Mechanisms Of Freezing Tolerance Research Articles

Hepatic transcriptome and gut microbiome provide insights into freeze tolerance in the high-altitude frog, Nanorana parkeri

Among amphibians, freeze tolerance is a low-temperature survival strategy that has been well studied in several species. One influence on animal health and survival under adverse conditions is the gut microbiome. Gut microbes can be greatly affected by temperature fluctuations but, to date, this has not been addressed in high-altitude species. Nanorana parkeri (Anura: Dicroglossidae) lives at high altitudes on the Tibetan plateau and shows a good freeze tolerance. In the present study, we addressed two goals: (1) analysis of the effects of whole body freezing on the liver transcriptome, and (2) assess modifications of the gut microbiome as a consequence of freezing. We found that up-regulated genes in liver were significantly enriched in lipid and fatty acid metabolism that could contribute to accumulating the cryoprotectant glycerol and raising levels of unsaturated fatty acids. The results suggest the crucial importance of membrane adaptations and fuel reserves for freezing survival of these frogs. Down-regulated genes were significantly enriched in the immune response and inflammatory response, suggesting that energy-consuming processes are inhibited to maintain metabolic depression during freezing. Moreover, freezing had a significant effect on intestinal microbiota. The abundance of bacteria in the family Lachnospiraceae was significantly increased after freezing exposure, which likely supports freezing survival of N. parkeri. The lower abundance of bacteria in the family Peptostreptococcaceae in frozen frogs may be associated with the hypometabolic state and decreased immune response. In summary, these findings provide insights into the regulatory mechanisms of freeze tolerance in a high-altitude amphibian at the level of gene expression and gut microbiome, and contribute to enhancing our understanding of the adaptations that support frog survival in high-altitude extreme environments.

Multi-Objective Real-Time Optimal Energy Management Strategy Considering Energy Efficiency and Flexible Torque Response for a Dual-Motor Four-Drive Powertrain

To exhaust the potential of energy efficiency and dynamic performance of the dual-motor four-drive powertrain, this study developed a multi-objective real-time optimal energy management strategy considering energy efficiency and flexible torque response. First, a theoretical analysis of energy loss and operating characteristics was performed to elucidate the energy-saving advantages and control challenges of the dual-motor four-drive powertrain. Second, an economic strategy based on the adaptive nonlinear particle swarm optimization (ANLPSO) and optimization freezing tolerance mechanism was devised to realize real-time optimal power distribution. Then, the pre-shifting recognition schedule and gradient torque recovery strategy were developed to achieve flexible torque response during gear shifting. Finally, smooth switching logic was created to assure a seamless transition between the two strategies. Numerous simulation results indicate that compared with the single-motor drive strategy, the proposed strategy can increase energy efficiency by 8.1%, 4.02%, and 9.49% under NEDC, WLTC, and CLTC, respectively. During shifting, the longitudinal acceleration and jerk of the proposed strategy are significantly superior to those of the original strategy, thereby enhancing the vehicle’s dynamic performance and ride comfort. The results of the drum experiment validate the efficacy of the proposed method for energy consumption optimization and torque coordination control in the actual vehicle environment.

Proteostasis in ice: the role of heat shock proteins and ubiquitin in the freeze tolerance of the intertidal mussel, Mytilus trossulus.

