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1.
紫花苜蓿(Medicago sativa)是全球性的栽培牧草, 因其秋眠性强弱而导致的抗寒性差异为深刻解析植物耐寒适应的进化生态学机制提供了一个很好的研究模式。作为多年生豆科植物, 秋眠性是紫花苜蓿适应晚秋日照缩短、温度降低, 以及提高越冬存活率的一种生长特性。秋眠性的形成与几千年来紫花苜蓿在全球的传播扩展和栽培利用有关, 长期处于不同的气候生境, 导致秋眠性的适应进化, 这为人类利用提供了丰富的遗传资源。根据秋眠性的强弱, 学术界目前将之分为11个等级。一般而言, 秋眠性的强弱影响了紫花苜蓿的低温驯化与越冬耐寒适应等过程, 导致不同品种间的抗寒性存在差异。迄今, 关于秋眠性的光温调控, 以及秋眠性影响低温驯化的生理生态过程研究较多, 而对相关的细胞信号转导与基因表达调控途径, 尤其是对秋眠性如何影响越冬紫花苜蓿抗冻蛋白作用等耐寒适应的分子机制, 尚知之甚少。针对目前研究中存在的问题, 该文提出了未来需要重点关注的科学问题。  相似文献   

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Abstract Water stress at a nonacclimating temperature (18–20°C) increased the cold hardiness of Medicagosativa L. (alfalfa) plants. This increased cold hardiness was retained when the previously water-stressed plants were cold acclimated (2–9°C) in the absence of water stress. Water stress during cold acclimation also increased cold hardiness. Alfalfa was demonstrated to suffer injury, measured as decreased growth following freezing, at sub-lethal temperatures. During cold acclimation the turgor potential (ψ) of watered plants increased, whereas the solute potential and the water content per unit dry weight decreased. The large positive psgrdap of acclimated plants indicates that the decreased water content per unit dry weight is related to an increased proportion of tissue dry matter rather than to tissue dehydration.  相似文献   

4.
To survive winters, woody perennials of temperate zones must enter into endodormancy. Resumption of spring growth requires sufficient exposure to low temperature (chill units, CUs) in winter (chilling requirement), which also plays a role in the development of cold hardiness (cold acclimation). Physiological studies on dormancy breaking have focused on identifying markers, such as appearance or disappearance of proteins in response to varying degrees of chill unit accumulation. However, whether these changes are associated with dormancy transitions or cold acclimation is not clear. In the present study, greenhouse-grown blueberry (Vaccinium section Cyanococcus) plants were used to address this question. Three blueberry cultivars, Bluecrop, Tifblue, and Gulfcoast having chilling requirement of approximately 1 200, 900 and 600 CUs, respectively, were first exposed to 4°C for long enough to provide chill units equivalent to one-half of their respective chilling requirement. This treatment was expected to result in cold acclimation. A fraction of plants was then subjected to a 15/12°C (light/dark) regime for 2 weeks, a treatment expected to be “dormancy-neutral” but cause deacclimation. Before and after each treatment, cold hardiness and dormancy status of floral buds were determined; proteins were extracted from the buds collected on the same sampling date, and separated by one-dimensional SDS-PAGE. Dehydrin-like proteins were identified by immunoblotting, using anti-dehydrin antiserum. Results indicate that the chilling treatment resulted in cold acclimation as indicated by increased bud hardiness in all three cultivars. Data also indicate a distinct accumulation of three dehydrin-like proteins of 65, 60, and 14 kDa during cold acclimation. The cold hardiness and levels of dehydrin proteins decreased during the exposure to 15/12°C for 2 weeks. Results also confirmed that this treatment had no negative effect on chill unit accumulation. Densitometric scans of protein gels indicated a close association between the abundance of dehydrins and degree of cold hardiness in these cultivars. In addition, levels of the dehydrin proteins and cold hardiness remained about the same between 100% and >100% satisfaction of chilling requirement. These results suggest that changes in dehydrin expression are more closely associated with cold hardiness than with dormancy transitions.  相似文献   

