Cold-acclimation induced changes in freezing tolerance and translatable RNA content in Citrus grandis and Poncirus trifoliata

Cold-acclimation-induced changes in freezing tolerance and translatable RNA content were compared in seedlings of a relatively cold sensitive citrus species, Citrus grandis L. Osb. cv. Thong Dee (pummelo), and the cold-hardy citrus relative, Poncirus trifoliata L. Raf. cv. Pomeroy (trifoliate orange). Cold acclimation of pummelo (10 days at 15°C followed by 4 weeks at 10°/5°C, day/night) resulted in a decrease in LT₅₀ from −6 to −8°C, while in trifoliate orange (acclimated for 7 weeks at 5°C), the LT₅₀ decreased from −9 to −18°C. Qualitative changes in the in vitro translation profile, revealed by two-dimensional gel electrophoresis, were observed following cold acclimation in both species. An mRNA for a large polypeptide (ca 160 kDa) was detected following cold acclimation of trifoliate orange. A similar change was not observed in pummelo following cold acclimation.     

Effects of acclimation and diapause on the thermal tolerance of the two-spotted spider mite Tetranychus urticae

The two-spotted spider mite, Tetranychus urticae, is a worldwide pest species that overwinters as diapausing females. Cold hardening is presumed to start during diapause development to ensure the successful overwintering of this species. To address this hypothesis, we compared cold tolerance between non-diapausing and diapausing females. We measured supercooling point (SCP) and survival to acute cold stress by exposing the mites at a range of sub-zero temperatures (from −4 to −28 °C for 2 h). The mean SCPs of non-diapausing and diapausing females were −19.6±0.5 and −24.7±0.3 °C respectively, and freezing killed the mites. Diapausing females were significantly more cold tolerant than non-diapausing ones, with LT₅₀ of −19.7 and −13.3 °C, respectively. Further, we also examined the effects of cold acclimation (10 d at 0 or 5 °C) in non-diapausing and diapausing females. Our findings indicated that diapause decreased SCP significantly, while cold acclimation had no effect on the SCP except for non-diapausing females that were acclimated at 5 °C. Acclimation at 5 °C enhanced survival to acute cold stress in diapausing and non-diapausing females, with LT₅₀ of −22.0 and −17.1 °C, respectively. Altogether, our results indicate that T. urticae is a chill tolerant species, and that diapause and cold acclimation elevate cold hardiness in this species.     

Physiological effects of temperature and growth regulators on foliar chlorophyll,soluble protein,and cold hardiness in citrus

Leaf chlorophyll (Chl, A, B) and total soluble protein were assayed in greenhouse-grown 1.5-year-old trees of 2 citrus types, trifoliate orange (Poncirus trifoliata (L.) Raf.) and sour orange (Citrus aurantium L.) exposed to 12 h (day/night) photoperiods in growth chambers under high (30°/21°C, day/night; noncold-hardening) and low (16°/5°C; cold-hardening) temperature regimes. Trees were sprayed 2 × per week for 5 weeks with one of the following solutions at 100 M: napthaleneacetic acid (NAA), paclobutrazol (2RS, 3RS)-1-(4-chlorophenyl-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol) (PPP333), benzyl-adenine (BA), abscisic acid (ABA), gibberellic acid (GA₃), minerals only (N, P, K, S, Ca, Mg) and BA (+) minerals. NAA, PP333, ABA and GA₃ decreased Chl A, B and soluble protein in both citrus types under cold-hardening conditions in contrast to increases with the use of BA and BA (+) minerals especially in trifoliate orange. Both BA and GA₃ increased Chl A, B and protein synthesis under high temperature in both citrus types. Under noncold-hardening temperatures, GA₃ enhanced Chl A, B but sharply reduced foliar protein concentration. Dieback of both cultivars following exposure to temperatures down to –6.7°C was decreased 7% by NAA sprays during noncold-hardening temperatures. Cold tolerance of noncoldhardened trifoliate orange trees was also improved with ABA and PP333. Foliar sprays of NAA (sour orange) and PP333 and BA (+) minerals (trifoliate) increased cold tolerance of cold-hardened trees by 8%. Results indicate that spray applications of growth regulators influence physiological factors associated with foliar functioning and cold tolerance in citrus during different temperature regimes.Summary Growth promoters (BA) and inhibitors (NAA) have the potential to promote cold hardines through either a strong stimulatory effect on foliar physiology or a marked inhibition of growth in general. This suggests that each growth regulator may possess an independent role in the cold-hardiness phenomenon and may also interact with physiological processes other than soluble protein and chlorophyll metabolism. The relationship between soluble protein levels in citrus foliage and the degree of cold hardiness remains uncertain and is essentially unresolved pending more specific qualitative research.University of Florida Agricultural Experiment Station Series No. 7446.This paper reports the results of research only. Mention of a trademark of a proprietary product does not constitute a recommendation for use by the U.S. Department of Agriculture to the exclusion of other products that may also be suitable.     

