POST MORTEM CHANGES IN EXTRACTABLE PROTEIN AND IN TISSUE pH IN THE GUINEA PIG BRAIN

—Guinea pigs were killed by asphyxiation with nitrogen and the soluble proteins were extracted from the brain at various times post mortem. The quantity of extractable brain protein decreased by 21 per cent when the animals remained at room temperature for 2 h post mortem. This decrease was not a consequence of extensive proteolysis or variations in blood volume but was probably a result of precipitation. After death, the pH of the brain fell rapidly to a minimum of ～6CE4 within about 35 min. Examination of the patterns of brain proteins after acrylamide gel electrophoresis showed a concomitant decrease in the content of several protein bands.     

Protease activity during rice leaf senescence

A protease activity was detected in rice (Oryza sativa L. cv. Ratna) leaves that hydrolysed hemoglobin more efficiently than bovine serum albumin. The activity was high when the enzyme was extracted and assayed with tris-maleate buffer [tris (hydroxymethyl) methyl amino-maleate] pH 7.0 rather than with water or with citrate-phosphate buffer pH 7.0. The enzyme had a strong dependence on sulfhydryl groups for its activity without which it was inaotive. The pH optimum was 7.0 and the temperature optimum was 40 °C. Protease activity expressed per unit leaf fresh weight (absolute activity) increased only little during senescence of detached rice leaves while the same activity expressed per unit soluble protein content (specific activity) increased by a greater factor (about 5 times) than absolute activity. Total and soluble protein content decreased during the senescence of detached leaves. Benzimidazole (10-3M) and kinetin (0.5xl0-5M) treatment arrested the increase of the protease activity and the deorease in the protein content during detached leaf senescence. It was indicative that protease protein was more stable than the bulk of other proteins in senescing leaves.     

Electrophoretic studies of the relationship of peroxidases,polyphenol oxidase,and indoleacetic acid oxidase to cold tolerance of alfalfa

Two cultivars of alfalfa (Medicago sativa L.), cold-tolerant Vernal and cold-sensitive Sonora, were grown under summer, winter, and dehardening environments to investigate the relationship of soluble proteins and enzyme activity and solubility characteristics to cold tolerance.Evaluations of cold-tolerance levels developed in crown and root samples were compared with results of soluble protein analyses and were in agreement with previously reported observations. Soluble protein content was associated with increases in cold tolerance and related to the environment from which samples were obtained; however, the degree of protein differences within samples of the same cultivar as well as between the two cultivars seemed to be influenced by the type of extractant used.Polyacrylamide disc gel electrophoresis of the extracted soluble proteins was performed on the basis of equal dry weights and equal quantities of protein. Amido black straining of gels indicated mainly quantitative changes and slight qualitative differences in component bands influenced by environment and extractant.Gels assayed for peroxidase, polyphenol oxidase, and indoleacetic acid oxidase enzymes exhibited mainly quantitative differences in constitutive isoenzyme components of both cultivars which were associated with environmental changes. Enzyme activities generally increased in winter, as cold tolerance and soluble protein content increased, and decreased during dehardening. The few qualitative differences in isoenzyme bands that were detected, appeared to be influenced by cultivar, environment, extractant, or substrate specificity differences.Variation in isoenzyme components between cultivars was maximum in summer samples, and minimum in winter samples, suggesting that overall reaction rates or activities of individual isoenzymes, preceding or during hardening, could be a limiting factor in cold-tolerance development.     

Effect of myocardial stunning on thiol status,myofibrillar ATPase and troponin I proteolysis

To investigate the mechanism underlying postischemic contractile dysfunction (myocardial stunning) we examined myocardial sulfhydryl group content, myofibrillar Ca²⁺-dependent Mg²⁺-ATPase activity and protein profile after global ischemia and reperfusion. The Langerdorff-perfused rabbit hearts were subjected to 15 min normothermic ischemia followed by 10 min reperfusion and myofibrils were isolated from homogenates of left ventricular tissues. Depressed contractile function during reperfusion was accompanied by a decrease in total sulfhydryl group content. However, myofibrillar protein profile was unchanged and Western immunoblotting analysis showed no significant differences in troponin I immunoreactive bands between control and stunned hearts. Likewise, myofibrillar Mg²⁺-ATPase activity was unaltered after ischemia and reperfusion. We conclude that myocardial stunning is not caused by altered myofibrillar function and protein degradation but may be partly due to the oxidative modification of as yet undefined proteins.     

