration. This increase was inhibited by KCN, indicating that the protein is not a CN-resistant alternative oxidase. Unlike plant mitochondrial uncoupling proteins such as PUMP, the uncoupling action of CSP 310 did not depend on the presence of free fatty acids in the incubation medium. We propose that the mechanism of the uncoupling action of CSP 310 differs from that of other known plant uncoupling systems and that the CSP 310 uncoupling system is a third uncoupling system in cereals.     

Non-phosphorylating bypass of the plant mitochondrial respiratory chain by stress protein CSP 310

Recently, it has been reported that the cold-stress protein CSP 310, discovered in the cytoplasm of cold-resistant winter cereals, causes uncoupling of oxidative phosphorylation during cold stress. To understand how the uncoupling mechanism of CSP differs from that of cyanide-insensitive alternative oxidase and plant mitochondrial uncoupling protein, we determined the effect of respiratory-chain inhibition on winter wheat (Triticum aestivum L. cv. Zalarinka) mitochondria. Our data show a possible involvement of stress protein CSP 310 in mitochondrial electron transport in winter wheat. CSP 310 shunts electrons around the main cytochrome pathway of the mitochondrial respiratory chain, i.e. electron flow bypasses ubiquinone and complex III via CSP 310 to complex IV.     

Complex I of winter wheat mitochondria respiratory chain is the most sensitive to uncoupling action of plant stress-related uncoupling protein CSP 310

The influence of stress uncoupling protein CSP 310 on functional stability of different mitochondrial respiratory chain complexes was analysed using various substrates of the tricarboxylic acid cycle. Complex I was the most sensitive to CSP 310 uncoupling action whilst other complexes were more stabile. It is proposed that the key point of CSP 310 uncoupling action is complex I of plant mitochondrial respiratory chain.     

The role of different plant seedling shoots mitochondrial uncoupling systems in thermogenesis during low-temperature stress

A difference was found between the temperature of control and heat-treated winter wheat and pea seedlings shoots during low temperature stress. Functioning of three thermogenic mitochondrial systems was established: (i) alternative cyanide-resistant oxidase, (ii) plant uncoupling mitochondrial protein and (iii) stress protein CSP 310 and these three caused the higher temperature of winter wheat control shoots. In peas only two thermogenic systems, the alternative cyanide-resistant oxidase and plant uncoupling mitochondrial proteins were found.     

Influence of CSP 310 and CSP 310-like proteins from cereals on mitochondrial energetic activity and lipid peroxidation in vitro and in vivo

Background  

The development of chilling and freezing injury symptoms in plants is known to frequently coincide with peroxidation of free fatty acids. Mitochondria are one of the major sources of reactive oxygen species during cold stress. Recently it has been suggested that uncoupling of oxidation and phosphorylation in mitochondria during oxidative stress can decrease ROS formation by mitochondrial respiratory chain generation. At the same time, it is known that plant uncoupling mitochondrial protein (PUMP) and other UCP-like proteins are not the only uncoupling system in plant mitochondria. All plants have cyanide-resistant oxidase (AOX) whose activation causes an uncoupling of respiration and oxidative phosphorylation. Recently it has been found that in cereals, cold stress protein CSP 310 exists, and that this causes uncoupling of oxidation and phosphorylation in mitochondria.     

Nature of the ligand bound to uncoupling CSP310 protein

It was shown that, in preparations of winter rye (Secale cereale L.) and winter wheat (Triticum aestivum L.), in proteins immunochemically related to a cold shock protein CSP310, and also in purified CSP310 from winter rye and triticale (Triticosecale X.), nucleic acid was present. Treatments with DNase and RNase showed that this nucleic acid was RNA. This protein-bound RNA was detected in the preparation of constitutively synthesized but not stress-induced protein. Stress-induced CSP310 bound high-molecular RNA in vitro at both 26 and 0°C, but it did not bind DNA. The data obtained permit an assumption that, during low-temperature stress, constitutively synthesized CSP310 with a low uncoupling capacity releases RNA and transits to a stress-induced form with a high uncoupling capacity.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 216–220.Original Russian Text Copyright © 2005 by Kolesnichenko, Tauson, Zykova, Klimenko, Grabelnykh, Pobezhimova.This revised version was published online in April 2005 with a corrected cover date.     