The bay mussel, Mytilus trossulus, is an animal that can survive extracellular ice formation. Depending on air and ocean temperatures, freeze tolerant intertidal organisms, like M. trossulus, may freeze and thaw many times during the winter. Freezing can cause protein denaturation, leading to an induction of the heat shock response with expression of chaperone proteins like the 70kDa heat shock protein (HSP70), and an increase in ubiquitin-conjugated proteins. There has been little work on the mechanisms of freeze tolerance in intertidal species, limiting our understanding of this survival strategy. Additionally, this limited research has focused solely on the effects of single freezing events, but the act of repeatedly crossing the freezing threshold may present novel physiological or biochemical stressors that have yet to be discovered. Mytilus are important ecosystem engineers and provide habitat for other intertidal species, thus understanding their physiology under thermal extremes is important for preserving shoreline health. We predicted that repeated freeze exposures would increase mortality, upregulate HSP70 expression, and increase ubiquitin conjugates in mussels, relative to single, prolonged freeze exposures. Mytilus trossulus from Vancouver, Canada were repeatedly frozen for a combination of 1 × 8h, 2 × 4h, or 4 × 2h. We then compared mortality, HSP70 expression, and the quantity of ubiquitin-conjugated proteins across experimental groups. We found a single 8-h freeze caused significantly more mortality than repeated freeze-thaw cycles. We also found that HSP70 and ubiquitinated protein was upregulated exclusively after freeze-thaw cycles, suggesting that freeze-thaw cycles offer a period of damage repair between freezes. This indicates that freeze-thaw cycles, which happen naturally in the intertidal, are crucial for M. trossulus survival in sub-zero temperatures.

Biochemical and Transcriptional Responses in Cold-Acclimated and Non-Acclimated Contrasting Camelina Biotypes under Freezing Stress.

Cold-acclimated and non-acclimated contrasting Camelina (Camelina sativa L.) biotypes were investigated for changes in stress-associated biomarkers, including antioxidant enzyme activity, lipid peroxidation, protein, and proline content. In addition, a well-known freezing tolerance pathway participant known as C-repeat/DRE-binding factors (CBFs), an inducer of CBF expression (ICE1), and a cold-regulated (COR6.6) genes of the ICE-CBF-COR pathway were studied at the transcriptional level on the doubled-haploid (DH) lines. Freezing stress had significant effects on all studied parameters. The cold-acclimated DH34 (a freezing-tolerant line) showed an overall better performance under freezing stress than non-acclimated plants. The non-cold-acclimated DH08 (a frost-sensitive line) showed the highest electrolyte leakage after freezing stress. The highest activity of antioxidant enzymes (glutathione peroxidase, superoxide dismutase, and catalase) was also detected in non-acclimated plants, whereas the cold-acclimated plants showed lower enzyme activities upon stress treatment. Cold acclimation had a significantly positive effect on the total protein and proline content of stressed plants. The qRT-PCR analysis revealed significant differences in the expression and cold-inducibility of CsCBF1-3, CsICE1, and CsCOR6.6 genes among the samples of different treatments. The highest expression of all CBF genes was recorded in the non-acclimated frost-tolerant biotype after freezing stress. Interestingly a significantly higher expression of COR6.6 was detected in cold-acclimated samples of both frost-sensitive and -tolerant biotypes after freezing stress. The presented results provide more insights into freezing tolerance mechanisms in the Camelina plant from both a biochemical point of view and the expression of the associated genes.

Involvement of glucose in freeze tolerance in the Japanese tree frog Hyla japonica.

Glycerol and aquaporin 9 (aquaglyceroporin) are known to be involved in freeze tolerance in the Japanese tree frog Hyla japonica. However, the regulatory mechanisms of freeze tolerance in this species have not been fully elucidated. In the present study, we focused on the inter- and intracellular dynamics of glucose to analyze the role of glucose and glucose-related proteins such as transporter and metabolic enzymes in freeze tolerance. Serum glucose concentrations were compared among the frogs that were nonhibernating, hibernating, and thawed after freezing at -4°C for 6hr. Serum concentrations of glucose in thawed frogs were significantly higher than those in hibernating and nonhibernating, active frogs. Periodic acid-Schiff staining showed that the accumulation of glycogen in the hepatocytes increased before hibernation and decreased after freezing and thawing. Quantitative RT-PCR analysis using the liver showed that, compared with active frogs, the type 2 glucose transporter gene (glut2) was upregulated in frozen frogs, the liver glycogen phosphorylase gene (pygl) was upregulated in frozen or thawed frogs, and the type 2 glycogen synthase gene (gys2) was upregulated in hibernating frogs. Immunohistochemistry of liver sections showed that, compared with nonhibernating frogs, Glut2 proteins were clearly increased most likely on the plasma membrane of hepatocytes in hibernating frogs and further increased by freezing, then decreased after thawing. These results suggest the possibility that glucose acts as a cryoprotectant in H. japonica.