Frost hardening and photosynthetic performance of Scots pine (Pinus sylvestris L.) needles. I. Seasonal changes in the photosynthetic apparatus and its function

Photosynthetic CO₂ uptake, the photochemical efficiency of photosystem II, the contents of chlorophyll and chlorophyll-binding proteins, and the degree of frost hardiness were determined in three-year-old Scots pine (Pinus sylvestris L.) trees growing in the open air but under controlled daylength. The following conditions were compared: 9-h light period (short day), 16-h light period (long day), and natural daylength. Irrespective of induction by short-day photoperiods or by subfreezing temperatures, frost hardening of the trees was accompanied by a long-lasting pronounced decrease in the photosynthetic rates of one-year-old needles. Under moderate winter conditions, trees adapted to a long-day photoperiod, assimilated CO₂ with higher rates than the short-day-treated trees. In the absence of strong frost, photochemical efficiency was lower under short-day conditions than under a long-day photoperiod. Under the impact of strong frost, photochemical efficiency was strongly inhibited in both sets of plants. The reduction in photosynthetic performance during winter was accompanied by a pronounced decrease in the content of chlorophyll and of several chlorophyll-binding proteins [light-harvesting complex (LHC)IIb, LHC Ib, and a chlorophyll-binding protein with MW 43 kDa (CP 43)]. This observed seasonal decrease in photosynthetic pigments and in pigment-binding proteins was irrespective of the degree of frost hardiness and was apparantly under the control of the length of the daily photoperiod. Under a constant 9-h daily photoperiod the chlorophyll content of the needles was considerably lower than under long-day conditions. Transfer of the trees from short-day to long-day conditions resulted in a significantly increased chlorophyll content, whereas the chlorophyll content decreased when trees were transferred from a long-day to a short-day photoperiod. The observed changes in photosynthetic pigments and pigment-binding proteins in Scots pine needles are interpreted as a reduction in the number of photosynthetic units induced by shortening of the daily light period during autumn. This results in a reduction in the absorbing capacity during the frost-hardened state. Received: 3 March 1997 / Accepted: 16 July 1997     

Reorganization of chloroplast ultrastructure associated with low-temperature hardening of Arabidopsis plants

When following low-temperature acclimation (5 days at 2°C) of cold-resistant plants of Arabidopsis (Arabidopsis thaliana Heynh. (L.), ecotype Columbia) in relation to the changes in chloroplast ultrastructure, we registered the high efficiency of hardening and the ability of hardened plants to lower a threshold of frost damage by about 3°C. During hardening, the area of grana in the chloroplasts more than doubled, with considerably increased numbers of thylakoids per granum and thylakoids per chloroplast. The rate of apparent photosynthesis decreased to lesser extent than the rate of dark respiration, as a result the content of soluble sugars increased fourfold, ensuring an adaptive reorganization of metabolism, which enabled the hardened plants to survive even at below-zero temperatures (up to ?7°C). The authors conclude that a considerable increase in the number of thylakoids in the chloroplasts helps maintain photosynthesis at low above-zero temperatures and is a prerequisite for the accumulation of soluble sugars in Arabidopsis leaves.     