The comparison of uncoupling activity of constituently synthesised and stress-induced forms of winter rye stress uncoupling protein CSP 310

A difference between the uncoupling action of constituently synthesised and stress-induced forms of winter rye stress uncoupling protein CSP 310 on winter wheat mitochondria in vitro was found from the initiation of incubation. The uncoupling activity of CSP 310 depended on its concentration in the incubation media. The addition of anti-CSP 310 antiserum to isolated mitochondria from stressed winter rye shoots caused coupling of oxidation and phosphorylation. Western-blot analysis did not locate dehydrins K-segment in CSP 310 subunits.     

The Effect of CSP310 on Lipid Peroxidation and Respiratory Activity in Winter Wheat Mitochondria

Certain concentrations of the cold-shock protein (CSP310) were shown to induce systems of lipid peroxidation (POL) in winter wheat (Triticum aestivum L.) mitochondria in vitro. The process of nonenzymatic POL turned out to be the most sensitive to the presence of CSP310 in the incubation medium. The maximum induction of the enzymatic POL occurred at a higher CSP310 concentration. Wheat, maize (Zea mays L.), and elymus (Elymus sibiricus L.) proteins, which are immunochemically related to CSP310, did not manifest prooxidant properties, and, moreover, the elymus proteins had a clear-cut antioxidant effect. At the same time, these proteins uncoupled oxidation and phosphorylation to a far lesser extent than the winter rye (Secale cereale L.) CSP310. During low-temperature stress, the activation of uncoupling systems of wheat mitochondria by pyruvate, linoleic acid, and CSP310 was accompanied by an increase in oxygen consumption by seedlings and a decrease in the POL level.     

Plant stress uncoupling protein CSP 310 caused thermogenesis in winter wheat mitochondria in vitro

The addition of plant stress uncoupling protein CSP 310 has been found to increase thermogenesis in isolated winter wheat mitochondriain vitro during cold stress. On the other hand, the addition of anti-CSP 310 antiserum has been found to decrease thermogenesis in isolated winter wheat mitochondria. This result corresponds well to data about the influence of mitochondria incubation with CSP 310 and anti-CSP 310 antiserum on their energetic activity. It is supposed that CSP 310 uncouples the oxidative phosphorylation in mitochondria that causes thermogenesis during cold stress.     

The search for proteins with immunochemical affinity to plant stress proteins at cold-adapted endemic Baikal fishes

The search for proteins with immunochemical affinity to plant stress proteins in endemic Baikal fishes shows the presence of proteins, immunochemically related to plant heat-stabile proteins and plant uncoupling protein CSP 310. Western blotting showed that among the native cytoplasmic proteins of endemic Baikal fishes there are proteins immunochemically related to heat-stabile plant proteins with molecular weights about 480, 200-290, 150, 140 and about 90-100kD. SDS-electrophoresis showed the presence of polypeptides with molecular weights 23, 17 and 14kD in all species investigated and an additional 35kD polypeptide in Cottocomephorus grewingki. The search for polypeptides with immunochemical affinity to plant stress uncoupling protein CSP 310 in endemic Baikal fishes shows the presence of a 14kD polypeptide, immunochemically related to it.     

Winter wheat mitochondria functioning in vitro in the presence of calcium ions and stress uncoupling CSP310 protein

The effects of different Ca²⁺ concentrations on winter wheat (Triticum aestivum L.) functioning and cytochrome c release after organelle incubation with cold-shock protein with a mol. wt of 310 kD or after cold shock were studied. Low (1–5 μM) and high (25–50 μM) Ca²⁺ concentrations inhibited mitochondrial respiration in control seedlings, whereas 10 μM Ca²⁺ enhanced respiration in state 4 and reduced indices characterizing coupling (respiratory control (RC) and ADP: O ratio). At concentrations of 6–20 and 50 μM, Ca²⁺ ions suppressed CSP310 uncoupling effect, which reduced the rate of respiration and an increase in the RC and ADP: O ratio. Low-temperature stress and exogenous CSP310 induced cytochrome c leakage from winter wheat mitochondria both in the absence of Ca²⁺ and in the presence of its low concentrations.     

Overexpression of plant uncoupling mitochondrial protein in transgenic tobacco increases tolerance to oxidative stress

An Arabidopsis thaliana cDNA clone encoding a plant uncoupling mitochondrial protein (AtPUMP1) was overexpressed in transgenic tobacco plants. Analysis of the AtPUMP1 mRNA content in the transgenic lines, determined by Northernblot, revealed variable levels of transgene expression. Antibody probing ofWestern blots of mitochondrial proteins from three independent transgenic lines showed significant accumulation of AtPUMP1 in this organelle. Overproduction of AtPUMP1 in transgenic tobacco plants led to a significantincrease in tolerance to oxidative stress promoted by exogenous hydrogen peroxide as compared to wild-type control plants. These results provide thefirst biological evidence for a role of PUMP in protection of plant cells against oxidative stress damage.     