Photoprotection contributes to freezing tolerance as revealed by RNA-seq profiling of rhododendron leaves during cold acclimation and deacclimation over time.

Cold acclimation (CA) and deacclimation (DA), which are often accompanied by changes in freezing tolerance (FT), carbohydrates and hormones, are crucial for winter survival, especially under global warming. Plants with weak CA and premature DA caused by warm winters and/or unseasonal warm spells can be easily injured by adverse reactions to cold. Thus, understanding the molecular mechanisms of FT is imperative. In this study, we used high-throughput RNA-seq to profile the CA and DA of leaves of overwintering Rhododendron "Miyo-no-Sakae" over time; these leaves do not undergo dormancy but do undergo photoprotection during CA, and they do not grow during DA. Using Mfuzz and weighted gene coexpression network analysis, we identified specific transcriptional characteristics in each phase of CA and DA and proposed networks involving coexpressed genes and physiological traits. In particular, we discovered that the circadian rhythm is critical for obtaining the strongest FT, and high expression of circadian rhythm-related genes might be linked to sugar accumulation during winter. Furthermore, evergreen leaves exhibited robust photoprotection during winter, as revealed by high values of nonphotochemical quenching, high expression of transcripts annotated as "early light-induced proteins", loss of granum stacks and destacking of thylakoids, all of which were alleviated during DA. The strong requirement of photoprotection could be the reason for decreased abscisic acid (ABA) and jasmonic acid (JA) contents during CA, and decreases in ABA and JA contents may contribute to decreases in lignin content. Our data suggest that the molecular mechanisms of FT in overwintering leaves are unique, which may be due to the high requirements for photoprotection during winter.

Antioxidant and non-specific immune defenses in partially freeze-tolerant Xizang plateau frogs, Nanorana parkeri.

The Xizang plateau frog Nanorana parkeri can tolerate brief and partial freezing of their body. To determine the significant role of antioxidant defense and non-specific immune defense in freezing survival of this species, we assayed parameters of oxidative damage, antioxidant defense and non-specific immune enzymes during freezing exposure (-2°C for 12h) in five organs (heart, brain, liver, kidney, and skeletal muscle). The results showed that freezing led to a significant rise in the content of malondialdehyde (MDA) and carbonyl groups (CG) in brain, liver and kidney tissues. The activities of superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) increased significantly in brain and liver tissues with an augmentation of total antioxidant capacity (T-AOC). Apparent increments in muscle SOD activity and liver GST activity were also observed during freezing exposure. Vitamin C content significantly decreased in liver and kidney but a significant increase occurred in brain. Activities of non-specific immune enzymes, acid phosphatase (ACP) and alkaline phosphatase (AKP), were also assessed. ACP activity was significantly reduced in all five tissues tested whereas AKP activity decreased significantly in four tissues but rose in brain. In summary, freezing is accompanied by oxidative stress in the high-altitude frog, N. parkeri, as documented by increases in the content of MDA and CG in tissues. Freezing exposure also induced tissue-specific changes in the antioxidant defenses showing that activation of antioxidant systems is a part of the survival strategy of this in a high-altitude frog during freezing. Such up-regulation of antioxidant enzymes suggests a particularly important role for them in the liver and brain, serving as an anticipatory mechanism to deal with the ROS challenge during freeze/thaw episodes. Our findings contribute to extending the current understanding of the mechanisms of freeze tolerance in high-altitude frogs.