Dormancy development during cold hardening of in vitro cultured Malus domestica Borkh. plants in relation to their frost resistance and cryotolerance

The development of dormancy, frost resistance and cryotolerance of in vitro apple plants (Malus domestica Borkh.), cv. Greensleeves during their exposure to cold hardening was studied. In vitro cultures were cold hardened at 4°C under a short photoperiod up to 25?weeks. The dormancy status, non-structural saccharides, proline, water content and frost resistance were evaluated for optimization of cryopreservation. According to regrowth tests, in vitro cultures exhibited endogenous dormancy after the maximal frost resistance was reached. The highest regeneration ability of shoot tips after cryopreservation by encapsulation–dehydration method coincided with the period of the plant’s dormant state and maximum of frost resistance. All studied saccharides and proline exhibited the maximal values at the beginning of cold hardening and/or the dormancy phase. Contrary to the accumulation of saccharides and proline, water content showed the inverse time behaviour. According to these results, the cold hardening-induced endodormancy, high frost resistance and accumulation of saccharides and proline are the important prerequisites for the successful cryopreservation of shoot tips of in vitro grown apple plants.     

Oryzalin-Induced Changes in Water Status and Cytoskeleton Proteins of Winter Wheat Seedlings upon Cold Acclimation and ABA Treatment

The effect of oryzalin (a specific inhibitor of tubulin polymerization in plant cells) on water retention by the leaves and roots of winter wheat (Triticum aestivum L.) seedlings was studied. The cultivars differing in their frost resistance were compared after their acclimation to low temperature (3°C for 3 or 7 days) and after treatment with ABA. In control untreated plants, oryzalin reduced the water-retaining capacity (WRC) of leaves and roots. Both hardening and ABA lowered the effect of the inhibitor on WRC in leaves, whereas their effects on water retention by roots were opposite, i.e., hardening weakened and ABA intensified the effect of oryzalin. Oryzalin-induced reduction of WRC decreased in the following sequence of cultivars: weakly frost resistant moderately frost resistant highly frost resistant. It was more pronounced in the leaves than in the roots, the latter being characterized by the lower WRC and lower frost resistance. After three-day-long hardening of plants, an additive effect of hypothermia and ABA on oryzalin-induced decrease in WRC of leaves and the lack of such effect in the roots were observed. The immunochemical analysis of the composition and content of cytoskeletal proteins with Western blotting showed that in the leaves the actin/tubulin ratio was higher than in the roots. The treatment of nonacclimated plants with ABA lowered the content of - and -tubulins and actin in roots but did not affect the level of actin in leaves. Hardening negated the effects of ABA on cytoskeletal proteins. Oryzalin produced the greatest inhibitory effect on WRC and an increase in frost resistance in ABA-treated plants in the experiments with leaves of the weakly frost resistant cultivar before and after hardening. Organ- and cultivar-specific and ABA-mediated dependence of WRC on cytoskeletal proteins and microtubules and microfilaments formed by them is supposed to result from their effect on the state of intracellular water and water permeability of the plasma membrane. In the course of cold acclimation of plants and upon their treatment with ABA, this dependence was more distinctly expressed in leaves than in roots, and especially in the plants of the weakly frost resistant cultivar.     

Interrelations between photoperiod, frost hardiness and sulfhydryl groups in cabbage

Hardening of SD (8 and 12 hrs) and LD (18- and 24-hr photoperiods) cabbage plants in stages at temperatures starting with + 5° and ending with − 3° led to the following changes:

Soluble protein plus nonprotein N showed a net increase only in the SD plants. In both SD and LD plants, it decreased to a minimum toward the end of the first stage of hardening, increased to a maximum in the second stage. The degree of this change was proportional to the photoperiod. These changes were mainly due to the proteins.

As in previous investigations, SH content rose during the first 1 to 2 weeks, but only in the SD plants. This rise was primarily due to the protein fraction. After the first 1 to 2 weeks both SD and LD plants showed a decrease in SH content, most pronouncedly during the second stage of hardening.

Nonprotein SH content was very low and decreased during hardening in both SD and LD plants. Nonprotein SS increased during the first stage of hardening. Total nonprotein SH+2SS rose to a maximum during the first stage of hardening, paralleling both osmotic potential and hardiness. During the second stage the total decreased, in spite of the rise in hardiness. These results held true for both SD and LD plants.

These results are all explainable on the basis of the SHSS theory of frost resistance.