5.
Fall dormancy (FD) in alfalfa (Medicago sativa L.) can be described using 11 FD ratings, is widely used as an important indicator of stress resistance, productive performance and spring growth. However, the contrasting growth strategies in internode length and internode number in alfalfa cultivars with different FD rating are poorly understood. Here, a growth chamber study was conducted to investigate the effect of FD on plant height, aboveground biomass, internode length, and internode number in alfalfa individuals in the early growth stages. In order to simulate the alfalfa growth environment in the early stage, 11 alfalfa cultivars with FD ratings from one to 11 were chosen and seeded at the greenhouse, and then were transplanted into an artificial growth chamber. The experimental design was a randomized complete block in a split-plot arrangement with three replicates. Plant height, above-ground biomass, internode length, and internode number were measured in early growth stage in all individuals. Our findings showed that plant height and the aboveground biomass of alfalfa did not significantly differ among 11 different FD rated cultivars. Also, internode length and internode number positively affected plant height and the aboveground biomass of alfalfa individuals and the average internode length significantly increased with increasing FD rating. However, internode number tended to sharply decline when the FD rating increased. Moreover, there were no correlations, slightly negative correlations, and strongly negative correlations between internode length and internode number in alfalfa individuals among the three scales, including within-FD ratings, within-FD categories and inter-FD ratings, respectively. Therefore, our results highlighted that contrasting growth strategies in stem elongation were adopted by alfalfa with different FD ratings in the early growth stage. Alfalfa cultivars with a high FD rating have longer internodes, whereas more dormant alfalfa cultivars have a larger number of internodes. There were tradeoffs between internode length and internode number in response to FD in alfalfa, which reflected certain scale-dependence.  相似文献   

6.
Cold hardiness and biochemical changes were investigated in winter and summer pupae of the cabbage armyworm Mamestra brassicae at the diapause and post-diapause stages under temperature acclimation. Diapause pupae were successively acclimated to 25, 20 and then 10 degrees C (warm-acclimated group). Pupae at the diapause and post-diapause stages were successively acclimated to 5, 0, -5 and then -10 degrees C (cold-acclimated groups). Supercooling point values in winter and summer pupae remained constant regardless of the diapause stages and acclimated temperatures. Warm-acclimated pupae at the diapause stage did not survive the subzero temperature exposure, whereas, cold-acclimated pupae achieved cold hardiness to various degrees. Winter pupae were more cold hardy than summer pupae, and pupae at the post-diapause stage were more cold hardy than those at the diapause stage. Trehalose contents in winter pupae rose under cold acclimation. Summer pupae accumulated far lower trehalose contents than winter pupae, with the maximal level occurring in winter pupae at the post-diapause stage. Glycogen content remained at a high level in diapause pupae after warm acclimation, whereas it decreased after cold acclimation. Alanine, the main free amino acid in haemolymph after cold acclimation, increased at lower temperatures in both diapause and post-diapause pupae, but the increase was greater in the diapause pupae. These results suggest that cold hardiness is more fully developed in winter pupae than in summer pupae, and cold acclimation provides higher cold hardiness in winter pupae at the post-diapause stage than at the diapause stage.  相似文献   

7.
Cold injury is frequently seen in the commercially important shrub Hydrangea macrophylla but not in Hydrangea paniculata. Cold acclimation and deacclimation and associated physiological adaptations were investigated from late September 2006 to early May 2007 in stems of field-grown H. macrophylla ssp. macrophylla (Thunb.) Ser. cv. Blaumeise and H. paniculata Sieb. cv. Kyushu. Acclimation and deacclimation appeared approximately synchronized in the two species, but they differed significantly in levels of mid-winter cold hardiness, rates of acclimation and deacclimation and physiological traits conferring tolerance to freezing conditions. Accumulation patterns of sucrose and raffinose in stems paralleled fluctuations in cold hardiness in both species, but H. macrophylla additionally accumulated glucose and fructose during winter, indicating species-specific differences in carbohydrate metabolism. Protein profiles differed between H. macrophylla and H. paniculata, but distinct seasonal patterns associated with winter acclimation were observed in both species. In H. paniculata concurrent increases in xylem sap abscisic acid (ABA) concentrations ([ABA](xylem)) and freezing tolerance suggests an involvement of ABA in cold acclimation. In contrast, ABA from the root system was seemingly not involved in cold acclimation in H. macrophylla, suggesting that species-specific differences in cold hardiness may be related to differences in [ABA](xylem). In both species a significant increase in stem freezing tolerance appeared long after growth ceased, suggesting that cold acclimation is more regulated by temperature than by photoperiod.  相似文献   

