抗寒锻炼中不同抗寒性小麦细胞膜糖蛋白的细胞化学研究

本研究根据植物细胞的特点,修改了在动物和人体细胞方面立的酶标Con A的电镜细胞化学方法,成功地展现了2个不同抗寒性冬小麦品种幼苗在抗寒锻炼和脱锻炼过程中细胞膜系统上糖蛋白的分布动态,显示与Con A连接的标志酶-辣根过氧化物酶活性的反应产物呈颗粒状分散分布在质膜、内质网、核膜及液泡膜的一些部位上,揭示糖蛋白在冬小麦细胞膜系统上的分布似有其特定的位点。经抗寒锻炼后,强抗寒性品种燕大1817细胞内的糖蛋白在内质网和核膜上的分布量明显地增加;同时,几乎所有的胞间连丝通道中都有糖蛋白的分布。脱锻炼后,内质网和核膜上的糖蛋白分布量又减少,胞间连丝通道中的糖蛋白也消失,基本上回复到抗寒锻炼前的分布状态。抗寒性弱的冬小麦品种郑州39-1幼苗在同样的抗寒锻炼和脱锻炼过程中不产生这些明显的变化。这些结果说明,抗寒锻炼中内质网和核膜上糖蛋白分布量的增加,以及糖蛋白输入胞间连丝的动态变化是与植物抗寒力的提高和保持稳定密切相关的。     

小麦质膜及液泡膜的ATP酶活性在抗寒锻炼中的变化

采用磷酸铅沉淀的电镜细胞化学方法,对比观察了耐寒的冬小麦幼苗和不耐寒的春小麦幼苗在抗寒锻炼过程中质膜及液泡膜ATP酶活性的变化,结果指出:冬小麦质膜ATP酶在抗寒锻炼过程中获得了耐低温的特性,以致在适于锻炼的低温(3℃)下,能保持高的活性反应;同时液泡膜在锻炼过程中形成高的ATP酶活性。春小麦幼苗在低温锻炼中不能提高抗寒力,其质膜ATP酶在锻炼过程中也不能发展抗寒性能,因而在3℃低温培育下不表现明显的活性;同时液泡膜也不产生ATP酶活性。这些结果进一步证明,在抗寒锻炼中,冬小麦质膜耐低温特性的形成,以及在液泡膜上产生高的ATP酶活性,是与植株抗寒力的提高有密切关系的重要特性。     

人体内cAMP与cGMP动态平衡机理的探讨

cAMP(环磷酸腺苷)与cGMP(环磷酸鸟苷)作为“第二信使”参与激素的作用。 Goldberg等根据在一些分化的细胞(心肌、平滑肌、血小板和中枢神经细胞)中观察到cAMP和cGMP对细胞代谢反应和生理功能的調节作用是相反的,浓度的变化是相反而相关的,提出了双向控制学说和阴阳学说,似乎和我国中医学的“阴阳”相符。他们认为这一对阴阳物质能调节细胞的酶反应和细胞的生理效应。正常代谢和生理反应就是这一对阴阳物质的动态平衡的结果。为了探讨cAMP与cGMP     

基于CFTR的胞浆内第二信使cAMP检测方法的建立*

目的: 建立一种基于CFTR可敏感检测胞浆内第二信使cAMP的检测方法。方法: 构建CFTR和YFP-H148Q/I152L真核表达载体,应用脂质体转染法构建共表达CFTR和YFP-H148Q/I152L的FRT细胞,倒置荧光显微镜观察其表达情况,流式细胞仪检测细胞纯度;荧光淬灭动力学实验验证细胞模型的有效性;荧光淬灭动力学实验验证细胞模型可筛选CFTR调节剂;放射免疫法检测加入CFTR激活剂后细胞内的cAMP浓度。结果: 倒置荧光显微镜下观察到CFTR表达在细胞膜上,YFP-H148Q/I152L表达于胞浆中;成功构建共表达CFTR和YFP-H148Q/I152L的FRT细胞模型;荧光变化斜率值与CFTR调节剂浓度成剂量依赖关系,该模型可筛选CFTR调节剂;荧光变化斜率值可反映胞浆内cAMP浓度,该模型可敏感检测胞浆内cAMP浓度。结论: 此细胞模型可以高效敏感检测胞浆内第二信使cAMP浓度,为cAMP信号相关靶点的研究提供一种简便快捷的方法。     

