Comparison of Low Temperature Responses of Chilling-Sensitive and Chilling-Tolerant Rice Mitochondrial Membrane

After low temperature (4℃ 48 h) treatment oxidative phosphorylation efficiency (ADP/O) and respiratory control ratio (RCR) of mitochondria isolated from chilling- sensitive rice (Qiu Guang, Zao Jin) shoots were lowered and ultrastructure of mitochon- dria in the rice shoots were obviously changed, but no similar changes were observed in the mitochondria from chilling-tolerant rice (Jin Geng 44, Jin Geng 60) shoots after chilling treatment. A “break” at 18℃ had been observed in the Arrhenius plot for succinic oxidase activity by seedling mitochondria of chilling-sensitive rice, while that of mitochondria from chilling-telerant rice shoots exhibited a continuous linear unuction with temperatures between 5–30℃.     

Phospholipid Biosynthesis and Fatty Acid Content in Relation to Chilling Injury during Germination of Seeds

The proportion of labeled ¹⁴C-glycerol incorporated into phospholipids and the fatty acid composition of three phospholipids in germinating seeds and seedlings of chilling-sensitive lima beans (Phaseolus lunatus L.) and chilling-resistant broad beans (Vicia faba L.) and peas (Pisum sativum L.) at 10 and 25 C were determined. During the imbibition of seeds (first 24 hours), lima beans were sensitive to chilling injury at 10 C and a higher proportion of label was incorporated into phosphatidylethanolamine and phosphatidylglycerol than in broad beans and peas. Broad beans and peas incorporated a higher proportion of label into phosphatidylcholine. The oleic acid content of phosphatidylcholine was higher and linolenic acid content was lower in peas and broad beans than in lima beans at 10 and 25 C. The unsaturated to saturated fatty acid ratio was much higher for the chilling-resistant seeds than for the chilling-sensitive ones. In the seedling stage, the proportion of label incorporated into the four major phospholipids was similar in the three species regardless of temperature treatment. The fatty acid content of the phospholipids examined was not different in the three species in the seedling stage.     

Lipid-protein interactions in thylakoid membranes of chilling-resistant and -sensitive plants studied by spin label electron spin resonance spectroscopy

Lipid-protein interactions in thylakoid membranes from lettuce, pea, tomato, and cucumber have been studied using spin-labeled analogues of the thylakoid membrane lipid components, monogalactosyl diglyceride and phosphatidylglycerol. The electron spin resonance spectra of the spin-labeled lipids all consist of two components, one corresponding to the fluid lipid environment in the membranes and the other to the motionally restricted lipids interacting with the integral membrane proteins. Comparison of the spectra from the same spin label in thylakoid membranes from different plants shows that the overall lipid fluidity in the membranes decreases with chilling sensitivity. Spectral subtraction has been used to quantitate the fraction of the membrane lipids in contact with integral membrane proteins. Thylakoid membranes of cucumber, a typical chilling-sensitive plant, have been found to have a higher proportion of motionally restricted lipids and a different lipid selectivity for lipid-protein interaction, as compared with those of pea, a typical chilling-resistant plant. This correlation with chilling sensitivity holds generally for the different plants studied. It seems likely that the chilling sensitivity in thylakoid membranes is not determined by lipid fluidity alone, but also by the lipid-protein interactions which could affect protein function in a more direct manner.     

Roles of the plasma membrane and the cell wall in the responses of plant cells to freezing

