Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Differentiation of freezing tolerance and vernalization responses in Cruciferae exposed to a low temperature

High levels of freezing tolerance were induced in leaves of different Cruciferae species including Brassica napus, Arabidopsis thaliana, Barbarea vulgaris, Thlaspi arvenses and Descurainia sophia by low-temperature acclimation. Concomitantly, the amount of total RNA doubled in these three species. Analyses of methylation patterns and dosage of rRNA genes were carried out to determine whether or not alterations occur in this DNA during development of freezing tolerance. Hybridizations of Southern transfers with an rDNA probe revealed two additional EcoRI sites in purified DNA isolated from freezing-tolerant leaves of winter B. napus cv Jet Neuf and D. sophia (both of which require low temperatures for vernalization), but not in isolates of A. thaliana or spring B. napus cv Topas. An increase of rDNA cistrons was also observed in both B. napus cv Jet Neuf and D. sophia but not in A. thaliana or B. napus cv Topas upon cold acclimation. These results suggest that low temperature induced amplification of rDNA and the differential methylation of EcoRI sites may possibly be related to the vernalization process but may not be related to the development of freezing tolerance. However, the higher activity of RNA polymerase (2.5 times more) observed upon cold acclimation may explain the concomitant increase in total RNA and may be related to the development of freezing tolerance in the Cruciferae.     

Lithium decreases cold-induced microtubule depolymerization in mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter [1991] Plant Physiol 97: 175-181). The objective of this study was to gain insight into the question of whether microtubules depolymerize as a direct response to environmental stresses or as an indirect response to cellular changes that accompany the stresses. Leaf sections of spinach were treated with Li⁺ before and during exposure to low temperature. Treatment with Li⁺ decreased the amount of microtubule depolymerization in cells subjected to low temperature, relative to a nontreated control, raising the possibility that the microtubules in these cells may not be inherently cold labile. Rather, microtubule depolymerization may be in response to cold-induced changes in concentration of cytoplasmic components.     

Dehydrins: A commonalty in the response of plants to dehydration and low temperature

Among proteins that accumulate in plants in response to dehydrative forces or low temperature, dehydrins (late embryogenesis abundant [ Lea ] D11 family) have been the most commonly observed. Dehydrins are composed of several typical domains joined together in a few characteristic patterns, with numerous minor permutations. These domains include one or more putative amphipathic a -helix forming consensus regions, a phosphorylatable tract of Ser residues, and an N-terminal consensus sequence. Lesser conserved domains are also present at various positions, particularly between the putative a -helix forming domains, where they may occur as tandem repeats. This medley of permutations is mirrored by a wide size range of dehydrin polypeptides from less than 100 to nearly 600 amino acid residues. As of yet, the fundamental biochemical mode of action of dehydrins has not been demonstrated, but a number of immunolocalization and cell fractionation studies have established that dehydrins can be located in the nucleus or cytoplasm. Furthermore, it appears that these proteins associate with macromolecules ranging from nucleoprotein complexes in the nucleus to an endomembrane sheath in the cytoplasm. At present, all observations are consistent with a hypothesis that dehydrins are surfactants capable of inhibiting the coagulation of a range of macromolecules, thereby preserving structural integrity.     

Far-red light-induced changes in intracellular potentials of spinach mesophyll cells: interaction with red light

In green plants, the large bioelectric changes that photosynthetically active light stimulates make it difficult to observe electrical potential changes related to phytochrome photoconversion. As a first step towards distinguishing between photosynthetic and phytochrome effects, we showed that red light enhances far-red stimulated intracellular potential changes in spinach (Spinacia oleracea) leaf mesophyll cells.

For a dark-adapted leaf, the response to far-red light increased during the first 10 to 30 exposures of 2.5 minutes, after which it was constant. The intracellular potential depolarized by an average of 0.3 millivolts during each 2.5-minute far-red light period, and returned to the resting value during each subsequent dark period. Continuous supplementary red light (at 1-5% of the fluence rate of the far-red light that stimulated the depolarizations) increased the response to far-red 2- to 3-fold. Supplementary red light did not amplify the response to alternating 702 nanometers light and dark periods. The Emerson enhancement effect thus does not seem to explain amplification of the response to 730 nanometers light by supplementary red light. This does not prove that photosynthetic pigments are not involved in some other way.

     

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.     

不同小麦品种对低温胁迫的反应及抗冻性评价

以济麦19、济麦21、济南17等15个冬小麦品种为材料,对其在低温胁迫条件下功能叶和叶鞘超氧化物歧化酶(SOD)活性、过氧化物酶(POD)活性、过氧化氢酶(CAT)活性、丙二醛(MDA)含量与可溶性蛋白含量等生理指标进行测定,以功能叶各项指标的抗冻系数作为衡量抗冻性的指标,利用主成分分析、聚类分析对其抗冻性进行综合评价。低温胁迫条件下,不同冬小麦品种起身拔节期功能叶和叶鞘中SOD活性、POD活性和CAT活性均不同程度地上升,MDA含量和可溶性蛋白含量均下降。通过主成分分析和聚类分析,将15个冬小麦品种划分为3类:济麦19、山农8355属强抗冻类型;山农664、泰山9818、济麦21、济麦22、烟农24、烟农19、烟农21、汶农6号、鲁麦21、济南17属中度抗冻类型;其余3个品种(泰山23、聊麦18、临麦2号)属弱抗冻类型。     

Effects of glycine hydroxamate, carbon dioxide, and oxygen on photorespiratory carbon and nitrogen metabolism in spinach mesophyll cells