植物寒害和抗寒机制中膜与蛋白质研究的进展

低温对细胞膜体系的损伤是植物寒害的重要机制。膜体系的稳定性与植物的抗寒性成正相关,但不同的细胞膜体系对细胞外结冰的敏感程度是不同的。抗寒锻炼中膜磷脂的生物合成与抗寒力的发展有密切关系,但不是抗寒力发展的前提条件,可能是对发展高水平的抗寒力起作用;而膜脂脂肪酸不饱和度的增加是植物对低温生长的反应,与抗寒性无立直接关系。近年来膜脂 — 膜蛋白相互关系的研究引起研究者们的重视,已在多种植物低温锻炼中观察到抗寒特异蛋白质合成与基因表达均有所改变,并发现抗寒力的诱导主要是在转录水平上的调控。     

植物寒害和抗寒机制中膜与蛋白质研究的进展

低温对细胞膜体系的损伤是植物寒害的重要机制。膜体系的稳定性与植物的抗寒性成正相关,但不同的细胞膜体系对细胞外结冰的敏感程度是不同的。抗寒锻炼中膜磷脂的生物合成与抗寒力的发展有密切关系,但不是抗寒力发展的前提条件,可能是对发展高水平的抗寒力起作用;而膜脂脂肪酸不饱和度的增加是植物对低温生长的反应,与抗寒性无直接关系。近年来膜脂-膜蛋白相互关系的研究引起研究者们的重视,已在多种植物低温锻炼中观察到抗寒特异蛋白质合成与基因表达均有所改变,并发现抗寒力的诱导主要是在转录水平上的调控。     

冬小麦(Triticum aestivumL)分蘖节细胞内三磷酸腺甙酶活性的细胞化学研究

采用磷酸铅沉淀的细胞化学方法,对冬小麦分蘖节细胞内的三磷酸腺甙酶(ATP-ase)活性进行了亚显微结构的定位,并试验了ATP-ase 在5℃低温下的反应活性。结果指出:无论在一般采用的常温(22℃)反应条件下,或是在5℃的低温反应中,在小麦分蘖节细胞的质膜、胞间连丝、核仁,染色质以及液泡中,均显示出很高的ATP-ase 的活性反应;在造粉体周围的细胞质以及细胞间隙内,也观察到ATP-ase 活性反应的磷酸铅沉淀物;而在线粒体、质体和内质网上,则一般未表现出ATP-ase 的活性。对于ATP-ase 活性在小麦分蘖节细胞各部位的定位情况及其可能的功能意义,以及ATP-ase 在5℃低温下的高活性反应同冬小麦植株在低温下抗寒锻炼的关系,进行了讨论。     

小鼠艾氏腹水癌细胞内3'''',5''''-环腺苷酸(cAMP)的免疫荧光细胞化学定位和外源性cAMP对癌细胞内cAMP的影响

本工作用显示cAMP的免疫荧光细胞化学方法观察了小鼠艾氏腹水癌细胞内cAMP的分布,并发现经外源性cAMP及氨茶碱处理后,有使小鼠艾氏腹水癌细胞内cAMP荧光增强的效应。cAMP主要分布在胞质内。实验组动物从接种癌细胞的次日起,每日从腹腔注射2毫克的cAMP和1毫克的氨茶碱,发现在接种后第9天,大多数癌细胞胞质内的cAMP荧光增强,以核周区胞质内的荧光最显著。进一步证实了我室以前用生化测定癌细胞内cAMP含量的结果。本文并对显示cAMP的免疫荧光细胞化学方法进行了讨论,用cAMP荧光染色反应的特异性鉴定证明了方法的可靠性。     

