Mass spectrometric approach for identifying putative plasma membrane proteins of Arabidopsis leaves associated with cold acclimation

Although enhancement of freezing tolerance in plants during cold acclimation is closely associated with an increase in the cryostability of plasma membrane, the molecular mechanism for the increased cryostability of plasma membrane is still to be elucidated. In Arabidopsis, enhanced freezing tolerance was detectable after cold acclimation at 2 degrees C for as short as 1 day, and maximum freezing tolerance was attained after 1 week. To identify the plasma membrane proteins that change in quantity in response to cold acclimation, a highly purified plasma membrane fraction was isolated from leaves before and during cold acclimation, and the proteins in the fraction were separated with gel electrophoresis. We found that there were substantial changes in the protein profiles after as short as 1 day of cold acclimation. Subsequently, using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS), we identified 38 proteins that changed in quantity during cold acclimation. The proteins that changed in quantity during the first day of cold acclimation include those that are associated with membrane repair by membrane fusion, protection of the membrane against osmotic stress, enhancement of CO2 fixation, and proteolysis.     

Shivering and non-shivering thermogenesis in a marsupial,the eastern barred bandicoot (Perameles gunnii)

We investigated the metabolic rate of the Tasmanian marsupial, the eastern barred bandicoot, Perameles gunnii, before and after acclimation to cold temperature (5 °C) for a 2-week period. Although body temperature did not change significantly, we observed a significant increase in the metabolic rate (MR) when measured at 5 °C before and after cold acclimation. Nor-epinephrine had a significant effect on the metabolic rate when measured in the thermoneutral zone and when measured at 5 °C after cold acclimation; however, there was no significant increase when measured at 5 °C before cold acclimation. Nor-epinephrine also resulted in a small but significant decrease in body temperature. Electromyography (EMG) measurements were obtained before and after cold acclimation during shivering. Shivering decreased after two weeks of cold exposure indicating that the bandicoot had acclimated to that temperature. Nor-epinephrine (NE) significantly reduced shivering before but not after cold acclimation. The metabolic rate and shivering decreased in the adult eastern barred bandicoot after acclimation at 5 °C and nor-epinephrine had similar effects to cold acclimation. Our findings of minor changes in thermal conductance suggest that insulation differences were unlikely explanations for our results. These experiments indicate that this marsupial is able to increase its heat production by non-shivering thermogenesis.     

异色瓢虫成虫冷驯化反应及体内几种酶活力的相关变化

为明确冷驯化反应对异色瓢虫Harmonia axyridis (Pallas) 实验种群成虫耐寒性及其生殖能力的影响, 本研究测定了成虫低温存活率、过冷却点(supercooling point, SCP)、体内含水量及雌虫繁殖能力等。结果表明: 冷驯化（在5℃下诱导3 d, 5 d）后, 成虫再在-5℃下暴露3 d的存活率由对照（预先未进行冷驯化）的46%分别提高至60%和67%, 而诱导10 d后的存活率(51%)反而下降。冷驯化效应在其成虫转移至饲养条件下7 d后就消失。随着低温诱导时间的延长过冷却点及体内含水量均呈现下降趋势, 短时间(5, 10 d)的诱导不能使成虫的SCP明显降低, 但可以使含水量极显著下降。冷驯化后异色瓢虫雌虫产卵前期延长; 虽然冷驯化对雌虫首次产卵量没有影响, 但是随着诱导时间的延长连续观察72 h内单头雌虫累计产卵量却降低。冷驯化过程中成虫体内几种酶活力的检测结果表明: 两种细胞保护酶超氧化物歧化酶(SOD)与过氧化氢酶(CAT)活性升高, 与新陈代谢有关的乳酸脱氢酶(LDH)及Na⁺, K⁺-ATP酶活性却降低。结果显示, 低温胁迫前异色瓢虫成虫经过不同时间的诱导后有可能提高其低温抵抗能力, 而且冷驯化诱导成虫耐寒性增加是一种复杂的生理生化过程, 这一过程对其生存和繁殖具有重要的适应意义。     

Cold hardiness in summer and winter diapause and post-diapause pupae of the cabbage armyworm, Mamestra brassicae L. under temperature acclimation

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.     