Mapping freezing tolerance QTL in alfalfa: based on indoor phenotyping

BackgroundWinter freezing temperature impacts alfalfa (Medicago sativa L.) persistence and seasonal yield and can lead to the death of the plant. Understanding the genetic mechanisms of alfalfa freezing tolerance (FT) using high-throughput phenotyping and genotyping is crucial to select suitable germplasm and develop winter-hardy cultivars. Several clones of an alfalfa F1 mapping population (3010 x CW 1010) were tested for FT using a cold chamber. The population was genotyped with SNP markers identified using genotyping-by-sequencing (GBS) and the quantitative trait loci (QTL) associated with FT were mapped on the parent-specific linkage maps. The ultimate goal is to develop non-dormant and winter-hardy alfalfa cultivars that can produce extended growth in the areas where winters are often mild.ResultsAlfalfa FT screening method optimized in this experiment comprises three major steps: clone preparation, acclimation, and freezing test. Twenty clones of each genotype were tested, where 10 samples were treated with freezing temperature, and 10 were used as controls. A moderate positive correlation (r ~ 0.36, P < 0.01) was observed between indoor FT and field-based winter hardiness (WH), suggesting that the indoor FT test is a useful indirect selection method for winter hardiness of alfalfa germplasm. We detected a total of 20 QTL associated with four traits; nine for visual rating-based FT, five for percentage survival (PS), four for treated to control regrowth ratio (RR), and two for treated to control biomass ratio (BR). Some QTL positions overlapped with WH QTL reported previously, suggesting a genetic relationship between FT and WH. Some favorable QTL from the winter-hardy parent (3010) were from the potential genic region for a cold tolerance gene CBF. The BLAST alignment of a CBF sequence of M. truncatula, a close relative of alfalfa, against the alfalfa reference showed that the gene’s ortholog resides around 75 Mb on chromosome 6.ConclusionsThe indoor freezing tolerance selection method reported is useful for alfalfa breeders to accelerate breeding cycles through indirect selection. The QTL and associated markers add to the genomic resources for the research community and can be used in marker-assisted selection (MAS) for alfalfa cold tolerance improvement.

Frozen in the dark: interplay of night-time activity of xanthophyll cycle, xylem attributes, and desiccation tolerance in fern resistance to winter.

While most ferns avoid freezing as they have a tropical distribution or shed their fronds, wintergreen species in temperate and boreoalpine ecosystems have to deal with sub-zero temperatures. Increasing evidence has revealed overlapping mechanisms of desiccation and freezing tolerance in angiosperms, but the physiological mechanisms behind freezing tolerance in ferns are far from clear. We evaluated photochemical and hydraulic parameters in five wintergreen fern species differing in their ability to tolerate desiccation. We assessed frond freezing tolerance, ice nucleation temperature and propagation pattern, and xylem anatomical traits. Dynamics of photochemical performance and xanthophyll cycle were evaluated during freeze-thaw events under controlled conditions and, in selected species, in the field. Only desiccation-tolerant species, which possessed a greater fraction of narrow tracheids (<18 μm) than sensitive species, tolerated freezing. Frond freezing occurred in the field at -3.4 ± 0.9 °C (SD) irrespective of freezing tolerance, freezable water content, or tracheid properties. Even in complete darkness, maximal photochemical efficiency of photosystem II was down-regulated concomitantly with zeaxanthin accumulation in response to freezing. This was reversible upon re-warming only in tolerant species. Our results suggest that adaptation for freezing tolerance is associated with desiccation tolerance through complementary xylem properties (which may prevent risk of irreversible cavitation) and effective photoprotection mechanisms. The latter includes de-epoxidation of xanthophylls in darkness, a process evidenced for the first time directly in the field.

Genetic analysis of freezing tolerance in camelina [Camelina sativa (L.) Crantz] by diallel cross of winter and spring biotypes.