抗寒锻炼对冬小麦幼苗质膜Ca^2＋—ATPase的稳定作用

通过氯化铈（ＣｅＣｌ３）沉淀的电镜细胞化学方法,观察了抗寒锻炼对冬小麦（ＴｒｉｔｉｃｕｍａｅｓｔｉｖｕｍＬ．）幼苗质膜Ｃａ２＋ＡＴＰａｓｅ的稳定作用,主要结果是：（１）正常温度（２０℃）下生长的冬小麦幼苗（未经抗寒锻炼）,其质膜上有很强的Ｃａ２＋ＡＴＰａｓｅ活性反应;当经过－９℃３ｈ的低温处理后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性明显降低;在处理１２ｈ后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性进一步降低;当处理时间延长到２４ｈ,质膜的Ｃａ２＋ＡＴＰａｓｅ完全失活,同时细胞的超微结构受到破坏。（２）冬小麦幼苗在２℃低温下锻炼１５ｄ后,其质膜的Ｃａ２＋ＡＴＰａｓｅ活性高于未经抗寒锻炼的小麦幼苗。抗寒锻炼后的小麦幼苗在－９℃处理３ｈ后,质膜的Ｃａ２＋ＡＴＰａｓｅ活性与低温处理前相比无明显降低;经低温处理１２ｈ,质膜仍保持较高的Ｃａ２＋ＡＴＰａｓｅ活性,较同样低温处理（－９℃,１２ｈ）但未经抗寒锻炼的幼苗高;当－９℃低温处理２４ｈ后,质膜上仍可观察到Ｃａ２＋ＡＴＰａｓｅ的活性反应,而且细胞的超微结构也未受到破坏。结果表明,抗寒锻炼可提高冬小麦幼苗质膜Ｃａ２＋ＡＴＰａｓｅ在低温下的稳定性     

注射依赖cAMP的蛋白激酶催化亚基加速胚胎内细胞间连接通讯的发育(英文)

本研究以爪蟾胚胎内胚层细胞间连接通讯发育的时程为指标,观察了cAMP和依赖cAMP的蛋白激酶催化亚基对这一发育的影响。将依赖cAMP的蛋白激酶催化亚基注射入爪蟾胚胎四细胞期的每一个细胞可使该胚胎内胚层细胞间连接通讯的发育明显加快,提示在正常状态下这一发育的进程是受细胞内依赖cAMP的磷酸化水平的制约。但用双丁酰cAMP和磷酸二酯酶抑制剂对胚胎进行培育,或将cAMP和磷酸二酯酶抑制剂注射入胚胎细胞,对这一发育并无影响,可能是由于这些胚胎细胞内依赖cAMP的蛋白激酶的实际有效含量较低而cAMP含量较高所致。注射这种蛋白激酶催化亚基所诱导的胚胎细胞间连接通讯在注射后约8.5小时出现。这一时间比前人在哺乳动物细胞培养中用cAMP或必需的信使RNA诱导出细胞间连接通讯所需要的时间(2—4小时)长得多,提示在胚胎中依赖cAMP的磷酸化对细胞间连接通讯发育的作用是从RNA转录的水平上开始的。     

Dynamic thermal time model of cold hardiness for dormant grapevine buds

Background and Aims

Grapevine (Vitis spp.) cold hardiness varies dynamically throughout the dormant season, primarily in response to changes in temperature. The development and possible uses of a discrete-dynamic model of bud cold hardiness for three Vitis genotypes are described.

Methods

Iterative methods were used to optimize and evaluate model parameters by minimizing the root mean square error between observed and predicted bud hardiness, using up to 22 years of low-temperature exotherm data. Three grape cultivars were studied: Cabernet Sauvignon, Chardonnay (both V. vinifera) and Concord (V. labruscana). The model uses time steps of 1 d along with the measured daily mean air temperature to calculate the change in bud hardiness, which is then added to the hardiness from the previous day. Cultivar-dependent thermal time thresholds determine whether buds acclimate (gain hardiness) or deacclimate (lose hardiness).

Key Results

The parameterized model predicted bud hardiness for Cabernet Sauvignon and Chardonnay with an r² = 0·89 and for Concord with an r² = 0·82. Thermal time thresholds and (de-)acclimation rates changed between the early and late dormant season and were cultivar dependent but independent of each other. The timing of these changes was also unique for each cultivar. Concord achieved the greatest mid-winter hardiness but had the highest deacclimation rate, which resulted in rapid loss of hardiness in spring. Cabernet Sauvignon was least hardy, yet maintained its hardiness latest as a result of late transition to eco-dormancy, a high threshold temperature required to induce deacclimation and a low deacclimation rate.