Cold acclimation in Pinus sylvestris: Phospholipids in purified plasma membranes from needles of pine

Effects of cold acclimation on needles of Scots pine ( Pinus sylvestris L., Provenance Södra Ydre) were studied at the membrane level. Before and after a period of cold acclimation the plasma membranes were isolated from the needles by a aqueous polymer two-phase partition technique. Fatty acid composition of total lipids or of individual phospholipids from the plasma membrane showed that the plasma membrane fraction was different from the other microsomal fractions analyzed, especially the 18:2 levels of the individual phospholipids. Furthermore, the cold acclimation period did not result in a decreased saturation level in the plasma membranes. Different steps in cold acclimation reactions at the membrane level are discussed.     

Adaptive thermogenesis in Brandt's vole (Lasiopodomys brandti) during cold and warm acclimation

Brandt's voles (Lasiopodomys brandti) exposed to cold (5±1 °C) or warm (23±1 °C) showed some physiological and biochemical variations which might be important in adaptation to their environments. Cold acclimation induced increases in resting metabolic rate (RMR) and the serum triiodothyronine (T₃) level, the state-4 respiration of liver and muscle mitochondria were activated after 7 days when animals exposed to cold, and the activity of cytochrome c oxidase (COX) of liver and muscle mitochondria tended to rise with cold exposure. RMR and T₃ level decreased during warm acclimation. The state-4 respiration of liver mitochondria declined after 3 days and muscle after 7 days when animals exposed to warm, and the activities of COX of liver and muscle mitochondria tended to decrease with warm acclimation. The cold activation of liver and muscle mitochondrial respiration (regulated by T₃) was one of the cytological mechanisms of elevating RMR. Both state-4 respiration and COX activity of brown adipose tissue (BAT) mitochondria increased significantly during cold acclimation and decreased markedly after acclimated to warm. The uncoupling protein 1 (UCP1) contents in BAT increased after exposure to cold and decreased after warm acclimation. Nonshivering thermogenesis (NST) plays an important role in the process of thermoregulation under cold acclimation for Brandt's voles. Changes in thermogenesis is a important way to cold adaptation for Brandt's voles in natural environments.     

Monitoring expression profiles of Arabidopsis genes during cold acclimation and deacclimation using DNA microarrays

A comparative analysis of gene expression profiles during cold acclimation and deacclimation is necessary to elucidate the molecular mechanisms of cold stress responses in higher plants. We analyzed gene expression profiles in the process of cold acclimation and deacclimation (recovery from cold stress) using two microarray systems, the 7K RAFL cDNA microarray and the Agilent 22K oligonucleotide array. By both microarray analyses, we identified 292 genes up-regulated and 320 genes down-regulated during deacclimation, and 445 cold up-regulated genes and 341 cold down-regulated genes during cold acclimation. Many genes up-regulated during deacclimation were found to be down-regulated during cold acclimation, and vice versa. The genes up-regulated during deacclimation were classified into (1) regulatory proteins involved in further regulation of signal transduction and gene expression and (2) functional proteins involved in the recovery process from cold-stress-induced damages and plant growth. We also applied expression profiling studies to identify the key genes involved in the biosynthesis of carbohydrates and amino acids that are known to play important roles in cold acclimation. We compared genes that are regulated during deacclimation with those regulated during rehydration after dehydration to discuss the similarity and difference of each recovery process.Electronic Supplementary Material Supplementary materials are available for this article at     

Human thermoregulatory responses to cold air are altered by repeated cold water immersion