Camelina biotypes had different responses to freezing stress, which was mainly inherited by additive gene effects and can be reliably used in breeding programs and for a better understanding of freezing tolerance mechanisms in camelina plants. Camelina [Camelina sativa (L.) Crantz] is a frost-tolerant oilseed plant that is cultivated as an autumn crop in semi-arid regions. However, camelina establishment in these areas is limited by low temperatures in winter that results in decreased seed yield. In the present study, genetic basis of freezing tolerance (FT) in spring and winter biotypes of camelina was analyzed at seedling stage using a diallel cross experiment. The parents consisted of two winter doubled haploid (DH) lines with high (DH34 and DH31), two spring lines with medium (DH19 and DH26), and two spring lines with low FT (DH08 and DH91). For this purpose, the parents along with F1 entries were subjected to freezing stress and survival percentage, electrolyte leakage, and lethal temperature for 50% mortality (LT50) of the lines were measured. Results showed that although both additive and non-additive effects of the genes determine the FT, further analyses indicated that it was mainly controlled by the additive effects. Therefore, selection-based methods may be more efficient for improving FT in camelina genotypes. The results of specific combining ability (SCA) and heterosis analysis among various DH lines suggested that more tolerant cultivars of camelina could be developed by targeted crossings. When a tolerant winter line and a susceptible spring line were crossed, their progenies showed a higher FT compared with the progenies of a cross between two susceptible spring lines indicating FT is controlled by additive effects of the genes in camelina plants. These findings provided new insight into the genetic basis of freezing-related traits in camelina and could be used for more sophisticated breeding programs.

Evaluation of freezing tolerance in Actinidia germplasm based on relative electrolyte leakage

Cold stress in winter can have a disastrous effect on kiwifruit yield and affect geographical distribution. However, freezing tolerance in Actinidia genotypes remains largely unknown. Here, we report changes in metabolite content and enzyme activity in the shoots of Actinidia genotypes exposed to low-temperature stress (− 5 °C, − 10 °C, − 15 °C, − 20 °C, − 25 °C and − 30 °C). Moreover, the relative electrolyte leakage method was used to evaluate the freezing tolerance of kiwifruit germplasm; 51 genotypes from 16 species of Actinidia were evaluated in total. The data revealed that relative electrolyte leakage, proline (Pro), soluble protein, and catalase (CAT) activity changed with different low temperatures. Results showed that among 16 species, A. kolomikta, A. polygama, and A. arguta had lower LT50 than other species. A. arguta, originating from the northeast of China, exhibited stronger freezing resistance than the ones from other places. There was little difference in freezing tolerance between A. chinensis and A. deliciosa. These findings provide new insights into the freezing tolerance ability and mechanisms of kiwifruit and further contribute to our understanding of the relationship between freezing tolerance and geographic distribution.

Isoform Sequencing Provides Insight Into Freezing Response of Common Wheat (Triticum aestivum L.).

The objective of the study is to reveal the freezing tolerance mechanisms of wheat by combining the emerging single-molecule real-time (SMRT) sequencing technology PacBio Sequel and Illumina sequencing. Commercial semiwinter wheat Zhoumai 18 was exposed to −6°C for 4 h at the four-leave stage. Leaves of the control group and freezing-treated group were used to perform cDNA library construction. PacBio SMRT sequencing yielded 51,570 high-quality isoforms from leaves of control sample of Zhoumai 18, encoded by 20,366 gene loci. In total, 73,695 transcript isoforms, corresponding to 23,039 genes, were identified from the freezing-treated leaves. Compared with transcripts from the International Wheat Genome Sequencing Consortium RefSeq v1.1, 57,667 novel isoforms were discovered, which were annotated 21,672 known gene loci, as well as 3,399 novel gene loci. Transcriptome characterization including alterative spliced events, alternative polydenylation sites, transcription factors, and fusion transcripts were also analyzed. Freezing-responsive genes and signals were uncovered and proved that the ICE-ERF-COR pathway and ABA signal transduction play a vital role in the freezing response of wheat. In this study, PacBio sequencing and Illumina sequencing were applied to investigate the freezing tolerance in common wheat, and the transcriptome results provide insights into the molecular regulation mechanisms under freezing treatment.