8.
The effects of exogenous abscisic acid (ABA), low temperature, and seedling age on the content of tubulin, actin, and phosphorylated proteins and the structural organization of microtubules (MTs) in cells of different tissues and organs of winter wheat cultivars contrasting in cold hardiness were studied by immunocytochemical methods using monoclonal (against - and -tubulin and actin) and polyclonal (phosphothreonine) antibodies. The leaves and roots of five- and nine- day-old seedlings of three cultivars were characterized by unequal proportion of actin/tubulin proteins. ABA decreased the content of the cytoskeleton and the 60-kD phosphorylated proteins, thus promoting a decrease in the number of MTs and occurrence of a less branched network of weakly fluorescent tubulin components in the cells of the root differentiating zone (which is most responsible for the development of cold hardiness in wheat). Although the cold acclimation of plants (3°C, 7 days) did not change the level of tubulin and actin proteins, it evoked the spatial aggregation of MT, leading to formation of a dense network of tubulin cytoskeleton comprised of thick bundles of intensively fluorescent MTs. In the case of a combined action of the studied factors, low temperatures abolished the hormone effect described above, evoking an increase in the content of the cytoskeletal and 60-kD phosphorylated proteins and MT structures. We suggest that the ABA-induced decrease in the levels of proteins and MTs occurs at the initial stages of plant cold acclimation (3°C, 2-3 days). It may be the signal that triggers the processes of low-temperature adaptation. As the duration of cold acclimation increased (3°C, 7 days), the role of ABA in the formation of plant tolerance decreased. Apparently, in this case other hormone-independent mechanisms of frost hardiness development are triggered, in which the role of the cytoskeleton components and cytoskeleton-associated proteins increases.  相似文献   

9.
? Autumnal cold acclimation in conifers is a complex process, the timing and extent of which vary widely along latitudinal gradients for many tree species and reflect local adaptation to climate. Although previous studies have detailed some aspects of the metabolic remodelling that accompanies cold acclimation in conifers, little is known about global metabolic dynamics, or how these changes vary among phenotypically divergent populations. ? Using untargeted GC-MS metabolite profiling, we monitored metabolic dynamics during autumnal cold acclimation in three populations of Sitka spruce from the southern, central, and northern portions of the species range, which differ in both the timing and extent of cold acclimation. ? Latitudinal variation was evident in the nature, intensity, and timing of metabolic events. Early development of strong freezing tolerance in the northern population was associated with a transient accumulation of amino acids. By late autumn, metabolic profiles were highly similar between the northern and central populations, whereas profiles for the southern population were relatively distinct. ? Our results provide insight into the metabolic architecture of latitudinal adaptive variation in autumn acclimation and show that different mechanisms are the basis of early October cold hardiness and autumn-acclimated cold hardiness.  相似文献   

10.
昆虫耐寒性研究   总被引:33,自引:4,他引:33  
景晓红  康乐 《生态学报》2002,22(12):2202-2207
昆虫是变温动物,气候变化是造成种群季节消长的基本原因之一。尤其在不良的低温环境中,昆虫耐寒力的高低是其种群存在与发展的种要前提,昆虫对低温的适应能力及其机理也因而成为昆虫生态学和生物进化研究中的一个深受重视的问题,本文论述了与耐寒性直接相关的过冷却点昆虫的抗寒对策,明确了昆虫耐寒性的一些基本概念,一方面从环境影响昆虫的角度对耐寒性的一般规律,如季节性变化,地理变异快速冷驯化的作用等做了简要的概念括,另一方面阐述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等做了简要的概括,另一方面简单述了昆虫适应环境的生理生化机制,包括低分子量的抗冻物质的产生,冰核剂的作用及抗冻蛋白的功能等。强调昆虫与环境相互作用过程中的生态生理适应,并指出昆虫耐寒性应当与生活史中别的因素联系起来,这样才能对耐寒性有一个更加全面的理解。  相似文献   

