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.     

Subcellular localization of dehydrins in wheat plant seedlings subjected to low-temperature adaptation

Subcellular localization of dehydrins (dhn) in stem cell tissues of winter wheat seedlings (Triticulum aestivum L., cult. Irkutskaya ozimaya) was studied by immunoelectron microscopy. It was found that cold hardening at 4°C for 7 days resulted in a duplication of the dhn quantity in the cells as compared with control conditions (22°C). The maximum increase of the dhn content was observed in rough endoplasmic reticulum, mitochondria, cell walls, and intercellular spaces (3.8-, 3.0- and 2.8-fold, respectively); minimum increase was found in chloroplasts (1.4-fold). In the membrane compartments (mitochondria, rough endoplasmic reticulum, chloroplasts) low-temperature stress caused an increase of dhn quantity not only near membranes but also in the intermembrane space. A significant accumulation of dhn (2.5-fold) in the nucleus under low- temperature was found. We conclude that cold hardening of the plant induces accumulation and translocation of dhn to the regions of inter- and intracellular compartments that most require protection during the low-temperature stress.     

Cell-wall lectins during winter wheat cold hardening

The polypeptide composition and functional activity of cell-wall lectins from roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings during cold hardening were studied. Several phases of lectin activity changes were observed, which indicates their involvement in the development of general adaptation syndrome of the cell. After 0.5-h low-temperature treatment, marked alterations occurred in the profile of protein elution: lectins with mol wts of 78 and 42.5 kD disappeared and new ones with mol wts of 72, 69, 37, and 34.5 kD appeared. It was established that 17.5-and 69-kD lectins and most lectins eluted with glucose were arabinogalactan proteins (AGP), which permitted a supposition that these lectins were involved in the interaction between the cell wall and cytoskeleton. After 7-day-long hardening, total protein content reduced and lectins with mol wts of 69 and 37 kD disappeared, which corresponded to reduced lectin activity by the end of hardening. A transient appearance of 37-and 69-kD lectins, which are AGP, might indicate their involvement in the triggering the development of plant-cell defense responses.     

Energy state of spring and winter wheat during cold hardening. Soluble sugars and adenine nucleotides

Marked increases were found in the content of total soluble sugars, reducing sugars and ATP in winter wheat ( Triticum aestivum L. cv. Frederick) during cold hardening. The changes in soluble sugars and ATP of spring wheat ( T. aestivum L. cv. Glenlea) grown under similar conditions were less pronounced. The increase in ATP content during hardening of winter wheat was not associated with significant changes in the content of ADP or AMP. The adenylate energy charge did not change during hardening in either cultivar, but it was higher in the winter cultivar under both growth conditions. This difference could be related to the cold hardiness capacity of winter wheat.     

Seasonal Changes in the Composition and Content of Dehydrins in Winter Wheat Plants

Seasonal changes in the pattern and content of dehydrins in winter wheat (Triticum aestivum) plants grown under field and laboratory conditions were studied by one-dimensional PAGE and immunochemical methods. During hardening, plants accumulated dehydrin-like polypeptides with mol wts of 209, 196, 66, 50, and 41 kD. In winter, low-molecular-weight dehydrins with mol wts of 24, 22, 17, 15, and 12 kD were synthesized and accumulated as well. Their content dropped sharply in spring when plants became unhardened. Accumulation/disappearance of these proteins corresponded to the fluctuations in wintering plant frost tolerance before winter and in spring. It is assumed that both high- and medium-molecular-weight dehydrins are involved in plant stress responses and adaptation, whereas low-molecular-weight dehydrins are evidently involved only in the process of low-temperature adaptation.     

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.     

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.     

Regulation of RNA Synthesis by DNA-Dependent RNA Polymerases and RNases during Cold Acclimation in Winter and Spring Wheat

Chromatin DNA-dependent RNA polymerases and RNases activities were measured in winter and spring varieties to understand the overall regulation of RNA synthesis during cold acclimation. We found that total RNA polymerase activities were significantly higher in chromatin isolated from winter wheat compared to the spring wheat during the acclimation period. This increase was parallel to the increase in protein and RNA contents during hardening. The ratio of RNA polymerase I to RNA polymerase II activity was higher than 2 in winter wheat after 30 days of hardening compared, to a ratio of 0.90 under the nonhardening conditions. The increase in activity and the ratio of polymerase I to polymerase II was maintained after the separation of the enzymes from the template, suggesting that RNA synthesis is regulated in part at the enzyme level. On the other hand, the chromatin associated RNase activity decreased in both varieties during acclimation, indicating a nonspecific inhibition caused by low temperature rather than a selective genetic response associated with cold acclimation.     