In an effort to clarify the responses of a wide range of plant cells to freezing, we examined the responses to freezing of the cells of chilling-sensitive and chilling-resistant tropical and subtropical plants. Among the cells of the plants that we examined, those of African violet ( Saintpaulia grotei Engl.) leaves were most chilling-sensitive, those of hypocotyls in mungbean [ Vigna radiata (L.) R. Wilcz.] seedlings were moderately chilling-sensitive, and those of orchid [ Paphiopedilum insigne (Wallich ex Lindl.) Pfitz.] leaves were chilling-resistant, when all were chilled at -2 degrees C. By contrast, all these plant cells were freezing-sensitive and suffered extensive damage when they were frozen at -2 degrees C. Cryo-scanning electron microscopy (Cryo-SEM) confirmed that, upon chilling at -2 degrees C, both chilling-sensitive and chilling-resistant plant cells were supercooled. Upon freezing at -2 degrees C, by contrast, intracellular freezing occurred in Saintpaulia leaf cells, frost plasmolysis followed by intracellular freezing occurred in mungbean seedling cells, and extracellular freezing (cytorrhysis) occurred in orchid leaf cells. We postulate that chilling-related destabilization of membranes might result in the loss of the ability of the plasma membrane to act as a barrier against the propagation of extracellular ice in chilling-sensitive plant cells. We also examined the role of cell walls in the response to freezing using cells in which the plasma membrane had been disrupted by repeated freezing and thawing. In chilling-sensitive Saintpaulia and mungbean cells, the cells with a disrupted plasma membrane responded to freezing at -2 degrees C by intracellular freezing. By contrast, in chilling-resistant orchid cells, as well as in other cells of chilling-resistant and freezing-resistant plant tissues, including leaves of orchard grass ( Dactylis glomerata L.), leaves of Arabidopsis thaliana (L.) Heynh. and cortical tissues of mulberry ( Morus bombycis Koids.), cells with a disrupted plasma membrane responded to freezing by extracellular freezing. Our results indicate that, in the chilling-sensitive plants cells that we examined, not only the plasma membrane but also the cell wall lacked the ability to serve as a barrier against the propagation of extracellular ice, whereas in the chilling-resistant plant cells that we examined, not only the plasma membrane but also the cell wall acted as a barrier against the propagation of extracellular ice. It appears, therefore, that not only the plasma membrane but also the cell wall greatly influences the freezing behavior of plant cells.     

Composition and positional distribution of fatty acids in polar lipids from Chlorella ellipsoidea differing in chilling susceptibility and frost hardiness

The composition and positional distribution of fatty acids in the polar lipids from 4 strains of Chlorella differing in chilling susceptibility and frost hardiness were analyzed by enzymatic hydrolysis and gas-liquid chromatography. Analysis of the polar lipids from chilling-sensitive, chilling-resistant and chilling-sensitive revertant strains of Chlorella ellipsoidea IAM C-102 showed that the sum of palmitic and trans -3-hexadecenoic acid in phosphatidylglycerol (PG) is about 60% for the sensitive strains and 53% for the resistant strain. The sum of dipalmitoyl and 1-palmitoyl-2-( trans -3-hexadecenoyl) PG as estimated from the positional distribution of their fatty acids, is about 10% in the case of each of the three strains. The contents of unsaturated fatty acids in phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were higher in the resistant than in the sensitive strain. This suggests that unsaturation of fatty acids in not only PG but also PC and PE is involved in chilling sensitivity of Chlorella . On the other hand, lipid changes during the development of frost hardiness of C. ellipsoidea IAM C-27, a frost hardy strain, were examined. The results showed that fatty acids in most lipid classes are unsaturated in the hardening process but their degree of unsaturation is not greatly different from that of the chilling-resistant strain, suggesting that not only unsaturation of fatty acids in lipids but also other factors are necessary for the development of frost hardiness.     

顺磁探针16－NS和荧光探针DPH在DPPG交插脂双层中的行为研究

研究了１６—ＮＳ和ＤＰＨ在被山莨菪碱诱导形成交插脂结构的ＤＰＰＧ脂质体中的行为。在交插脂双层结构中,标记在脂酰链末端的１６—ＮＳ的序参数明显增大,即同标记在靠脂酰链头部的５—ＮＳ的序参数的差距缩小。这些颇磁探针在交插脂双层结构中的运动比在非交插脂双层结构中受到更大的限制。ＤＰＨ荧光强度在ＤＰＰＧ由非交插脂双层转变为交插脂双层结构时急剧下降。引起这种下降的原因可能是膜脂在从非交插脂双层结构向交插脂双层结构的转变过程中使得ＤＰＨ处于一个更为亲水的环境中。     