Thermal tolerance of European Sea Bass (Dicentrarchus labrax) juveniles acclimated to three temperature levels

This study was carried out to determine upper (CTMax) and lower (CTMin) thermal tolerance, acclimation response ratio (ARR) and thermal tolerance polygon of the European sea bass inhabiting the Iskenderun Bay, the most southeasterly part of the Mediterranean Sea, at three acclimation temperatures (15, 20, 25 °C). Acclimation temperature significantly affected the CTMin and CTMax values of the fish. At 0.3 °C min⁻¹ cooling or heating rate, CTMin ranged from 4.10 to 6.77 °C and CTMax ranged from 33.23 to 35.95 °C in three acclimation temperatures from 15 to 25 °C. Thermal tolerance polygon for the juveniles at the tested acclimation temperatures was calculated to be 296.14 °C². In general, the current data show that our sea bass population possesses acclimation response ratio (ARR) values (0.25-0.27) similar to some tropical species. The cold tolerance values attained for this species ranged from 4.10 to 6.77 °C, suggesting that cold winter temperatures may not pose danger during the culture of European sea bass in deep ponds or high water exchange rate systems. Upper thermal tolerance is more of a problem in the southern part of the Mediterranean as maximum water temperature in ponds may sometimes exceed 33-34 °C, during which underground cool-water should be used to lower ambient water temperature in the mid-summer. For successful culture of sea bass in ponds, temperature should be maintained around 25 °C throughout the year and this can be managed under greenhousing systems using underground well-waters, commonly available in the region.     

<Emphasis Type="Italic">Ptcorp</Emphasis> gene induced by cold stress was identified by proteomic analysis in leaves of <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.     

锌胁迫下小麦SSH文库的构建及初步分析

Zn是植物必需的微量元素,同时也是近年来造成环境污染的重金属元素之一.为了从基因表达水平揭示小麦Zn胁迫响应的分子机制,本研究利用抑制差减杂交(suppression subtractive hybridization,SSH)技术构建了Zn胁迫（0.5 mmol/L,1 mmol/L）下小麦的正反向SSH文库.从正反向文库中随机挑选阳性单克隆,并利用通用引物T7/Sp6对其进行验证. 结果显示,正反库中分别获得307和821个EST序列,其片段长度在200～1 000 bp之间,它们反映了Zn胁迫下特异响应的基因.利用BLASTn和BLASTx将这些EST序列进行比对分析,在正、反库中分别筛选出221和641个uniESTs,其中751个uniESTs被注释（包括正库中193个和反库中558个）.这些序列的功能主要涉及信号转导、抗氧化防御、转录与翻译、物质运输、核糖体结构、能量代谢,以及一些功能未知的基因.     

cDNA-AFLP analysis of salt-inducible genes expression in Chrysanthemum lavandulifolium under salt treatment

Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino is a halophyte species that belongs to the Asteraceae family, and the genus Chrysanthemum. It is one of the ancestors of C. × morifolium Ramatella. Understanding the tolerance mechanism associated with salt stress in C. lavandulifolium could provide important information for explaining the salt tolerance of higher plants and could also help enhancing breeding programs of cultivated Chrysanthemum. In this study, cDNA amplified fragment length polymorphism (cDNA-AFLP) was used to detect differential gene expression in leaves of C. lavandulifolium in response to NaCl treatment. The determination of membrane permeablility, peroxidase activity (POD), malon-dialdehyde (MDA), as well as proline and leaf chlorophyll contents under different NaCl concentrations showed that a 200 mM NaCl treatment was an optimal condition for the cDNA-AFLP experiment. Using this concentration during different times (0, 3 h, 12 h, 24 h and 48 h), we obtained 1930 cDNA fragments using 64 primers. After sequencing 234 randomly chosen cDNA clones and BLASTx analyzing, we got 129 expressed sequence tags (ESTs) which had no significant homology with other sequences, 85 ESTs were homologous to genes with known functions, whereas the rest of ESTs showed homology to unclassified or putative proteins. 25 ESTs that were similar to known functional genes involved in several abiotic and biotic stresses were confirmed by semi-quantitative RT-PCR and qRT-PCR. The expression patterns of these salt-responsive genes not only responded to salt stress but also to plant hormones, such as abscisic acid (ABA), and to other abiotic stresses such as drought and cold. These results indicate an extensive cross-talk among several stresses. Our results provide interesting information for further understanding the molecular mechanisms of salt tolerance in C. lavandulifolium.     

Thermal preference,tolerance and oxygen consumption of adult white shrimp Litopenaeus vannamei (Boone) exposed to different acclimation temperatures

Final temperature preferendum of white shrimp adults were determined with acute and gravitation methods. The final preferendum was similar, independent of method (26.2–25.6 °C). A direct relationship was determined between the critical thermal maxima values and the acclimation temperatures (P<0.05). The end point of Critical Thermal Maxima (CTMax) for adults was defined as the loss of righting response (LRR). The acclimation response ratio (ARR) for adults of white shrimp had an interval of 0.36–0.76, values that agreed with others obtained for crustaceans from tropical and subtropical climates. The oxygen consumption rates increased significantly (P<0.05) from 39.6 up to 90.0 mg O₂ kg⁻¹ h⁻¹ wet weight (w.w.) as the acclimation temperature increased from 20 to 32 °C. The range of temperature coefficient (Q₁₀) of the white shrimp between 23 and 26 °C was the lower 1.60. The results obtained in this work are discussed in relation to the species importance in the reproductive scope and maintenance of breeders.     

Thermal tolerance of Litopenaeus vannamei (Crustacea: Penaeidae) acclimated to four temperatures

Critical thermal minima (CTMin) and maxima (CTMax) values were determined for the Pacific white shrimp Litopenaeus vannamei post-larvae and juveniles at four different acclimation temperatures (15, 20, 25, and 30 °C). The CTMin of shrimp at these acclimation temperatures were 7.82, 8.95, 9.80, and 10.96 °C for post-larvae and 7.50, 8.20, 10.20, and 10.80 °C for juveniles, respectively, at 1 °C h⁻¹ cooling rate. The CTMax values were 35.65, 38.13, 39.91, and 42.00 °C for post-larvae and 35.94, 38.65, 40.30, and 42.20 °C for juveniles at the respective acclimation temperatures. Both acclimation temperature and size of the shrimp affected CTMin values of L. vannamei (P<0.01). Overall, juveniles displayed significantly lower CTMin values than the post-larvae (P<0.0001). However, the CTMax response by post-larvae and juveniles were not significantly different from each other and no interaction was determined between the acclimation temperature and development stage (P>0.01). The area of the thermal tolerance polygon over four acclimation temperatures (15, 20, 25, and 30 °C) for the post-larvae of L. vannamei was calculated to be 434.94 °C². The acclimation response ratio (ARR) values were high ranging from 0.35 to 0.44 for both post-larvae and juveniles. L. vannamei appears to be more sensitive to low temperatures than other penaeid species and its cold tolerance zone ranged from 7.5 to 11 °C. In successful aquaculture temperature must never fall below 12 °C to prevent mortalities. Upper thermal tolerance is less of a problem as in most subtropical regions maximum water temperature rarely exceeds 34 °C, but care should be given if shallow ponds with low water renewal rate are being used.     

