Cold-induced changes in the polysome pattern and protein synthesis in winter rye (Secale cereale) leaves

Cold-induced changes in the polysome pattern and protein synthesis were analyzed in winter rye, Secale cereale L. cv. Voima, during one week's cold stress treatment, which was performed by transferring the 7-day-old plants from the greenhouse (25°C, long-day conditions) to 3°C and a photoperiod of 10. 5 h. Freezing resistance determined by electrolyte leakage increased significantly upon cold stress starting from LT₅₀ value –5°C. and reaching –9°C on the day 7 of cold exposure. After 4 weeks at low temperature, plants reached an LT₅₀ of –12°C. The polysome content increased markedly during cold stress compared to the control plants. After 2 weeks of cold treatment the polysome content decreased to the same level as that in control plants. The size-class distribution of polysomes showed a high proportion of large protein synthesizing polysomes in cold-stressed plants. After 2 weeks the values were comparable to those in control plants. Cold-induced proteins were detected using ³⁵S-labelled methionine for in vitro translations. At least 2 new polypeptides, M_r 30000 and 18000, were induced on the first day of cold stress and continued to be expressed at low temperatures 4 weeks later.     

Ultrastructural changes arising from freezing of leaf blade cells of rye (Secale cereale): an investigation using freeze-substitution

The survival at sub-zero temperatures of leaf blade cells of rye ( Secale cereale L. cv. Voima), which had not been cold acclimated, was determined by measuring the efflux of ninhydrin-positive substances: 50% of the cells were dead at −4°C (LT₅₀) and none survived at −12°C or below. Examination of ultrastructural changes during cold hardening and freezing injury requires frozen tissues prepared for transmission electron microscopy without thawing. Specimens were prepared from leaf blade segments at room temperature, −4°C or −12°C by plunge freezing at 3 m s⁻¹ into a cooling medium at −170°C followed by freeze-substitution in acetone with OsO₄ fixation. Comparisons of room temperature specimens were made with those prepared by chemical fixation using glutaraldehyde/paraformaldehyde/tannic acid. On freezing to −12°C, the cells were severely dehydrated and distorted, the vacuoles severely shrunken and the cytoplasm and mitochondria disorganized whereas the chloroplasts were little affected. On freezing to −4°C, some cells were as disorganized as those at −12°C, others were relatively intact, and some showed evidence of intracellular ice crystal formation.     

Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Gibberellin status and responsiveness in shoots of tall and dwarf genotypes of diploid rye (Secale cereale)

The recessive dwarfing alleles of rye ( Secale cereale L.), ct1 and ct2 , caused a 35–55% reduction in the length of leaf 2 compared with corresponding tall lines grown at both 10°C and 20°C. The dwarf lines were 45–50% as responsive to applied GA₃ as the tall lines at 20°C but the absolute GA-responsiveness of the dwarfs was greater at 10°C than at 20°C. There was no significant difference in the contents of GA₁₉, GA₂₀, GA₂₉, GA₁, GA₃ and GA₈ in the leaf extension zone of tall and dwarf seedlings grown at 20°C. It was concluded that the mechanism whereby GA homeostasis is maintained is functional in both tall and dwarf lines despite marked differences in leaf extension rate. The recessive rye mutations may cause loss of function late in the GA-cell elongation pathway or, alternatively, indirectly affect GA-responsiveness in vegetative tissues. The genetic and physiological evidence indicates that ct1 and ct2 are unrelated to the GA-insensitive Rht genes in hexaploid bread wheat.     

Aluminium toxicity in rye (Secale cereale): root growth and dynamics of cytoplasmic Ca2+ in intact root tips

Aluminium (Al) toxicity in rye (Secale cereale L.), an Al-resistant crop, was examined by measuring root elongation and cytoplasmic free activity of calcium ([Ca2+]cyt) in intact root apical cells. Measurement of [Ca2+]cyt, was achieved by loading a Ca2+-sensitive fluorescent probe. Fluo-3/AM ester, into root apical cells followed by detection of intracellular fluorescence using a confocal laser scanning microscope. After 20 min of exposure to 50 microM Al (pH 4-2) a slight increase in [Ca2+]cyt of root apical cells was observed, while the response of [Ca2+]cyt to 100 microM Al (pH 4.2) was faster and larger ([Ca2+]cyt increased by 46% in 10 min). Increases in [Ca2+]cyt were correlated with inhibition of root growth, generally measurable after 2 h. Addition of 400 microM malic acid (pH 4.2) largely ameliorated the effect of 100 microM Al on [Ca2+]cyt in root apical cells and protected root growth from Al toxicity. These results suggest that an increase in [Ca2+]cyt in root apical cells in rye is an early effect of Al toxicity and is followed by the secondary effect on root elongation.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