顺磁探针16－NS和荧光探针DPH在DPPG交插脂双层中的行为研究

研究了１６—ＮＳ和ＤＰＨ在被山莨菪碱诱导形成交插脂结构的ＤＰＰＧ脂质体中的行为。在交插脂双层结构中,标记在脂酰链末端的１６—ＮＳ的序参数明显增大,即同标记在靠脂酰链头部的５—ＮＳ的序参数的差距缩小。这些颇磁探针在交插脂双层结构中的运动比在非交插脂双层结构中受到更大的限制。ＤＰＨ荧光强度在ＤＰＰＧ由非交插脂双层转变为交插脂双层结构时急剧下降。引起这种下降的原因可能是膜脂在从非交插脂双层结构向交插脂双层结构的转变过程中使得ＤＰＨ处于一个更为亲水的环境中。     

Mechanism of the chilling-induced decrease in proton pumping across the tonoplast of rice cells

The ATP-generated proton pumping across tonoplast vesicles from chilling-sensitive Boro rice (Oryza sativa L. var. Boro) cultured cells was markedly decreased by chilling at 5 degrees C for 3 d. The membrane fluidity of core hydrophobic and surface hydrophilic regions of the lipid bilayer was measured by steady-state fluorescence depolarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium 1,6-diphenyl-1,3,5-hexatriene and by electron spin resonance spectroscopy of 16- and 5-doxyl stearic acid, respectively. The fluidity of the surface region of the lipid bilayer of the tonoplast vesicles decreased by chilling. The fluidity of the surface region of the liposomes and the proton pumping across the reconstituted proteoliposomes with tonoplast H+-ATPase decreased with increasing content of the glycolipids. The proton pumping across chimera proteoliposomes was reduced by chilling only when it was reconstituted in the presence of tonoplast glycolipids from chilled Boro cells. These data suggest that the reduction in ATP-generated proton pumping across the tonoplast by chilling is due to the decrease in the fluidity of the surface region of the lipid bilayer of the tonoplast, which is caused by the changes in glycolipids.     

Differential responses of antioxidative system to chilling and drought in four rice cultivars differing in sensitivity

Responses of antioxidative defense systems to chilling and drought stresses were comparatively studied in four cultivars of rice (Oryza sativa L.) differing in sensitivity, two of them (Xiangnuo no. 1 and Zimanuo) are tolerant to chilling but sensitive to drought and the other two (Xiangzhongxian no. 2 and IR50) are tolerant to drought but sensitive to chilling. The seedlings of rice were transferred into growth chamber for 5 d at 8 degrees C as chilling treatment, or at 28 degrees C as control, or at 28 degrees C but cultured in 23% PEG-6000 solution as drought stress treatment. Under drought stress the elevated levels of electrolyte leakage, contents of H(2)O(2) and total thiobarbituric acid-reacting substances (TBARS) in Xiangzhongxian no. 2 and IR50 are lower than those in Xiangnuo no. 1 and Zimanuo. On the contrary, Xiangnuo no. 1 and Zimanuo have much lower level of electrolyte leakage, H(2)O(2) and TBARS than Xiangzhongxian no. 2 and IR50 under chilling stress. Activities of antioxidant enzymes (superoxide dismutase (SOD), catalase, and ascorbate-peroxidase (APX)) and contents of antioxidants (ascorbaic acid and reduced glutathione) were measured during the stress treatments. All of them were enhanced greatly until 3 d after drought stress in the two drought-tolerant cultivars, or after chilling stress in the two chilling-tolerant cultivars. They all were decreased at 5 d after stress treatments. On the other hand, activities of antioxidant enzymes and contents of antioxidants were decreased greatly in the drought-sensitive cultivars after drought stress, or in the chilling-sensitive cultivars after chilling stress. The results indicated that tolerance to drought or chilling in rice is well associated with the enhanced capacity of antioxidative system under drought or chilling condition, and that the sensitivity of rice to drought or chilling is linear correlated to the decreased capacity of antioxidative system.     