Drivers of plasticity in freeze tolerance in the intertidal mussel Mytilus trossulus.

Freezing is an extreme stress to living cells, and so freeze-tolerant animals often accumulate protective molecules (termed cryoprotectants) to prevent the cellular damage caused by freezing. The bay mussel, Mytilus trossulus, is an ecologically important intertidal invertebrate that can survive freezing. Although much is known about the biochemical correlates of freeze tolerance in insects and vertebrates, the cryoprotectants that are used by intertidal invertebrates are not well characterized. Previous work has proposed two possible groups of low-molecular weight cryoprotectants in intertidal invertebrates: osmolytes and anaerobic byproducts. In our study, we examined which group of candidate cryoprotectants correlate with plasticity in freeze tolerance in mussels using 1H NMR metabolomics. We found that the freeze tolerance of M. trossulus varies on a seasonal basis, along an intertidal shore-level gradient, and with changing salinity. Acclimation to increased salinity (30ppt compared with 15ppt) increased freeze tolerance, and mussels were significantly more freeze tolerant during the winter. Mussel freeze tolerance also increased with increasing shore level. There was limited evidence that anaerobic byproduct accumulation was associated with increased freeze tolerance. However, osmolyte accumulation was correlated with increased freeze tolerance after high salinity acclimation and in the winter. The concentration of most low molecular weight metabolites did not vary with shore level, indicating that another mechanism is likely responsible for this pattern of variation in freeze tolerance. By identifying osmolytes as a group of molecules that assist in freezing tolerance, we have expanded the known biochemical repertoire of the mechanisms of freeze tolerance.

The lizard abides: cold hardiness and winter refuges ofLiolaemus pictus argentinusin Patagonia, Argentina

In environments where the temperature periodically drops below zero, it is remarkable that some lizards can survive. Behaviorally, lizards can find microsites for overwintering where temperatures do not drop as much as the air temperature. Physiologically, they can alter their biochemical balance to tolerate freezing or avoid it by supercooling. We evaluated the cold hardiness of a population of Liolaemus pictus argentinus Müller and Hellmich, 1939 in the mountains of Esquel (Patagonia, Argentina) during autumn. Additionally, we assessed the thermal quality (in degree-days) of potential refuges in a mid-elevation forest (1100 m above sea level (asl)) and in the high Andean steppe (1400 m asl). We analyzed the role of urea, glucose, total proteins, and albumin as possible cryoprotectants, comparing a group of lizards gradually exposed to temperatures lower than 0 °C with a control group maintained at room temperature. However, we found no evidence to support the presence of freeze tolerance or supercooling mechanisms in this species as related to the analyzed metabolites. Instead, the low frequency of degree-days below 0 °C and temperatures never lower than −3 °C in potential refuges suggest that L. p. argentinus might avoid physiological investments (such as supercooling and freeze tolerance) by behaviorally selecting appropriate refuges to overcome cold environmental temperatures.

O/W Pickering Emulsion Templated Organo-hydrogels with Enhanced Mechanical Strength and Energy Storage Capacity.

The increased quantities of fat in plants could allow the cells to inhibit the growth of ice and thus prevent the damages of their tissue structure in winter. In view of the structural buildup of freezing tolerance mechanism, here we presenteda facile way of employing O/W Pickering emulsion as a template to produce the freestanding organo-hydrogels with increased mechanical stability and energy storage capacity. The oil droplets stabilized by cellulose nanofibrils were dispersed in the alginate polymer network that cross-linked with Ca2+, which resulted in homogeneous and closely packed microstructures. The prepared organo-hydrogels could maintain original gel structure under frozen conditions and had extraordinary mechanical performance. It could endure compressive stress up to 35 KPa (at 50% strain) and the elastic modulus was around 72 KPa, while the solid content of polysaccharides was only about 0.75%. By using our comprehensive strategy, organo-hydrogels with higher volumes of oil phase exhibited an enhanced cold storage capacity. For alginate hydrogel, it took 8 min when the temperature rose from 0 to 5 °C, while for the organo-hydrogel with oil volume of 30%, it took about 24 min. After 34 min, the inner temperature of alginate hydrogel was close to 25 °C, and about 70 min were needed for the temperature oforgano-hydrogel to reach 25 °C. This kind of gel materials with complementary heteronetworks not only will have potential applications in cold chain logistics, but also can be applied in other fields with unusual functions.