     

The effect of CCC on the nitrogen compounds content in rape plants and their frost hardiness. Relation to the conditions of day-length and temperature

Both CCC and cold (5°C) treatment gave rise to an increased content of the water-soluble proteins in leaf tissue of the winter rape, irrespective of the day length. This effect was accompanied by a decrease of the insoluble nitrogen compounds content, mainly under theLD (the 16 hour day). The applied retardant also stimulated the consumption of the structural compounds induced by low temperature. Low temperature treatment hardened plants more distinctly than the CCC application. The frost hardening effect of CCC andSD (the 8 hour day) was manifested only at 20°C and it disappeared at 5°C. Changes in frost hardiness were not correlated with the changes in the reducing sugar content and in the reducing ability of the studied tissue. The coincident effect of CCC and cold on the reducing sugar content was observed underSD conditions.     

Changes in vitellin and other yolk proteins during embryonic development in the silkworm, Bombyx mori

Changes in the amounts of vitellin and other yolk proteins of the eggs of the silkworm, Bombyx mori were investigated during embryonic development using polyacrylamide gel electrophoresis and immunotitration techniques. In the newly laid eggs, soluble proteins were separated into at least nine bands after electrophoresis. The major band was identified as vitellin, accounting for about 40% of the total proteins. The four predominant bands including vitellin exhibited the same mobility as the proteins of haemolymph, but one other major band was specific to the eggs, accounting for about 20% of the proteins.During embryonic differentiation 6–7 days after oviposition, the total protein content did not decrease and the banding patterns and their relative concentrations remained unchanged as a whole. However the concentration of the egg specific protein steadily decreased. During subsequent larval differentiation until hatching, the total proteins were utilized to about 50% of the initial levels: the rapid degradation was observed in almost every species of proteins.An immunotitration experiment further demonstrated that vitellin was not utlilized during embryonic differentiation but was consumed markedly during larval differentiation. However, about 30% of initial level was reserved in the newly hatched larvae. Such a prolonged persistence of vitellin is discussed in relation to protein metabolism during embryonic development in silkworms.     

Effect of nitrogen supply on frost resistance, nitrogen metabolism and carbohydrate content in white clover (Trifolium repens)

Effects of mineral nitrogen (2, 4, 6 and 8 m M NH₄NO₃) and nodulation with Rhizobium on frost hardiness in seedlings of white clover ( Trifolium repens ) have been studied. Seedlings of a population from Bodø (67°N lat.) were grown in Leonard jars under controlled conditions in a phytotron. For induction of frost hardening, plants were first exposed to 12 h photoperiod conditions for 2 weeks at 18°C, then for 2 weeks at 6°C and finally for 2 weeks at 0.5°C. Frost hardiness after treatments at 6 and 0.5°C was significantly enhanced by increasing nitrogen supply and was positively correlated with total nitrogen content of the stolons. Frost hardiness of nodulated plants correlated to the tissue nitrogen concentration. Content of soluble proteins in stolons decreased during hardening at 6°C but did not change during treatment at 0.5°C. There were minor changes in total amount of free amino acids during hardening. Both absolute and relative amounts of proline and arginine increased, and those of asparagine decreased during hardening. Absolute amounts of all free amino acids increased with increasing nitrogen supply, but the changes during hardening were similar in all treatments. There was a significant increase in the content of soluble carbohydrates during hardening. However, this increase was inversely related to nitrogen supply.     

CaCl2对金叶女贞茎抗寒性和电阻抗图谱参数的影响

为探明CaCl₂处理对金叶女贞(Ligustrum vicaryi)茎抗寒性和电阻抗图谱参数的影响,找出抗寒性和电阻抗图谱参数的关系,2006年9月15日开始对金叶女贞当年生扦插苗进行CaCl₂处理,对照为清水;分别用电阻抗图谱法和电导法估测其抗寒性,并进行比较分析。结果表明：CaCl₂处理能在抗寒锻炼初期使金叶女贞茎的抗寒性提高3.5℃,金叶女贞茎的抗寒性与未冷冻处理茎的弛豫时间τ、冷冻处理后茎的胞外电阻率r_e、含水量之间均存在较高的相关性。     