A moderate change in temperature induces changes in fatty acid composition of storage and membrane lipids in a soil arthropod

A moderate change in ambient temperature can lead to vital physiological and biochemical adjustments in ectotherms, one of which is a change in fatty acid composition. When temperature decreases, the composition of membrane lipids (phospholipid fatty acids) is expected to become more unsaturated to be able to maintain homeoviscosity. Although different in function, storage lipids (triacylglycerol fatty acids) are expected to respond to temperature changes in a similar way. Age-specific differences, however, could influence this temperature response between different life stages. Here, we investigate if fatty acid composition of membrane and storage lipids responds similarly to temperature changes for two different life stages of Orchesella cincta. Juveniles and adults were cold acclimated (15 °C → 5 °C) for 28 days and then re-acclimated (5 °C → 15 °C) for another 28 days. We found adult membranes had a more unsaturated fatty acid composition than juveniles. Membrane lipids became more unsaturated during cold acclimation, and a reversed response occurred during warm acclimation. Membrane lipids, however, showed no warm acclimation, possibly due to the moderate temperature change. The ability to adjust storage lipid composition to moderate changes in ambient temperature may be an underestimated fitness component of temperature adaptation because fluidity of storage lipids permits accessibility of enzymes to energy reserves.     

Combined effects of temperature and salinity on critical thermal minima of pacific white shrimp Litopenaeus vannamei (Crustacea: Penaeidae)

Critical thermal minima (CTMin) were determined for the Pacific white shrimp Litopenaeus vannamei juveniles from four different acclimation temperatures (15, 20, 25, and 30 °C) and salinities (10‰, 20‰, 30‰, and 40‰). The lowest and highest CTMin of shrimp ranged between 7.2 °C at 15 °C/30‰ and 11.44 °C at 30 °C/20‰ at the cooling rate of 1 °C h⁻¹. Acclimation temperature and salinity, as well as the interaction of both parameters, had significant effects on the CTMin values of L. vannamei (P<0.01). Yet, the results showed a much more profound effect of temperature on low thermal tolerance of juveniles. Only 40‰ salinity had an influence on the CTMin values (P<0.01). As the acclimation temperature was lowered from 30 to 15 °C thermal tolerance of the shrimp significantly increased by 3.25–4.14 °C. The acclimation response ratio (ARR) of the Pacific white shrimp exposed to different combinations of salinity and temperature ranged between 0.25 and 0.27. When this species is farmed in sub-tropical regions, its pond water temperature in the over-wintering facilities (regardless of the water salinity level) must never fall below 12 °C throughout the cold season to prevent mortalities.     

Effects of acclimation temperature on thermal tolerance, locomotion performance and respiratory metabolism in Acheta domesticus L. (Orthoptera: Gryllidae)

The effects of acclimation temperature on insect thermal performance curves are generally poorly understood but significant for understanding responses to future climate variation and the evolution of these reaction norms. Here, in Acheta domesticus, we examine the physiological effects of 7-9 days acclimation to temperatures 4 °C above and below optimum growth temperature of 29 °C (i.e. 25, 29, 33 °C) for traits of resistance to thermal extremes, temperature-dependence of locomotion performance (jumping distance and running speed) and temperature-dependence of respiratory metabolism. We also examine the effects of acclimation on mitochondrial cytochrome c oxidase (CCO) enzyme activity. Chill coma recovery time (CRRT) was significantly reduced from 38 to 13 min with acclimation at 33-25 °C, respectively. Heat knockdown resistance was less responsive than CCRT to acclimation, with no significant effects of acclimation detected for heat knockdown times (25 °C: 18.25, 29 °C: 18.07, 33 °C: 25.5 min). Thermal optima for running speed were higher (39.4-40.6 °C) than those for jumping performance (25.6-30.9 °C). Acclimation temperature affected jumping distance but not running speed (general linear model, p = 0.0075) although maximum performance (U_MAX) and optimum temperature (T_OPT) of the performance curves showed small or insignificant effects of acclimation temperature. However, these effects were sensitive to the method of analysis since analyses of T_OPT, U_MAX and the temperature breadth (T_BR) derived from non-linear curve-fitting approaches produced high inter-individual variation within acclimation groups and reduced variation between acclimation groups. Standard metabolic rate (SMR) was positively related to body mass and test temperature. Acclimation temperature significantly influenced the slope of the SMR-temperature reaction norms, whereas no variation in the intercept was found. The CCO enzyme activity remained unaffected by thermal acclimation. Finally, high temperature acclimation resulted in significant increases in mortality (60-70% at 33 °C vs. 20-30% at 25 and 29 °C). These results suggest that although A. domesticus may be able to cope with low temperature extremes to some degree through phenotypic plasticity, population declines with warmer mean temperatures of only a few degrees are likely owing to the limited plasticity of their performance curves.     