How crickets become freeze tolerant: The transcriptomic underpinnings of acclimation in Gryllus veletis

Some ectotherms can survive internal ice formation. In temperate regions, freeze tolerance is often induced by decreasing temperature and/or photoperiod during autumn. However, we have limited understanding of how seasonal changes in physiology contribute to freeze tolerance, and how these changes are regulated. During a six week autumn-like acclimation, late-instar juveniles of the spring field cricket Gryllus veletis (Orthoptera: Gryllidae) become freeze tolerant, which is correlated with accumulation of low molecular weight cryoprotectants, elevation of the temperature at which freezing begins, and metabolic rate suppression. We used RNA-Seq to assemble a de novo transcriptome of this emerging laboratory model for freeze tolerance research. We then focused on gene expression during acclimation in fat body tissue due to its role in cryoprotectant production and regulation of energetics. Acclimated G. veletis differentially expressed >3000 transcripts in fat body. This differential expression may contribute to metabolic suppression in acclimated G. veletis, but we did not detect changes in expression that would support cryoprotectant accumulation or enhanced control of ice formation, suggesting that these latter processes are regulated post-transcriptionally. Acclimated G. veletis differentially regulated transcripts that likely coordinate additional freeze tolerance mechanisms, including upregulation of enzymes that may promote membrane and cytoskeletal remodelling, cryoprotectant transporters, cytoprotective proteins, and antioxidants. Thus, while accumulation of cryoprotectants and controlling ice formation are commonly associated with insect freeze tolerance, our results support the hypothesis that many other systems contribute to surviving internal ice formation. Together, this information suggests new avenues for understanding the mechanisms underlying insect freeze tolerance.

First evidence of freezing tolerance in a resurrection plant: insights into molecular mobility and zeaxanthin synthesis in the dark.

The photoprotective mechanisms of desiccation tolerance and freezing tolerance and their relation to molecular mobility (cell vitrification) were assessed in a single model: the exceptional subalpine and resurrection plant Ramonda myconi. Dehydrated leaves showed a drop in maximal photochemical efficiency of PSII (Fv/Fm) accompanied by synthesis of zeaxanthin (Z), even in the dark, which was limited by cell vitrification after complete desiccation. The recovery of Fv/Fm after a severe drying treatment (7 days at 50% relative humidity) confirmed the tolerance of R. myconi leaves to desiccation. In winter, R. myconi plants showed a highly dynamic component of photoinhibition. Interestingly, the potential activity of the enzyme violaxanthin de‐epoxidase (VDE) occurred at −7°C, below the freezing temperature range of the leaves (−2 ± 2°C) and even in the dark. This suggests that, in nature, the enzyme can still be active in frozen leaves, as long as they are above the glass transition temperature. The drop in Fv/Fm and increase in Z were reversible upon rehydration and thawing, respectively, but were not perfectly matched, suggesting that both Z‐independent and Z‐dependent forms of sustained dissipation are occurring. Overall, our data reinforce the light‐independent activity of the VDE enzyme under stress and suggest that Z‐accumulation could occur in darkness in a scenario when temperatures drop dramatically in the night under natural conditions.

Population-specific transcriptional differences associated with freeze tolerance in a terrestrial worm.