11.
昆虫是变温动物,种群的繁衍面临如何安全度过漫长而寒冷的冬季的挑战.为了安全越冬,昆虫必须适应冬季的低温,增强耐寒能力是昆虫的一种重要的季节适应机制.近年来,关于昆虫耐寒性的研究不断开展,研究内容涉及影响耐寒性的因素、耐寒性的机制等方面.影响昆虫耐寒性的因素包括环境因素,如气候的季节变化、纬度或海拔的差异等,以及昆虫自身的发育阶段、滞育发生等.  相似文献   

12.
We analyzed changes in populations of translatable mRNAs occurringin crowns of the cold-tolerant alfalfa (Medicago sativa L.)cv. Apica (CT) and the cold-sensitive cv. CUF-101 (CS) aftertheir acclimation at low nonfreezing temperatures and at subzerotemperatures. Both cultivars showed very similar translationprofiles under all treatments. Low temperatures induced significantchanges in the populations of translatable mRNAs. We observeda relationship between the accumulation of cold-regulated (COR)translation products and freezing tolerance within cultivars.Moreover, at least three COR translation products were specificto the CT and might be related to hardiness potential in alfalfa.Whereas extension of the cold acclimation period at 2C reducedcold tolerance, incubation at subzero temperatures increasedor maintained freezing tolerance. This increased hardiness wasassociated with enhanced translation of COR polypeptides andalso with the appearance of new translatable mRNAs. This is,to our knowledge, the first report of altered gene expressionin plants incubated at subzero temperatures. Marked changesin populations of translatable mRNAs at temperatures below freezingmight be related to previous reports that alfalfa achieves maximumhardiness under snow cover when the soil has frozen. Translationin the presence of [3H]glycine showed that a large proportionof the COR genes encode for glycine-rich proteins (GRPs) andthat some of the GRPs are specific to the CT. (Received May 29, 1992; Accepted October 13, 1992)  相似文献   

13.
Abstract.  1. Cold tolerance is a fundamental adaptation of insects to high latitudes. Flexibility in the cold hardening process, in turn, provides a useful indicator of the extent to which polar insects can respond to spatial and temporal variability in habitat temperature.
2. A scaling approach was adopted to investigate flexibility in the cold tolerance of the high Arctic collembolan, Hypogastrura tullbergi , over different time-scales. The cold hardiness of animals was compared from diurnal warming and cooling phases in the field, and controlled acclimation and cooling treatments in the laboratory. Plasticity in acclimation responses was examined using three parameters: low temperature survival, cold shock survival, and supercooling points (SCPs).
3. Over time-scales of 24–48 h, both field animals from warm diurnal phases and laboratory cultures from a 'warm' acclimation regime (18 °C) consistently showed greater or equivalent cold hardiness to animals from cool diurnal phases and acclimation regimes (3 °C).
4. No significant evidence was found of low temperature acclimation after either hours or days of low temperature exposure. The cold hardiness of H. tullbergi remained 'seasonal' in character and mortality throughout was indicative of the summer state of acclimatization.
5. These data suggest that H. tullbergi employs an 'all or nothing' cryoprotective strategy, cold hardening at seasonal but not diel-temporal scales.
6. It is hypothesised that rapid cold hardening offers little advantage to these high Arctic arthropods because sub-zero habitat temperatures during the summer on West Spitsbergen are rare and behavioural migration into soil profiles offers sufficient buffering against low summer temperatures.  相似文献   