The effects of repeated cold water immersion on thermoregulatory responses to cold air were studied in seven males. A cold air stress test (CAST) was performed before and after completion of an acclimation program consisting of daily 90-min cold (18 degrees C) water immersion, repeated 5 times/wk for 5 consecutive wk. The CAST consisted of resting 30 min in a comfortable [24 degrees C, 30% relative humidity (rh)] environment followed by 90 min in cold (5 degrees C, 30% rh) air. Pre- and postacclimation, metabolism (M) increased (P less than 0.01) by 85% during the first 10 min of CAST and thereafter rose slowly. After acclimation, M was lower (P less than 0.02) at 10 min of CAST compared with before, but by 30 min M was the same. Therefore, shivering onset may have been delayed following acclimation. After acclimation, rectal temperature (Tre) was lower (P less than 0.01) before and during CAST, and the drop in Tre during CAST was greater (P less than 0.01) than before. Mean weighted skin temperature (Tsk) was lower (P less than 0.01) following acclimation than before, and acclimation resulted in a larger (P less than 0.02) Tre-to-Tsk gradient. Plasma norepinephrine increased during both CAST (P less than 0.002), but the increase was larger (P less than 0.004) following acclimation. These findings suggest that repeated cold water immersion stimulates development of true cold acclimation in humans as opposed to habituation. The cold acclimation produced appears to be of the insulative type.     

The effect of repeated mild cold water immersions on the adaptation of the vasomotor responses

There are several types of cold adaptation based on the alteration of thermoregulatory response. It has been thought that the temperature of repeated cold exposures during the adaptation period is one of the factors affecting the type of cold adaptation developed. This study tested the hypothesis that repeated mild cold immersions would induce an insulative cold adaptation but would not alter the metabolic response. Seven healthy male participants were immersed to their xiphoid process level repeatedly in 26°C water for 60 min, 3 days every week, for 4 weeks. During the first and last exposure of this cold acclimation period, the participants underwent body immersion tests measuring their thermoregulatory responses to cold. Separately, they conducted finger immersion into 5°C water for 30 min to assess their cold-induced vasodilation (CIVD) response before and after cold acclimation. During the immersion to xiphoid process, participants showed significantly lower mean skin temperature and skin blood flow in the forearm post-acclimation, while no adaptation was observed in the metabolic response. Additionally, blunted CIVD responses were observed after cold acclimation. From these results, it was considered that the participants showed an insulative-type of cold acclimation after the repeated mild cold immersions. The major finding of this study was the acceptance of the hypothesis that repeated mild cold immersion was sufficient to induce insulative cold adaptation but did not alter the metabolic response. It is suggested that the adaptation in the thermoregulatory response is specific to the response which is repeatedly stimulated during the adaptation process.     

Electron Microscopic Cytochemical Study of Cyclic Adenosine Monophosphate Phosphodiesterase Activity During Gold Acclimation of Wheat Seedlings

Comparative studies on localization and distribution of cyclic adenosine monophosphate phosphodiesterase (cAMP-PDEase) activity in the young leaf cells of two different cold resistant wheat (Triticum aestivurn L.) varieties during cold acclimation were carried out by means of ultracytochemical method. The results indicated that the reaction products of cAMP- PDEase activity in two varieties of the seedlings grown at optimum temperature (20–25℃) were mainly localized at plasmalemma, nucleoli and chromatin. When the wheat seedlings were subjected to low temperature acclimation, cAM P-PDEase activity in the strong cold resistant variety Yanda 1817 seedlings was markedly decreased; however, little alteration of this enzyme activity was observed in the weak cold resistant variety Zhengzhou 39-1. cAMP-PDEase activity was recovered after deacclimation. The results suggested that changes in cAMP-PDEase activity during cold acclimation were closely related to the development of plant cold hardiness.     

大绒鼠冷驯化和脱冷驯化能量代谢特征的变化

通过测定冷驯化(5℃)到脱冷驯化(30℃)条件下,大绒鼠(Eothenomys miletus)的体重、摄入能、静止代谢率(RMR)、非颤抖性产热(NST)和血清瘦素含量等参数,探讨了血清瘦素浓度与能量收支的关系。结果表明,冷驯化可致大绒鼠体重下降,RMR、NST、摄入能升高,血清瘦素浓度降低;脱冷驯化后大绒鼠体重增加,RMR、NST、摄入能降低,血清瘦素浓度增加。血清瘦素含量与体重呈正相关,与RMR、NST、摄入能呈负相关。表明大绒鼠的体重、摄入能和产热能力具有较强的可塑性,且瘦素可能参与了大绒鼠适应冷驯化及恢复过程中的能量平衡和体重的调节。     