Conclusions

A robust model of grapevine bud cold hardiness was developed that will aid in the anticipation of and response to potential injury from fluctuations in winter temperature and from extreme cold events. The model parameters that produce the best fit also permit insight into dynamic differences in hardiness among genotypes.     

A Second-order Dynamic Model for the Frost Hardiness of Trees

The development of frost hardiness in forest trees is describedby a dynamic model in which the input variables are the prevailingenvironmental conditions and the developmental stage of trees.The assumption of the model is that for each temperature andphotoperiod there is a discrete stationary level of frost hardiness,which is attained if these environmental factors remain constant.The dependence of the stationary level on temperature and photoperiodis assumed to be piece-wise linear and additive. The rate ofacclimation, i.e. frost hardening or dehardening, is describedas a second-order dynamic process with two time constants, thesecond of which changes depending on the stage of the annualdevelopment of the trees. The frost hardiness model was calibratedand tested using experimental data from Douglas fir [Pseudotsugamenziesii var. glauca (Beissn.) Franco] seedlings. The resultssuggest that the second-order model describes the changes infrost hardiness better than the first-order model with onlyone time constant.Copyright 1995, 1999 Academic Press Acclimation, developmental stage, Douglas fir, dynamic model, frost hardiness, photoperiod, Pseudotsuga menziesii, temperature     

A Simulation Model for the Annual Frost Hardiness and Freeze Damage of Scots Pine

The changes in the frost hardiness of Scots pine were modelledby a dynamic model where the input variables were temperatureand photoperiod and the phase of annual development. The damagecaused by freezing was described by the sigmoidal relationshipbetween the relative needle damage and freezing temperature.The model simulations were carried out using temperature datafrom two sites in central Finland—Suonenjoki and Tampere.The validity of the frost hardiness model was tested with measuredfrost hardiness data from Suonenjoki. The effects of climaticwarming were also simulated by increasing temperature of thelong-term climatic data. Genotypic differences in chilling requirement,which determines the timing of the reduction of hardening competence,were included in the simulations. The simulated needle damageincreased as a result of climatic warming, and the differencesin the chilling requirement had a stronger effect on the amountof damage in the warmed climate than in the present climate.A large variation between years was found in the level of damage. Annual development; climatic change; dynamic model; freeze damage; frost hardiness,Pinus sylvestris ; Scots pine     

针叶树种抗寒性数学模型研究进展

介绍了几种针叶树种抗寒性动态模型的研究发展概况、不同模型的优势和局限,以及今后的研究展望.在已建立的数学模型中,初期研究只考虑温度作为影响抗寒性的环境因子,引入了抗寒性固定水平概念;由于这类模型对抗寒性的预测存在明显误差,之后的研究假设抗寒性固定水平是温度和光周期加性影响的结果,并考虑了抗寒性年发育阶段对环境的响应.加性模型受到研究人员的重视,并对加性原理进行了实际检验.有研究表明,温度和光周期对欧洲赤松苗不同器官的影响不是累加的,而是存在交互作用.因此,抗寒性数学模型需要不断地发展和完善.     

Quantitative Analysis of Cold Hardening and Dehardening in Lolium

The change in cold hardiness of three Lolium multiflorum Lam.varieties was followed in plants exposed to hardening or dehardeningconditions at a range of temperatures. Hardening and dehardeningwere analysed as quantitative processes dependent upon temperatureand time. Their time courses changed exponentially to an asymptotewhich was logistically related to temperature. Both componentsof the model were fitted simultaneously. Parameters of biologicalinterest, such as the initial rates of hardening and dehardeningfor a given temperature and the percentage of the process completedin a given time, were derived and compared for varieties ofcontrasting hardiness. The analysis demonstrated the importanceof dehardening in determining hardiness. Similar results wereobtained when the model was applied to hardening and dehardeningdata for Lolium perenne L. The potential of this quantitative analysis for distinguishingbetween possible mechanisms of cold hardiness is discussed andfurther experiments required to characterise the kinetics ofhardening and dehardening more fully are identified. Lolium multiflorum, Lolium perenne, cold hardening, cold dehardening, processes, model, varieties     

Tolerance to multiple climate stressors: a case study of Douglas‐fir drought and cold hardiness

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Genetic analysis of frost hardiness traits in tuber-bearingSolanum species