Effects of acclimation and acute temperature change on specific dynamic action and gastric processing in the green shore crab, Carcinus maenas

The effects of temperature acclimation and acute temperature change were investigated in postprandial green shore crabs, Carcinus maenas. Oxygen uptake, gut contractions and transit rates and digestive efficiencies were measured for crabs acclimated to either 10 °C or 20 °C and subsequently exposed to treatment temperatures of 5, 15, or 25 °C. Temperature acclimation resulted in a partial metabolic compensation in unfed crabs, with higher oxygen uptake rates measured for the 10 °C acclimated group exposed to acute test temperatures. The Q₁₀ values were higher than normal, probably because the acute temperature change prevented crabs from fully adjusting to the new temperature. Both the acclimation and treatment temperature altered the characteristics of the specific dynamic action (SDA). The duration of the response was longer for 20 °C acclimated crabs and was inversely related to the treatment temperature. The scope (peak oxygen consumption) was also higher for 20 °C acclimated crabs with a trend towards an inverse relationship with treatment temperature. Since the overall SDA (energy expenditure) is a function of both duration and scope, it was also higher for 20 °C acclimated crabs, with the highest value measured at the treatment temperature of 15 °C. The decline in total SDA after acute exposure to 5 and 25 °C suggests that both cold stress and limitations to oxygen supply at the temperature extremes could be affecting the SDA response. The contractions of the pyloric sac of the foregut region function to propel digesta through the gut, and contraction rates increased with increasing treatment temperature. This translated into faster transit rates with increasing treatment temperatures. Although pyloric sac contractions were higher for 20 °C acclimated crabs, temperature acclimation had no effect on transit rates. This suggests that a threshold level in pyloric sac contraction rates needs to be reached before it manifests itself on transit rates. Although there was a correlation between faster transit times and the shorter duration of the SDA response with increasing treatment temperature, transit rates do not make a good proxy for calculating the SDA characteristics. The digestive efficiency showed a trend towards a decreasing efficiency with increasing treatment temperature; the slower transit rates at the lower treatment temperatures allowing for more efficient nutrient absorption. Even though metabolic rates of 10 °C acclimated crabs were higher, there was no effect of acclimation temperature on digestive efficiency. This probably occurred because intracellular enzymes and digestive enzymes are modulated through different control pathways. These results give an insight into the metabolic and digestive physiology of Carcinus maenas as it makes feeding excursions between the subtidal and intertidal zones.     

Response of juvenile diamond-backed terrapins (Malaclemys terrapin) to an aquatic thermal gradient

In many ectotherms, selection of environmental thermal niches may positively affect growth, nutrient assimilation rates, immune system function, and ultimately survival. Temperature preference in some turtle species may be influenced by environmental conditions, including acclimation temperature. We tested for effects of acclimation temperature (22 °C, 27 °C) on the selected temperature and movement patterns of 14 juvenile Malaclemys terrapin (Reptilia: Emydidae) in an aquatic thermal gradient of 14–34 °C and in single-temperature (22 °C, 27 °C) control tests. Among 8–10 month old terrapins, acclimation temperature influenced activity and movement patterns but did not affect temperature selection. In thermal gradient and single-temperature control tests, turtles acclimated to 27 °C used more tank chambers and relocated between chambers significantly more frequently than individuals acclimated to 22 °C. However, acclimation temperature did not affect temperature selection: both 22- and 27 °C-acclimated turtles selected the warmest temperature (34 °C), and avoided the other temperatures available, during thermal gradient tests. These results suggest that young M. terrapin are capable of detecting small temperature increments and prefer warm temperatures that may positively influence growth and metabolism.