The Relationship of Carbohydrates to Cold Acclimation of Hedera helix L. cv. Thorndale

While both the total sugar content and cold hardiness greatly increased during artificial cold acclimation, no direct parallelism was demonstrated. In fact, plants hardened in the dark exhibited an increase in hardiness during the period when the total sugar content declined, furthermore, while there was evidence for an accumulation of sugars (especially sucrose) incorporation of ¹⁴C indicated that all of the isolated fractions were in a dynamic state. Dehardening, paralleled by a large increase in the starch content, was more rapid in the light than in the dark. Furthermore, in stems the sugar content increased after 3 days of dehardening but declined after 7 days. This increase may represent the release of sugars from a previously unextractable form such as a glycoprotein complex. Starvation experiments indicate that photosynthates produced during the cold acclimation period are preferentially used during cold acclimation rather than reserve carbohydrates. This was also indicated by the smaller amount of starch hydrolysis in plants hardened in the light. Thus, while there appears to be a role for carbohydrates in the cold acclimation process, the lack of parallelism between sugar content and hardiness may be interpreted as indicating 1) cold acclimation is not merely an accumulation of sugars or an osmotic effect per se, and 2) under normal conditions, the level of carbohydrates is not limiting the rate or degree of cold acclimation.     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Changes in carbohydrates and freezing tolerance during cold acclimation of red raspberry cultivars grown in vitro and in vivo

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.     

Adaptation to cold and proteomic responses of the psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031

There is considerable interest in the use of psychrotrophic bacteria for food biopreservation and in the understanding of cold adaptation mechanisms. The psychrotrophic biopreservative Lactococcus piscium strain CNCM I-4031 was studied for its growth behavior and proteomic responses after cold shock and during cold acclimation. Growth kinetics highlighted the absence of growth latency after cold shock, suggesting a very high promptness in cold adaptation, a behavior that has never been described before for lactic acid bacteria (LAB). A comparative proteomic analysis was applied with two-dimensional gel electrophoresis (2-DE), and upregulated proteins were identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Both cold shock and cold acclimation triggered the upregulation of proteins involved in general and oxidative stress responses and fatty acid and energetic metabolism. However, 2-DE profiles and upregulated proteins were different under both conditions, suggesting a sequence of steps in cold adaptation. In addition, the major 7-kDa Csp protein was identified in the L. piscium CNCM I-4031 genome but was not cold regulated. The implication of the identified cold shock proteins and cold acclimation proteins in efficient cold adaptation, the possible regulation of a histidyl phosphocarrier protein, and the roles of a constitutive major 7-kDa Csp are discussed.     

Alterations in Detergent-Resistant Plasma Membrane Microdomains in Arabidopsis thaliana During Cold Acclimation

Microdomains in the plasma membrane (PM) have been proposedto be involved in many important cellular events in plant cells.To understand the role of PM microdomains in plant cold acclimation,we isolated the microdomains as detergent-resistant plasma membranefractions (DRMs) from Arabidopsis seedlings and compared lipidand protein compositions before and after cold acclimation.The DRM was enriched in sterols and glucocerebrosides, and theproportion of free sterols in the DRM increased after cold acclimation.The protein-to-lipid ratio in the DRM was greater than thatin the total PM fraction. The protein amount recovered in DRMsdecreased gradually during cold acclimation. Cold acclimationfurther resulted in quantitative changes in DRM protein profiles.Subsequent mass spectrometry and Western blot analyses revealedthat P-type H⁺-ATPases, aquaporins and endocytosis-related proteinsincreased and, conversely, tubulins, actins and V-type H⁺-ATPasesubunits decreased in DRMs during cold acclimation. Functionalcategorization of cold-responsive proteins in DRMs suggeststhat plant PM microdomains function as platforms of membranetransport, membrane trafficking and cytoskeleton interaction.These comprehensive changes in microdomains may be associatedwith cold acclimation of Arabidopsis.     