The inheritance of frost hardiness and cold acclimation potential traits was studied in three segregating populations derived from a cross betweenSolanum commersonii Dun. PI 243503 (cmm) andSolanum cardiophyllum Lindl., PI 184762 (cph), two parental genotypes with contrasting frost hardiness and cold acclimation potential. The levels of frost hardiness and cold acclimation potential were expressed as the LT₅₀, the temperature at which 50% of the cells in leaf discs were killed, as measured by the ion leakage method, following a controlled freeze test There was considerable variation in both frost hardiness and cold acclimation potential in all three segregating populations (F₁ F₁ xcmm, and F₁ xcph). Frost hardiness and cold acclimation potential were not correlated, suggesting that these two traits are under independent genetic control. The analysis of generation means indicated that the variation for both traits could be best explained by an additive-dominance model, with additive gene effects the most important Broad-sense heritability was 0.73 and 0.74 in the F₁ population, for frost hardiness and cold acclimation potential, respectively, and was 0.85 for either trait in the F₁ xcmm population, indicating that these two traits are highly inheritable. Our results suggest that it should be possible to incorporate the frost hardiness and cold acclimation traits from S.commersonii into cultivated potato species.     

Inheritance of autumn frost hardiness in Pinus sylvestris L. seedlings

Summary Inheritance of frost hardiness was analysed making use of a 12×12 incomplete factorial mating design. Owing to space limitations only 59 families could be tested in four experiments. To link the four experiments, some families were common to two or more experiments. The seedlings were grown in climate chambers under conditions inducing autumn hardening. The plants were exposed to a freezing temperature of –10 °C for three hours at night lengths of 11–13 h. A statistical model was developed for analyses of variance of our data. The genetic variation and the variation due to the cultivation regimes during autumn hardening were of the same magnitude. The additive effects were the most important ones for induction of frost damage. No interaction following long-distance crossing was noted. Mixed model equations were used for ranking of the parents. The results obtained support a polygenic inheritance of frost hardiness. The large within-population variation offers good opportunities for hardiness breeding.     

Changes in Frost Hardiness of Stem Cortical Tissues of Cornus stolonifera Michx. after Recovery from Water Stress

Moderate water stress increases frost hardiness in many woody plants but little attention has been given to changes in hardiness after recovery from water stress. Tests were carried out to examine how much water stress-induced frost hardiness remained when plants were rewatered under different day length regimes. Red osier dogwood plants (Cornus stolonifera Michx.) were water-stressed at normal growing temperatures in long day (LD) or short day (SD) conditions, exposed to 6 nights of freezing temperatures, and then returned to normal growing conditions with full water supply. Water-stressed plants gained an additional 8 to 10 C of hardiness. The amount of freeze-induced hardiness in both stressed and control plants was not significant (approximately 2 C) and was not affected by photoperiod. When plants were kept in or transferred to LD, they lost nearly all of their water stress-induced hardiness within 7 days after rewatering. Water-stressed plants in SD lost the least amount of hardiness (5 C) when rewatered. In dogwood, water stress is an effective way to increase hardiness temporarily, but the photoperiod has a large effect on the retention of the acquired hardiness.     

The Role of Light in Cold Acclimation of Hedera helix L. var. Thorndale

The role of light in cold acclimation of Hedera helix L. var. Thorndale appears to differ from that reported for winter annuals. Although light greatly enhances the degree of hardiness attained, cold acclimation is not obligatorily linked to a light requirement. Photoperiods, varying from 8 to 24 hours, received during the cold acclimation period were equally effective in promoting maximum hardiness. Relatively low light intensities and short photoperiods stimulated maximum hardiness, and proportional increases in hardiness in response to increased photoperiods were demonstrated only in stems of prestarved plants. Exclusion of CO₂ and high concentrations of photosynthetic inhibitors decreased hardiness, but in no instance was hardiness reduced to the level of the dark control. The data are only compatible with a photosynthetic role of light if it is assumed that only a small portion of the total photosynthates are required to elicit maximum hardiness. Alternatively, the light stimulation which was elicited by low light intensities, short photoperiods, in the absence of CO₂, and in the presence of photosynthetic inhibitors, may be a light signal similar to a phytochrome response.