The effects of added glycine hydroxamate on the photosynthetic incorporation of ¹⁴CO₂ into metabolites by isolated mesophyll cells of spinach (Spinacia oleracea L.) was investigated under conditions favorable to photorespiratory (PR) metabolism (0.04% CO₂ and 20% O₂) and under conditions leading to nonphotorespiratory (NPR) metabolism (0.2% CO₂ and 2.7% O₂). Glycine hydroxamate (GH) is a competitive inhibitor of the photorespiratory conversion of glycine to serine, CO₂ and NH₄⁺. During PR fixation, addition of the inhibitor increased glycine and decreased glutamine labeling. In contrast, labeling of glycine decreased under NPR conditions. This suggests that when the rate of glycolate synthesis is slow, the primary route of glycine synthesis is through serine rather than from glycolate. GH addition increased serine labeling under PR conditions but not under NPR conditions. This increase in serine labeling at a time when glycine to serine conversion is partially blocked by the inhibitor may be due to serine accumulation via the “reverse” flow of photorespiration from 3-P-glycerate to hydroxypyruvate when glycine levels are high. GH increased glyoxylate and decreased glycolate labeling. These observations are discussed with respect to possible glyoxylate feedback inhibition of photorespiration.     

Comparative experiments on temperature responses of bryophytes: assimilation,respiration and freezing damage

Abstract

(1) Temperature-net assimilation and temperature-respiration curves based on manometric measurements at high carbon dioxide concentrations are presented for twenty-three mosses and five hepatics.

(2) In most of the species, the optimum temperature for net assimilation under the experimental conditions was about 25°–30°C and the temperature compensation point about 35°–40°C.

(3) Substantially lower optima and maxima were found in Orthothecium rufescens, Plagiopus oederi, Acrocladium trifarium, Fontinalis squamosa, Nardia compressa and Hookeria lucens.

(4) Several northern and montane species (e.g. Anthelia julacea, Andreaea nivalis, Rhacomitrium lanuginosum) did not differ substantially from the majority of lowland species in the response of net assimilation to temperature. Some substantial differences were found between species of differing habitats.

(5) Most of the mosses and leafy liverworts tested withstood rapid cooling to ?5°C for 6 hr. They are evidently protected from intracellular freezing at normal rates of cooling by the withdrawal of water to form extracellular ice.

(6) Conocephalum conicum, Targionia hypophylla and Pellia epiphylla were killed by rapid cooling to ?5°C.

(7) Plagiochila spinulosa and Myurium hebridarumwithstood periods of 1–2 weeks at ?5°C. Survival of bryophytes for long periods of low temperatures appears to be principally a matter of desiccation resistance.     

A comparison of low temperature growth vs low temperature shifts to induce resistance to photoinhibition in spinach (Spinacia oleracea)

Plants of Spinacia oleracea L. cv. Savoy grown under cold-hardening (5°C) and nonhardening (16°C) conditions were exposed to a photoinhibitory irradiance of 1300 μmol rrr^: m-² S-¹ 5°C for 12 h. Plants grown at 5°C exhibited a greater resistance to photoinhibition at low temperature in comparison to plants grown at 16°C as measured by the photochemical efficiency of photosyslem II. In contrast, tuily expanded leaves of plants grown at 16°C and then shifted to 5°C for 10 days did not exhibit increased resistance to photoinhibition. This was observed irrespective of the phoioperiod experienced during the shift to a lower temperature. Furthermore, spinach grown at 16°C and subsequently exposed to a stepped, daily decrease in temperature from 16 to 1°C over 10 days w ith a concomitant reduction in photoperiod. also did not exhibit any change in susceptibility to photoinhibition. Thus, a decrease in photoperiod accompanied by either an abrupt or stepped low temperature shift cannot induce increased resistance to photoinhibition. This confirms the hypothesis that growth and development at cold-hardening temperature are absolute requirements for the acquisition of resistance to photoinhibition at low temperature.     

Metabolic responses to low temperature in fish muscle

For most fish, body temperature is very close to that of the habitat. The diversity of thermal habitats exploited by fish as well as their capacity to adapt to thermal change makes them excellent organisms in which to examine the evolutionary and phenotypic responses to temperature. An extensive literature links cold temperatures with enhanced oxidative capacities in fish tissues, particularly skeletal muscle. Closer examination of inter-species comparisons (i.e. the evolutionary perspective) indicates that the proportion of muscle fibres occupied by mitochondria increases at low temperatures, most clearly in moderately active demersal species. Isolated muscle mitochondria show no compensation of protein-specific rates of substrate oxidation during evolutionary adaptation to cold temperatures. During phenotypic cold acclimation, mitochondrial volume density increases in oxidative muscle of some species (striped bass Morone saxatilis, crucian carp Carassius carassius), but remains stable in others (rainbow trout Oncorhynchus mykiss). A role for the mitochondrial reticulum in distributing oxygen through the complex architecture of skeletal muscle fibres may explain mitochondrial proliferation. In rainbow trout, compensatory increases in the protein-specific rates of mitochondrial substrate oxidation maintain constant capacities except at winter extremes. Changes in mitochondrial properties (membrane phospholipids, enzymatic complement and cristae densities) can enhance the oxidative capacity of muscle in the absence of changes in mitochondrial volume density. Changes in the unsaturation of membrane phospholipids are a direct response to temperature and occur in isolated cells. This fundamental response maintains the dynamic phase behaviour of the membrane and adjusts the rates of membrane processes. However, these adjustments may have deleterious consequences. For fish living at low temperatures, the increased polyunsaturation of mitochondrial membranes should raise rates of mitochondrial respiration which would in turn enhance the formation of reactive oxygen species (ROS), increase proton leak and favour peroxidation of these membranes. Minimisation of mitochondrial oxidative capacities in organisms living at low temperatures would reduce such damage.