温度逆境交叉适应对葡萄叶片膜脂过氧化和细胞钙分布的影响

 研究了高温锻炼对低温胁迫下和低温锻炼对高温胁迫下葡萄(Vitis vinifera)叶片中丙二醛（MDA）、谷胱甘肽(GSH)和抗坏血酸(AsA)含量变化以及细胞中Ca2+分布的影响。结果表明: 高（低）温胁迫使正常生长的叶片丙二醛含量升高, GSH和AsA含量下降,低（高）温锻炼预处理能减少MDA含量,提高GSH和AsA含量,抑制了由于温度胁迫引起MDA含量升高和GSH和AsA下降趋势。常温下葡萄叶肉细胞的Ca2+主要分布于液泡、细胞间隙中;高温胁迫和低温胁迫后,细胞质中聚集大量Ca2+沉淀颗粒,液泡中和细胞间隙Ca2+沉淀颗粒减少,叶绿体超微结构被破坏,Ca2+稳态平衡遭到破坏。高温锻炼后细胞质出现大量的Ca2+沉淀颗粒,主要来源于细胞间隙,低温锻炼后细胞质也出现大量的Ca2+沉淀颗粒,主要来源于液泡,两者的叶绿体超微结构都完整;高温锻炼的叶片经过低温胁迫和低温锻炼的叶片经过高温胁迫后,细胞间隙和液泡内Ca2+沉淀颗粒增加,细胞质中Ca2+沉淀颗粒很少,叶绿体较完整,Ca2+稳态平衡得以维持。推测高低温锻炼能够通过Ca2+启动抗逆基因表达和维持细胞中Ca2+稳态平衡来交叉适应低高温的胁迫。     

The effect of photoperiod on changes in plasma, glucose, cholesterol, and free fatty acids during cold acclimation in frogs

1. Many previous experiments on cold acclimation in frogs were carried out on animals maintained in total darkness. I tested the effect of photoperiod on changes in plasma FFA, glucose, and cholesterol during cold acclimation. 2. Hematocrit levels were lower whereas plasma FFA levels were higher in frogs kept in total darkness during cold acclimation. 3. Plasma glucose and plasma cholesterol levels were not affected by photoperiod during cold acclimation.     

Cold acclimation of Arabidopsis thaliana results in incomplete recovery of photosynthetic capacity, associated with an increased reduction of the chloroplast stroma

The effects of short-term cold stress and long-term cold acclimation on the light reactions of photosynthesis were examined in vivo to assess their contributions to photosynthetic acclimation to low temperature in Arabidopsis thaliana (L.) Heynh.. All photosynthetic measurements were made at the temperature of exposure: 23 degrees C for non-acclimated plants and 5 degrees C for cold-stressed and cold-acclimated plants. Three-day cold-stress treatments at 5 degrees C inhibited light-saturated rates of CO2 assimilation and O2 evolution by approximately 75%. The 3-day exposure to 5 degrees C also increased the proportion of reduced QA by 50%, decreased the yield of PSII electron transport by 65% and decreased PSI activity by 31%. In contrast, long-term cold acclimation resulted in a strong but incomplete recovery of light-saturated photosynthesis at 5 degrees C. The rates of light-saturated CO2 and O2 gas exchange and the in vivo yield of PSII activity under light-saturating conditions were only 35-40% lower, and the relative redox state of QA only 20% lower, at 5 degrees C after cold acclimation than in controls at 23 degrees C. PSI activity showed full recovery during long-term cold acclimation. Neither short-term cold stress nor long-term cold acclimation of Arabidopsis was associated with a limitation in ATP, and both treatments resulted in an increase in the ATP/NADPH ratio. This increase in ATP/NADPH was associated with an inhibition of PSI cyclic electron transport but there was no apparent change in the Mehler reaction activity in either cold-stressed or cold-acclimated leaves. Cold acclimation also resulted in an increase in the reduction state of the stroma, as indicated by an increased total activity and activation state of NADP-dependent malate dehydrogenase, and increased light-dependent activities of the major regulatory enzymes of the oxidative pentose-phosphate pathway. We suggest that the photosynthetic capacity during cold stress as well as cold acclimation is altered by limitations at the level of consumption of reducing power in carbon metabolism.