Plant stress-related uncoupling protein CSP 310 caused lipid peroxidation in winter wheat mitochondria under chilling stress

The effect of CSP 310 on lipid peroxidation in winter wheat mitochondria was studied by the measurement of primary lipid peroxidation products - dienic conjugates. It was found that some concentrations of CSP 310 caused lipid peroxidation in isolated winter wheat mitochondria in all systems investigated at different concentrations during chilling stress.     

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.     

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.     

The comparison of proteins with immunochemical affinity to stress protein 310 kD in cytoplasmatic proteins of winter rye,winter wheat,Elymus and maize

The search for proteins, immunochemically related to winter rye CSP 310 among the native cytoplasmatic proteins of a number of cultivated cereals with different tolerance to low temperatures—maize, winter wheat and winter rye and the very low temperature tolerant wild grass—Elymus sibiricus was carried out. Western blotting showed that among the native cytoplasmatic proteins of all species investigated there are proteins immunochemically related to CSP 310 protein with molecular weights about 230 and about 140–110 kD. Proteins with molecular weights about 480 and 310 kD were found in significant amounts only in winter rye. In E. sibiricus proteins with molecular weights 380–320 kD were present but these were not present among the cytoplasmatic protein spectra of the other species. In each case the proteins immunochemically related to CSP 310 consisted of different combinations of two types of 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.     

Differential Expression of Proteins in Response to Molybdenum Deficiency in Winter Wheat Leaves Under Low-Temperature Stress

Molybdenum (Mo) is an essential micronutrient for plants. To obtain a better understanding of the molecular mechanisms of cold resistance enhanced by molybdenum application in winter wheat, we applied a proteomic approach to investigate the differential expression of proteins in response to molybdenum deficiency in winter wheat leaves under low-temperature stress. Of 13 protein spots that were identified, five spots were involved in the light reaction of photosynthesis, five were involved in the dark reaction of photosynthesis, and three were highly involved in RNA binding and protein synthesis. Before the application of cold stress, four differentially expressed proteins between the Mo deficiency (?Mo) vs. Mo application (+Mo) comparison are involved in carbon metabolism and photosynthetic electron transport. After 48 h of cold stress, nine differentially expressed proteins between the ?Mo vs. +Mo comparison are involved in carbon metabolism, photosynthetic electron transport, RNA binding, and protein synthesis. Under ?Mo condition, cold stress induced a more than twofold decrease in the accumulation of six differential proteins including ribulose bisphosphate carboxylase large-chain precursor, phosphoglycerate kinase, cp31BHv, chlorophyll a/b-binding protein, ribulose bisphosphate carboxylase small subunit, and ribosomal protein P1, whereas under +Mo condition cold stress only decreased the expression of RuBisCO large subunit, suggesting that Mo application might contribute to the balance or stability of these proteins especially under low-temperature stress and that Mo deficiency has greater influence on differential protein expression in winter wheat after low-temperature stress. Further investigations showed that Mo deficiency decreased the concentrations of chlorophyll a, chlorophyll b, and carotenoids; the maximum net photosynthetic rate; the apparent quantum yield; and carboxylation efficiency, even before the application of the cold stress, although the decrease rates were greater after 48 h of cold treatment, which is consistent with changes in the expressions of differential proteins in winter wheat under low-temperature stress. These findings provide some new evidence that Mo might be involved in the light and dark reaction of photosynthesis and protein synthesis.     

Accumulation of dehydrin-like proteins in the mitochondria of cereals in response to cold, freezing, drought and ABA treatment

Background

Dehydrins are known as Group II late embryogenesis abundant proteins. Their high hydrophilicity and thermostability suggest that they may be structure stabilizers with detergent and chaperone-like properties. They are localised in the nucleus, cytoplasm, and plasma membrane. We have recently found putative dehydrins in the mitochondria of some cereals in response to cold. It is not known whether dehydrin-like proteins accumulate in plant mitochondria in response to stimuli other than cold stress.