Effects of Frost Hardening and Dehardening on Photosynthetic Electron Transport and Fluorescence Properties in Isolated Chloroplasts of Pinus silvestris

Photosynthetic electron transport and low-temperature fluorescence emission properties have been analyzed in isolated chloroplasts during the course of frost hardening and dehardening of Pinus silvestris L. Both the partial electron-transport reactions (H₂O DPIP and Asc./DPIP NADP) and the overall electron transport (H₂O — NAPD) showed decreasing capacities during the course of hardening. Upon exposing the plants to ?5°C and high irradiance a block in the electron-transport chain between the two photosystems developed, whereas the partial reactions still showed activities. The decrease in activity of PSl was accompanied by a decrease in P700 content, as determined by light oxidation of P700, which indicates a correlation between the two changes. Hardening also induced changes in the in vivo chlorophyll organization. During the course of hardening the fluorescence emission bands F692 and F726 decreased relative to F680. These changes were more pronounced if the plants were treated in high than in low irradiance. This suggests a greater destruction of the chlorophyll antennae in close association with the two photoreactions than in the so-called light-harvesting chlorophyll a/b antenna. During dehardening basically the reverse of the changes observed during hardening occurred. The recovery of secondary needles was complete, whereas primary needles only partly recovered.     

Studies of Frost Hardiness in Woody Plants. II. Effect of Temperature on Hardening

The effect of temperature on hardening was studied at temperatures ranging from 0° to −20° using twigs of willow and poplar. In October and in late April when the twigs are not very frost hardy, hardening at 0° produced a considerable increase in their frost hardiness, although the effectiveness of hardening at 0° decreased with a decrease in the environmental temperature. In twigs which could withstand continuous freezing without injury, hardening at −3° to −5° was most effective in increasing the frost hardiness of the twigs. Below −20°, only negligible increase was observed either in frost hardiness or sugar content.

The rate of starch to sugar conversion differed remarkably in different twig tissues. The starch in xylem was more slowly converted to sugar than that in the cortex. The optimum temperature for converting starch into sugar during frost hardening was also found to be −3° to −5°. In addition, the greater the effectiveness of the hardening treatment, the greater the rate of conversion from starch to sugar. The frost hardiness of a twig is closely related to the sugar content of the twig, especially in the xylem.

     

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. Effects of castration and androgen replacement

Four intrinsic soluble secretory proteins are synthesized in vitro by isolated seminal-vesicle mucosa from sexually mature guinea pigs. Newly synthesized specific proteins labelled with [¹⁴C]glycine and [¹⁴C]lysine were precipitated by using double-antibody immunoprecipitation techniques and their radioactivity was assessed. Rates of synthesis were determined on each of 5 days after castration. By 5 days after castration the wet weight of the epithelium decreased to 42% of intact control values; the absolute amount of specific protein synthesized in vitro after 60min incubation decreased to 28% and the 27500g cytoplasmic protein content decreased to 31%. Thus androgen deprivation leads to a decrease in general protein synthesis in vivo, as well as to a decrease in specific protein synthesis in vitro. Specific protein synthesis comprised 76% of the total protein formed in isolated tissue from animals 5 days after castration as compared with 99–100% in tissue from intact animals. At 72h after an injection of testosterone or dihydrotestosterone, seminal-vesicle epithelium wet weight, cytoplasmic protein content and capability for synthesizing specific proteins in vitro were restored to approx. 70% of normal values. At 72h after onset of therapy with 3α-androstanediol, both epithelium wet weight and cytoplasmic protein content had increased significantly, but without a corresponding increase in the capability of the isolated tissue to synthesize specific proteins. The soluble labelled proteins synthesized in vitro by isolated epithelium from intact animals during 60 or 120min incubation were essentially entirely immunoprecipitable, i.e. specific. In contrast, approx. 29% of all soluble protein newly synthesized by isolated epithelium from animals 5 days after castration was acid-precipitable, but not immunoprecipitable, i.e. `non-specific'. The injection of testosterone into castrated animals inhibited the synthesis of the non-specific fraction by isolated tissue. The effects of castration on the ultrastructure of guinea-pig seminal-vesicle epithelium are also presented.     