Phenolic substances as allelopathic agents arising during the degradation of rye (Secale cereale) tissues

Rye seedlings, tillering plants and crop residues were allowed to decompose in model incubation experiments. Young tissues gave rise to high concentrations of allelochemicals, whereas crop residues did not produce inhibitors. Seven phenolic acids were identified in the investigated materials; p-hydroxybenzoic protocatechuic, gallic, vanillic, syringic, p-coumaric, ferulic as well as benzoic acid. However, neither the level of these acids nor the total content of phenolic compounds corresponded to the level of phytotoxicity determined in bioassays. This demonstrated that, apart from phenolics, other unidentified water-soluble organic compounds were also responsible for the toxicity of rye decomposition products. The study was conducted within program CPBP 04.10.03. The study was conducted within program CPBP 04.10.03.     

X-ray microanalytical (EDX) investigation of potassium distributions in mesophyll cells of non-acclimated and cold-acclimated rye leaves

Electron probe X-ray microanalysis was used to analyse the effects of sub-zero temperatures on K⁺ distribution in compartments within non-acclimated and cold acclimated rye (Secale cereale L. cv Voima) leaf cells and to evaluate membrane leakage of ions caused by freezing-injury. The specimens were rapidly frozen from growing temperatures and from two different sub-zero temperatures (LT₅₀ and LT₁₀₀) to which the leaves had already been slowly cooled. Measurements were made in the cytoplasm, vacuole and cell walls in freeze-substituted mesophyll cells. At ambient temperatures, the mean K⁺ concentration in the cytoplasm (100 mol m^?3) differed significantly from that of the vacuole (49 mol m^?3) in the non-acclimated (NA) cells, while in cold acclimated (A) cells, the concentrations were similar (109 vs 93 mol m^?3, respectively). At LT₅₀ temperatures, the K⁺ concentration in NA-cells decreased significantly in the cytoplasm (59 mol m^?3) but increased in the cell walls. In the A-cells, on the other hand, the mean K⁺ concentration increased significantly (about three-fold) in all major compartments. At LT₁₀₀ temperatures, K⁺ concentrations in the cytoplasm and cell walls decreased when compared with corresponding LT₅₀ values in the A-cells but increased in the NA-cells. The increased potassium concentration in the cytoplasm of A-cells at LT₅₀ temperature is compatible with the observed cell shrinkage and an absence of plasma membrane damage. The decreased potassium concentration in the cytoplasm of NA-cells at LT₅₀ temperature is compatible with the slight cell shrinkage and suggests that the plasma membrane in these cells shows increased permeability due to freeze injury.     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

Effect of seabuckthorn leaf extracts on circulating energy fuels, lipid peroxidation and antioxidant parameters in rats during exposure to cold, hypoxia and restraint (C–H–R) stress and post stress recovery

The present study was carried out to study mechanism of adaptogenic activity of seabuckthorn leaf extract, administered orally in rats both in single and five doses at a dose of 100 mg/kg body weight 30 min prior to C–H–R exposure. The efficacy of the extract was studied on circulating energy fuels, lipid peroxidation and anti-oxidant parameters in rats on attaining the T_rec 23 °C during C–H–R exposure and after recovery (T_rec 37 °C) from C–H–R induced hypothermia. Single dose treatment in rats restricted rise in blood malondialdehyde (MDA) levels and decrease in glutathione (GSH) and catalase (CAT) levels. Both single and five doses also restricted the rise in serum free fatty acids (FFA) and lactate dehydrogenase (LDH) levels on attaining T_rec 23 °C during C–H–R exposure, suggesting more efficient utilization of FFA for energy production and better maintained cell membrane permeability. This suggested that the adaptogenic activity of the extract might be due to its anti-oxidative activity, maintained blood glucose levels, better utilization of FFA and improved cell membrane permeability.