14.
In vitro screening for cold hardiness of raspberry cultivars   总被引:1,自引:0,他引:1  
Raspberry (Rubus idaeus L.) cultivars ‘Festival’, ‘Titan’ and ‘Willamette’ were cultured in vitro on three different media: (A) MS medium supplemented with 1.0 mg l-1 BAP and 0.1 mg l-1 IBA, (B) MS medium without growth regulators, and (C) MS medium with reduced sucrose (10 g l-1), and exposed to different low temperature acclimation treatments: (1) control, no acclimation, (2) 1 week at +15 °C, 1 week at +2 °C, 24 h at -2 °C and 3 days at +2 °C, and (3) 2 weeks at +15 °C, 2 weeks at +2 °C, 24 h at −2 °C and 3 days at +2 °C. After acclimation, shoot moisture content was measured, and cold hardiness (LT50) was determined by controlled freezing. Shoot moisture content was generally lower on culture medium B compared to the other media, but not affected by acclimation treatment. In non-acclimated plants, medium composition had no effect on cold hardiness and no cultivar differences in hardiness were observed. After acclimation, plants on culture medium B were on average more cold hardy than on the other media. Acclimation treatment 3 on media A and B allowed the best discrimination between the hardy cultivar ‘Festival’ and less cold hardy ‘Titan’ and ‘Willamette’. When acclimation treatments were tested further using 11 raspberry cultivars with different levels of cold hardiness, discrimination between cultivars was satisfactory only after acclimation treatment 3 on culture medium B. This revised version was published online in June 2006 with corrections to the Cover Date.  相似文献   

15.
Abstract. Larvae of the hoverfiy Episyrphus balteatus (DeGeer) are important predators of aphids in the U.K. A large proportion of the U.K. population migrates south to warmer climes at the end of summer, but a small number are thought to overwinter in the U.K., with the mated female being the overwintering morph. The cold tolerance of adult flies was investigated to assess the overwintering potential of E. balteatus in the U.K. The high supercooling point (SCP) of -8.3 ± 0.7°C, and lethal temperature (LTemp30) of -9.1°C for acclimated females suggest that E. balteatus has limited cold hardiness. This was confirmed by experiments where, despite a strong acclimation response in both males and females, there was no long-term survival at 5, 0 or - 5°C. At 5°C, 90% of females had died after 10 days. The weak cold hardiness of adult E. balteatus was corroborated by field experiments which demonstrated a 100% mortality after 10 weeks' exposure to U.K. winter conditions. The ecological significance of this limited cold hardiness is discussed in relation to the overwintering abilities of E. balteatus in the U.K.  相似文献   

16.
The Role of Light in Cold Acclimation of Hedera helix L. var. Thorndale   总被引:3,自引:3,他引:0  
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.  相似文献   

17.
Changes in LT50 and carbohydrate levels in response to cold acclimation were monitored in vitro and in vivo in red raspberry ( Rubus idaeus L.) cultivars with different levels of cold hardiness. Entire micropropagated plantlets or shoot tips from 3 cultivars were harvested before, during and after cold acclimation. Cane samples from container-grown plants of 4 cultivars were harvested before and during cold acclimation and deacclimation. Samples were evaluated for cold hardiness (LT50) by controlled freezing, then analyzed for carbohydrates, including starch, sucrose, glucose, fructose and raffinose. Hardiness of cold-acclimated 'Muskoka' and 'Festival' was superior to that of 'Titan' or 'Willamette'. In vitro plantlets had higher levels of soluble carbohydrates on a dry weight basis and higher ratios of sucrose:(glucose+fructose) than the container-grown plants. Total soluble carbohydrates, primarily sucrose, accumulated during cold acclimation in both plantlets (33–56% relative increase) and plants (143–191% relative increase). Sucrose increased 124–165% in plantlets and 253–582% in container-grown plants during acclimation and declined rapidly to the level of control plants during deacclimation. Glucose and fructose also accumulated, but to a lesser extent than sucrose. Raffinose concentrations were very low, but increased significantly during cold acclimation. In vitro, genotype hardiness was related to the high concentrations of total soluble carbohydrates, sucrose and raffinose. In vivo, hardier genotypes had lower concentrations of starch than the less hardy genotypes. These results demonstrated the importance of soluble carbohydrates, especially sucrose, in cold hardening of red raspberry and that the in vitro conditions or controlled acclimation conditions do not necessarily reflect the phenomena observed in vivo.  相似文献   