Low temperature acclimation and treatment with exogenous abscisic acid induce common polypeptides in Arabidopsis thaliana (L.) Heynh

Summary Exogenously applied abscisic acid (ABA) induced frost hardening of Arabidopsis thaliana (L.) Heynh. The freezing tolerance of A. thaliana plantlets treated with ABA (15 mg/l) at a non-acclimating temperature (20 °C) appeared to increase even more rapidly than following a low temperature (4 °C) acclimation. Analysis of in vivo-labelled soluble proteins by two-dimensional gel electrophoresis revealed several low temperature — or ABA — induced proteins, which where not produced in non-acclimated plants. A subset of these proteins was induced by both low temperature and ABA treatments, suggesting that they might be directly involved in the frost hardening process in A. thaliana.     

Protein composition and architecture of the photosynthetic membranes from the cyanobacterium, Anacystis nidulans R2

The protein composition of the photosynthetic membrane from the cyanobacterium, Anacystis nidulans R2, was analyzed by acrylamide gel electrophoresis following solubilization with lithium dodecyl sulfate. Autoradiograms of ³⁵S-labelled membranes revealed over 90 bands by this procedure. The effect of solubilization conditions on protein resolution was analyzed by modifying temperature and sulfhydryl concentrations. Labelling cells with ⁵⁹Fe yielded nine iron-containing bands on these gels. Three of these bands, at 33, 19, and 14 kDa, were also heme proteins as determined by tetramethylbenzidine staining, and represent cytochromes f, b₆ and c-552, respectively. The remaining iron proteins are highly sensitive to solubilization conditions, especially the presence of 2-mercaptoethanol, and we suggest that these bands may be Fe-S proteins. Lactoperoxidase-catalyzed iodination of the membranes indicated that at least 41 proteins have surface-exposed domains. Some of the known proteins with external surfaces include cytochrome c-552 and the chlorophyll-binding proteins of Photosystems I and II. Neither cytochrome f nor b₆ appear to be accessible to external labelling. When this structural information was combined with the isolation of functional submembrane complexes, we constructed a topological model of the membrane. Using this model we have discussed the protein architecture of the cyanobacterial membrane.     

Some physiological and biochemical changes in marine eukaryotic red tide alga Heterosigma akashiwo during the alleviation from iron limitation

Some physiological and biochemical changes in the marine eukaryotic red tide alga Heterosigma akashiwo (Hada) were investigated during the alleviation from iron limitation. Chlorophyll a/carotenoid ratio increases as a result of iron alleviation. In vivo absorption spectra of iron-limited cells showed a chlorophyll (Chl) absorption peak at 630 nm, 2 nm blue-shifted from the normal position. Low-temperature fluorescence emission spectra of the cells have one prominent Chl emission peak at 685 nm. The cells showed a decrease in fluorescence yield from 685 nm band during alleviation from iron limitation. Low-temperature fluorescence excitation spectra and room-temperature fluorescence spectra indicated an efficient excitation energy transfer in the cells alleviated from iron limitation. Photosynthetic efficiency and carbohydrate content per cell increased after alleviation from iron limitation. Total protein decreased in iron-limited cells, while iron deficiency induced the appearance of specific soluble proteins (17 and 55 kDa).     

Physiological mechanisms of frost tolerance: Possible role of protein in plant adaptation to cold

Studies performed on winter rape plants(Brassica nnpus var.oleifera, cv. ‘Gór-czański’) revealed that cold treatment affected the cell membranes and led to the temporary increase in electrolytic leakage from a tissue. This was followed by the marked decrease of the electrolytic leakage in the course of hardening. Changes in membrane properties were accompanied by the promotion of soluble protein accumulation. Inhibition of protein accumulation by the cycloheximide treatment brought about wilting of plants under cold conditions. Possible role of soluble protein in protection of cells against secondary water stress caused by the coldinduced changes in membrane properties is suggested. Cold-induced changes in the electrophoretic pattern of soluble protein are described and discussed.