Enchytraeus albidus is a terrestrial earthworm widespread along the coasts of northern Europe and the Arctic. This species tolerates freezing of body fluids and survives winters in a frozen state. Their acclimatory physiological mechanisms behind freeze tolerance involve increased fluidity of membrane lipids during cold exposure and accumulation of cryoprotectants (glucose) during the freezing process. Gene regulatory processes of these physiological responses have not been studied, partly because no gene expression tools were developed. The main aim of this study was to understand whether the freeze tolerance mechanisms have a transcriptomic basis in E. albidus. For that purpose, first the transcriptome of E. albidus was assembled with RNAseq data. Second, two strains from contrasting thermal environments (Germany and Greenland) were compared by mapping barcoded RNAseq data onto the assembled transcriptome. Both of these strains are freeze tolerant, but Greenland is extremely freeze tolerant. Results showed more plastic responses in the Greenland strain as well as higher constitutive expression of particular stress response genes. These altered transcriptional networks are associated with an adapted homeostasis coping with prolonged freezing conditions in Greenland animals. Previously identified physiological alterations in freeze‐tolerant strains of E. albidus are underpinned at the transcriptome level. These processes involve anion transport in the hemolymph, fatty acid metabolism, metabolism, and transport of cryoprotective sugars as well as protection against oxidative stress. Pathway analysis supported most of these processes, and identified additional differentially expressed pathways such as peroxisome and Toll‐like receptor signaling. We propose that the freeze‐tolerant phenotype is the consequence of genetic adaptation to cold stress and may have driven evolutionary divergence of the two strains.

A novel cold-regulated gene from Phlox subulata, PsCor413im1, enhances low temperature tolerance in Arabidopsis

Low temperature stress adversely affects plant growth, development, and crop productivity. Analysis of the function of genes in the response of plants to low temperature stress is essential for understanding the mechanism of chilling and freezing tolerance. In this study, PsCor413im1, a novel cold-regulated gene isolated from Phlox subulata, was transferred to Arabidopsis to investigate its function under low temperature stress. Real-time quantitative PCR analysis revealed that PsCor413im1 expression was induced by cold and abscisic acid. Subcellular localization revealed that PsCor413im1-GFP fusion protein was localized to the periphery of the chloroplast, consistent with the localization of chloroplast inner membrane protein AtCor413im1, indicating that PsCor413im1 is a chloroplast membrane protein. Furthermore, the N-terminal of PsCor413im1 was determined to be necessary for its localization. Compared to the wild-type plants, transgenic plants showed higher germination and survival rates under cold and freezing stress. Moreover, the expression of AtCor15 in transgenic plants was higher than that in the wild-type plants under cold stress. Taken together, our results suggest that the overexpression of PsCor413im1 enhances low temperature tolerance in Arabidopsis.

Transcriptome Changes in Response to Cold Acclimation in Perennial Ryegrass as Revealed by a Cross‐Species Microarray Analysis

Freezing tolerance in perennial ryegrass (Lolium perenne L.) can be dramatically increased by a period of cold acclimation. To understand the mechanisms of cold acclimation and freezing tolerance in L. perenne, a cross‐species microarray study was conducted by using total RNA from cold‐acclimated and nonacclimated L. perenne to hybridize with Affymetrix Barley1 GeneChips from barley (Hordeum vulgare L.). Our results indicate that selection of proper background correction and normalization method is critical for the success of a barley–perennial ryegrass cross‐species microarray study. By using a specific three‐step normalization method, a total of 2870 (12.6%) genes out of approximately 22,000 genes on the barley gene chip were identified as differentially regulated by either 1 or 7 d of cold acclimation. Quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) analysis confirmed that the expression patterns of 14 selected genes are consistent with the signal intensity patterns observed in the microarray study, supporting the validity of using a cross‐species microarray in studying gene expression in species for which genomic resources remain scarce. Gene ontology analysis of differentially regulated genes indicates that different biological processes, cellular components, and molecular functions responded differently to cold acclimation in observable temporal patterns, suggesting that perennial ryegrass gradually acquires freezing tolerance by systematically regulating gene expression in an orderly manner.