A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

Experiments comparing the photosynthetic responses of a chilling-resistant species (Pisum sativum L. cv Alaska) and a chilling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site of the differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25°C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 5°C in 400 millimolar NaCl were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5°C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5°C). During wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant.     

Effect of Exogenous Cholesterol on the Fluidity of Mitochondrlal Membran from Rice Root Apex

The effect of exogenous cholesterol on the fluidity of mitochondrial membrane from riceroot apex was investigated using the DPH fluorescece probe (1,6-diphenyl-l, 3, 5,-hexatriene).Results showed that exogenous cholesterol, either being absorbed by root system during water culture of rice or being added directly to the prepared mitochondrial of control rice root apex, decreased the fluorescence intesity of probe in the mitochondrial membrane and reduced the fluorescence polarization as well as micro-viscosity, but increased the fluidity of the mitochondrial membrane in rice root apex.     

外源胆固醇对水稻根端线粒体膜流动性的影响

应用荧光探剂 1,6-二苯基-1,3,5-己三烯(DPH)观察外源胆固醇对水稻极端线粒体膜流动性的影响。结果表明,无论外源胆固醇通过水培根系吸收或是直接添加给予水稻根端线粒体,都能使DPH与线粒体结合后的荧光强度减弱,使荧光偏振度和微粘度降低,增加水稻根端线粒体膜流动性。     

Vitamin D3 administration increases the membrane fluidity of intestinal mitochondria

The fluorescence anisotropy in the mitochondria from vitamin D-treated chicks is significantly lower than that from the vitamin D-deficient animals with the inner core probe DPH. Surface membrane fluidity, measured with the probe TMA-DPH, shows no differences between the organelles of both groups. The fluorescence studies performed in mitochondrial subfractions revealed that cholecalciferol treatment induces a decrease of lipid order parameter S (DPH) in the mitochondrial inner membrane. These results pose the question of whether vitamin D3 participates in the regulation of physiological function of the intestinal mitochondria through changes in the physical properties of the membranes.     

Electron spin resonance studies of the effects of lipids on the environment of proteins in mitochondrial membranes

The physical state of mitochondrial membranes has been investigated by means of stearic acid spin labels and of a maleimide spin label covalently bound to protein sulfhydryl groups. Stearic acid spin labels 5-NS and 16-NS show that n-butanol enhances the lipid fluidity of mitochondrial membranes in the whole temperature range between 4 and 37 degrees C; the effects in the hydrophobic membrane core, probed by 16-NS, are already apparent at 10 mM butanol. In liposomes formed of mitochondrial phospholipids, a fluidizing effect appears only at much higher concentration. Such results are compatible with the idea that butanol destabilizes lipid-protein interactions. On the other hand, the ratio between weakly and strongly immobilized SH groups probed by maleimide spin label is only slightly affected in the temperature range of 4-37 degrees C by addition of high concentrations of n-butanol, indicating that the environments probed are stable to agents inducing fluidity changes in the lipids. There are, however, indications that the environment probed by maleimide is affected by lipids, since the spin label, when bound to lipid-depleted mitochondria, becomes more immobilized, reconstitution of such lipid-depleted membranes with phospholipids restores the original spectra.     

Chilling response of plants: importance of galactolipase, free fatty acids and free radicals

The chilling response of plants is complex and based on the interplay of two important metabolic processes--lipolytic degradation of membrane lipids and a set of oxidative reactions leading to lipid peroxidation and membrane damage evoked in chilling-sensitive (CS) plants subjected to low temperature and light. The effects of chilling of detached leaves and intact plants differ and are often neglected during experiments. In closely-related species, the activity of several constitutive enzymes (i.e. superoxide dismutase, ascorbate peroxidase and glutathione reductase) appears to be higher in chilling-tolerant (CT) than in CS species; while in several native, closely-related CS species, lipid acyl hydrolase (galactolipase) activity is higher than in CT species. Moreover, in chilling-insensitive (CI) plants, galactolipase activity is very low and is neither activated by detachment of leaves nor under stress conditions in growing plants. Dark and low-temperature treatments of detached leaves of CS species and post-chilling recovery of growing plants in the light activate galactolipase, which is responsible for the release of free fatty acids (FFA), the main substrates of peroxidation by lipoxygenase and free radicals. In several CS species, increased galactolipase activity is an important factor contributing to chilling susceptibility. Thus, it seems likely that enhancement of chilling tolerance may be achieved by genetically suppressing galactolipase in order to reduce both the degradation of chloroplast lipids and the level of released FFA, and thereby avoiding the deleterious action of their peroxidation products on plant tissues.     