18.
Juvenile trees of temperate and boreal regions cease growth and set buds in autumn in response to short day-lengths (SD) detected by phytochrome. Growth cessation and bud set are prerequisites for the development of winter dormancy and full cold hardiness. In this study we show that the SD-requirement for bud set and cold hardening can be overcome in hybrid aspen (Populus tremula L. × tremuloides Michx.) by low night temperature and inhibition of gibberellin (GA) biosynthesis. Bud set and increased cold hardiness were observed under normally non-inductive long day-length (LD) in wild-type plants, when exposed to low night temperature and paclobutrazol. In addition, the effect of PHYA overexpression could be overcome in transgenic plants, producing bud set and cold acclimation by treatment with: SD, low night temperature and paclobutrazol. After cold acclimation, the degree of bud dormancy was lower for wild-type plants prior treated with LD and transgenic plants (overexpressing PHYA), than SD-treated, wild-type plants. Thus, low night temperature in combination with reduced GA content induced bud set and promoted cold hardiness under normally non-inductive photoperiods in hybrid aspen, but was unable to affect development of dormancy. This might suggest separate signalling pathways from phytochrome regulating the induction of cold/cold hardiness and bud dormancy in hybrid aspen or alternatively, there might be one pathway that fails to complete its action in the transgenic and paclobutrazol treated plants.  相似文献   

19.
Fall dormancy results in decumbent, slow shoot growth of alfalfa (Medicago sativa L.) in autumn and reduced shoot regrowth rates after herbage removal in summer. Although fall dormancy is used to predict alfalfa adaptation, we possess a poor understanding of the biological mechanisms underlying fall dormancy. Our objective was to examine growth and carbohydrate metabolism of suspension cell cultures derived from contrasting alfalfa cultivars that genetically differed in fall dormancy. Suspension cells were grown in B5h media containing 2% sucrose. Cells derived from fall non-dormant plants accumulated sugars more rapidly after transfer to fresh media and to higher concentrations than did cells derived from fall dormant alfalfa cultivars. Dark respiration rates of cells derived from non-dormant plants were similar to those derived from fall dormant plants when growth was limited at low cell sugar concentrations. However, both cell growth and dark respiration rates increased in cells derived from non-dormant cultivars in response to greater cell sugar concentrations. High growth rates of cells derived from rapid growing, fall non-dormant alfalfa cultivars were associated with rapid sugar uptake and higher cell respiration rates when compared to cells derived from dormant alfalfa cultivars.  相似文献   

20.
We have studied the response of interphase and mitotic microtubule arrays in root meristem cells of spring and winter cultivars of wheat Triticum aestivum L. (Moskovskaya 35 and Moskovskaya 39) to cold stress (1 h at 0°C) and acclimation to cold (3–48 h at 0°C). We show that, in general, interphase microtubules are more resistant to cold then mitotic arrays in both cultivars. During cold stress, no changes are detected in the microtubule system of interphase cells of spring wheat, whereas the density of endoplasmic microtubules increases in interphase cells of winter wheat. During mitosis, the density of the kinetochore fibers of the spindle decreases in the cells of both cultivars, but it is prevailing in the cells of spring cultivar of wheat. During acclimation to cold, the disorganization of the cortical microtubule bundles and the enhanced growth of the endoplasmic microtubule network, which is comprised of microtubule converging centers, are observed in cells of both cultivars. However, the mitotic microtubule systems of winter and spring cultivars respond differently to cold acclimation. During prophase, a diffuse tubulin “halo,”followed by the assembly of microtubule converging centers, accumulate at the perinuclear area in the cells of winter wheat. In cells of spring cultivar, the prophase spindle is only detected during initial stages of cold acclimation. During metaphase, aberrant mitotic spindles, abnormal metaphase plates, and the excessive appearance of microtubule converging centers are observed in cells of both cultivars. Acclimation induces the disorganization of the phragmoplast and the formation of multiple microtubule converging centers during telophase in the cells of both cultivars. Microtubule converging centers are detected at the perinuclear area of daughter cells in winter wheat and in the cortical cytoplasm in spring wheat. The excessive formation of microtubule converging centers suggests the activation of microtubule assembly during prolonged exposure to low temperature. Our data also demonstrates common pathways of microtubule response to cold treatment (0°C).  相似文献   

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