Results

We have found five putative dehydrins in the mitochondria of winter wheat, rye and maize seedlings. Two of these polypeptides had the same molecular masses in all three species (63 and 52 kD) and were thermostable. Drought, freezing, cold, and exogenous ABA treatment led to higher accumulation of dehydrin-like protein (dlp) 63 kD in the rye and wheat mitochondria. Protein 52 kD was induced by cold adaptation and ABA. Some accumulation of these proteins in the maize mitochondria was found after cold exposition only. The other three proteins appeared to be heat-sensitive and were either slightly induced or not induced at all by all treatments used.

Conclusions

We have found that, not only cold, but also drought, freezing and exogenous ABA treatment result in accumulation of the thermostable dehydrins in plant mitochondria. Most cryotolerant species such as wheat and rye accumulate more heat-stable dehydrins than cryosensitive species such as maize. It has been supposed that their function is to stabilize proteins in the membrane or in the matrix. Heat-sensitive putative dehydrins probably are not involved in the stress reaction and adaptation of plants.     

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.     

Localization of Proteins Immunologically Related to Subunits of Stress 310-kD Protein in Winter Wheat Mitochondria

We studied the localization of polypeptides immunochemically related to subunits of cold-shock 310-kD protein from winter rye (Secale cerealeL.) in mitochondria and submitochondrial structures of winter wheat (Triticum aestivumL.) seedlings. Polypeptides were separated by SDS-PAGE and probed with the antibody against 310-kD protein from rye seedlings. Wheat mitochondria contained the following polypeptides cross-reacting with this antibody: 66, 60, 55, and 23 kD in the inner membrane; 60 and 58 kD in the outer membrane; and 66 and 55 kD in the matrix.     

水稻幼苗冷锻炼过程中钙的效应

冷锻炼处理提高了水稻（Ｏｒｙｚａ ｓａｔｉｖａ Ｌ．）幼苗叶片中抗氧化剂（还原型谷胱甘肽,ＧＳＨ;抗坏血酸,ＡｓＡ）含量和膜保护酶（超氧化物歧化酶,ＳＯＤ）的活性,同时也提高了可溶性蛋白质中热稳定蛋白的含量。ＣａＣｌ２ 浸种处理对上述冷锻炼的作用有加强的效果,且明显地提高了过氧化氢酶（ＣＡＴ）和过氧化物酶（ＰＯＤ）的活性。有无ＣａＣｌ２ 处理的冷锻炼处理均减轻冷胁迫引起的ＧＳＨ 及ＡｓＡ 含量、ＳＯＤ 活性及热稳定蛋白质含量的下降程度,有利于幼苗在恢复过程中ＧＳＨ、ＡｓＡ、ＣＡＴ、ＳＯＤ、ＰＯＤ及热稳定蛋白质水平迅速回升。结合ＣａＣｌ２ 处理的冷锻炼苗在冷胁迫恢复生长时增长迅速,且苗健壮浓绿,说明ＣａＣｌ２浸种对冷锻炼处理提高水稻幼苗的抗冷力有明显的促进作用,这与ＣａＣｌ２ 浸种结合冷锻炼能更有效的提高细胞膜保护能力有关     

PHYSIOLOGICAL AND BIOCHEMICAL MECHANISMS OF TEMPERATURE SHOCKS ON OVERWINTERING MATURE LARVAE OF PINE NEEDLE GALL MIDGE THECODIPLOSIS JAPONENSIS (DIPTERA: CE-CIDOMYIIDAE)*

Abstract The supercooling points of cold (-10C and -5C) and heat (37 C, 40 C and 45 C) shocked overwintering larvae were nearly the same as that of un-shocked ones (ca. -20C). Temperature shocks enhanced the ability to endure subzero temperature (- 15C, 3 h), and the cold shock treatment had more significant effect on maintaining larval survival than that of heat shock. It is the third insect that heat shock and cold shock enhanced its survival rate under low temperature simultaneously. A special stress protein (MW = 83 kD) was expressed under cold shock at -10 C and heat shock at 40 C or 45 C. It is also a few instances that a stress protein was expressed in the same insect under both heat shock and cold shock simultaneously. Meanwhile, the antioxidant system under different treatments was studied. Rapid cold hardening process had no oxidative stress because of the increase content of reduced glutathione and activity of glutathione reductase, but other treatments had.     

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.