Effects of Chilling Temperature on Photosynthesis and Photosynthate Transport in Flag Leaves of Hybrid Rice at Milky Stage

To understand the physiological reasons for poor yield of the second rice crop in southern China challenged by low temperature. The authors investigated the effects of chilling temperature on photosynthesis and the activity of fructose 1, 6-diphosphatase (FBPase) of flag leaves at milky stage using two hybrid rice varieties with different cold tolerance. The results indicated that chilling temperature caused decreases of photosynthetic efficiency and FBPase activity in detached flag leaves. The decline of photosynthetic efficiency and FBPase activity was greater in Shanyou-63 which is as cold sensitive as its parents than in Xiuyou-57 which is cold tolerant like its parents. The milky stage is the period of the fastest grain filling. The decreased the yield of the second rice crop caused by natural low temperature was associated with decline of photosynthesis and the abilities of adaption and adjustment of FBPase in attached flag leaves. The relationship between the effects of chilling temperature on photosynthesis and photosynthate transport and the yield formed by grain was discussed.     

Galactolipase activity and free fatty acid levels in chloroplasts of domestic and wild tomatoes with different chilling tolerance

In order to establish differences in the chilling sensitivity of domestic and wild Lycopersicon species, galactolipase (EC 3.1.1.26) activity, free fatty acid (FFA) level and Hill reaction activity were measured in chloroplasts isolated from control and cold treated leaves of L. esculentum Mill., cv. Norton, L. hirsutum Humb. and Bonpl., L. peruvianum var. glandulosum Mill. Galactolipase activity was higher in chloroplasts from Lycopersicon species with high chilling sensitivity than in chloroplasts of more chilling-resistant ones. A similar relationship was observed for FFA level in chloroplasts from both cold-stored and control leaves. Decrease in Hill reaction activity due to cold stress was greater in chloroplasts of more chilling-sensitive species. The changes are accompanied by a decline of photochemical activity. Considering the changes in the three parameters noted above, an increasing order of chilling tolerance was established: L. esculentum < L. hirsutum (700 m) < L. hirsutum (3100 m) < L. peruvianum (3400 m). It is suggested that measurements of galactolipase activity and FFA may be useful in an evaluation of differences in resistance to chilling injury of closely related species.     

The relevance of seed membrane lipids to imbibitional chilling effects

Imbibitional chilling injury during rehydration of seeds is particularly marked in soybean but not in pea. Following the previous reports that the deleterious effects of chilling on soybean germination are probably mediated at least in part through a loss of membrane integrity, differences in composition of membrane lipid components extracted from pea (chilling-insensitive) and soybean (chilling-sensitive) were examined. Comparative compositional analysis of phospholipids, fatty acids and free sterols revealed few disparities between the two species. When the membrane lipids were re-formed into liposomes, little difference in permeability was found with respect to KCl, glucose or glycerol, even at chilling temperatures. It is suggested that the difference in chilling sensitivity between pea and soybean is not related to compositional differences in the major lipid components of the seed membranes.     

缺锌对大鼠红细胞膜生物物理性质的影响

利用激光衍射仪,荧光分光光度法,ＥＳＲ等牲物理技术和缺锌大鼠模型,研究缺锌对大鼠红细胞膜生物物理性质的影响,实验结果表明缺锌使大鼠红细胞的变形能力增大,稳定性降低,红细胞膜脂的流动性和膜蛋白的运动性增加,支持了锌具有维持膜结构和功能和生理作用的理论;为膜结构和功能的改变是缺锌病理变化主要原因的假说提供了实验依据。