Alternative Oxidase and Uncoupling Protein: Thermogenesis Versus Cell Energy Balance

The physiological role of an alternative oxidase and an uncoupling protein in plant and protists is discussed in terms of thermogenesis and energy metabolism balance in the cell. It is concluded that thermogenesis is restricted not only by a lower-limit size but also by a kinetically-limited stimulation of the mitochondrial respiratory chain.     

Evidence that fasting can induce a selective loss of uncoupling protein from brown adipose tissue mitochondria of mice

The effects of fasting and refeeding on the concentration of uncoupling protein in brown adipose tissue mitochondria have been investigated in mice. Fasting mice for 48 h led to a large decrease in the total cytochrome oxidase activity of the interscapular brown fat pad. Mitochondrial GDP binding and the specific mitochondrial concentration of uncoupling protein also fell on fasting. After 24 h refeeding both GDP binding and the mitochondrial concentration of uncoupling protein were normalized, but there was no alteration in the total tissue cytochrome oxidase activity. Fasting appears to induce a selective loss of uncoupling protein from brown adipose tissue mitochondria, which is rapidly reversible on refeeding.     

Uncoupling effect of lauryl sulfate on mitochondria can be mediated by release of bound endogenous fatty acids

The mechanism of uncoupling by lauryl sulfate (LS) has been studied. The very fact that uncoupling by low concentration of LS (a strong acid) resembles very much that by fatty acids (weak acids) was used as an argument against the fatty acid cycling scheme of uncoupling where protonated fatty acids operate as a protonophore. We have found that rat liver and heart muscle mitochondria can be uncoupled by low (70 microM) LS concentration in a fashion completely arrested by the ATP/ADP antiporter inhibitor carboxyatractylate (CAtr). On the other hand, uncoupling by two-fold higher LS concentration is not sensitive to CAtr. Addition of oleate desensitizes mitochondria to low LS so that addition of bovine serum albumin becomes necessary to recouple mitochondria. The data are accounted for assuming that low LS releases endogenous fatty acids from some mitochondrial depots, and these fatty acids are responsible for uncoupling. As to high LS, it causes a nonspecific (CAtr-insensitive) damage to the mitochondrial membrane.     

Possible Basic and Specific Functions of Plant Uncoupling Proteins (pUCP)

Evidence has been provided that the plant uncoupling proteins (pUCP) play basic physiological roles similar to the other uncoupling protein subfamily members (mammalian UCP1,2,3,4 and BMCP) and are effective in the situations of slight uncoupling that leads to: (1) accelerated respiration and metabolic rates that are beneficial to plant growth and development; (2) decreased formation of reactive oxygen species in mitochondria; and, (3) mild thermogenesis, inevitably accompanying the previous two phenomena. Hypothetically, specific physiological roles of pUCP such as cut off of ATP synthesis could be manifested in connection with climacteric respiratory rise during fruit ripening, seed dormancy, and plant senescence. pUCP might also facilitate growth under low temperatures, e.g., during seed germination or in roots. The existence of these specific roles is suggested by the immunochemical and functional localization of pUCP in mitochondria of fruits, seeds and roots of various plant species.     

Overexpression of wheat mitochondrial uncoupling protein in rice plants confers tolerances to oxidative stresses promoted by exogenous hydrogen peroxide and low temperature

To obtain a better understanding of the function of mitochondrial uncoupling protein (UCP) in higher plants, the wheat gene for mitochondrial uncoupling protein (WhUCP) in rice was overexpressed by Agrobacterium-mediated transformation with a construct containing the WhUCP ORF under control of the 35S promoter. The transgenic rice plants showed a significant increase in tolerance against oxidative stress promoted by exogenous hydrogen peroxide at the seedling stage. The transgenic rice plants overexpressing WhUCP also exhibited greater tolerance against cold stress than did the wild-type plants. These results demonstrated that the mitochondrial UCP in higher plants is positively involved in the pathway for abiotic stress tolerance, probably through a decrease in cellular oxidative damage, and that controlled uncoupling by UCP could be used for improvement of stress tolerance in higher plants.Kenjirou Ozawa and Seiji Murayama are contributed equally to the work