Cold Acclimation Research Articles

Functional divergence in orthologous transcription factors: insights from AtCBF2/3/1 and OsDREB1C.

Despite traditional beliefs of orthologous genes maintaining similar functions across species, growing evidence points to their potential for functional divergence. C-repeat binding factors/dehydration-responsive element binding protein 1s (CBFs/DREB1s) are critical in cold acclimation, with their overexpression enhancing stress tolerance but often constraining plant growth. In contrast, a recent study unveiled a distinctive role of rice OsDREB1C in elevating nitrogen use efficiency (NUE), photosynthesis, and grain yield, implying functional divergence within the CBF/DREB1 orthologs across species. Here, we delve into divergent molecular mechanisms of OsDREB1C and AtCBF2/3/1 by exploring their evolutionary trajectories across rice and Arabidopsis genomes, regulatomes, and transcriptomes. Evolutionary scrutiny shows discrete clades for OsDREB1C and AtCBF2/3/1, with the Poaceae-specific DREB1C clade mediated by a transposon event. Genome-wide binding profiles highlight OsDREB1C's preference for GCCGAC compared to AtCBF2/3/1's preference for A/GCCGAC, a distinction determined by R12 in the OsDREB1C AP2/ERF domain. Cross-species multi-omic analyses reveal shared gene orthogroups (OGs) and underscore numerous specific OGs uniquely bound and regulated by OsDREB1C, implicated in NUE, photosynthesis, and early flowering, or by AtCBF2/3/1, engaged in hormone and stress responses. This divergence arises from gene gains/losses (∼16.7‒25.6%) and expression reprogramming (∼62.3‒66.2%) of OsDREB1C- and AtCBF2/3/1-regulated OGs during the extensive evolution following the rice-Arabidopsis split. Our findings illustrate the regulatory evolution of OsDREB1C and AtCBF2/3/1 at a genomic scale, providing insights on the functional divergence of orthologous transcription factors following gene duplications across species.

A prion-like domain is required for phase separation and chloroplast RNA processing during cold acclimation in Arabidopsis

Abstract Arabidopsis (Arabidopsis thaliana) plants can produce photosynthetic tissue with active chloroplasts at temperatures as low as 4°C, and this process depends on the presence of the nuclear-encoded, chloroplast-localized RNA-binding protein CP29A. In this study, we demonstrate that CP29A undergoes phase separation in vitro and in vivo in a temperature-dependent manner, which is mediated by a prion-like domain (PLD) located between the two RNA recognition motif (RRM) domains of CP29A. The resulting droplets display liquid-like properties and are found near chloroplast nucleoids. The PLD is required to support chloroplast RNA splicing and translation in cold-treated tissue. Together, our findings suggest that plant chloroplast gene expression is compartmentalized by inducible condensation of CP29A at low temperatures, a mechanism that could play a crucial role in plant cold resistance.

The function of brown adipose tissue at different sites of the body in Brandt's voles during cold acclimation

Ambient temperatures have great impacts on thermoregulation of small mammals. Brown adipose tissue (BAT), an obligative thermogenic tissue for small mammals, is localized not only in the interscapular depot (iBAT), but also in supraclavicular, infra/subscapular, cervical, paravertebral, and periaortic depots. The iBAT is known for its cold-induced thermogenesis, however, less has been paid attention to the function of BAT at other sites. Here, we investigated the function of BAT at different sites of the body during cold acclimation in a small rodent species. As expected, Brandt's voles (Lasiopodomys brandtii) consumed more food and reduced the body mass gain when they were exposed to cold. The voles increased resting metabolic rate and maintained a relatively lower body temperature in the cold (36.5 ± 0.27 °C) compared to those in the warm condition (37.1 ± 0.36 °C). During cold acclimation, the uncoupling protein 1 (UCP1) increased in aBAT (axillary), cBAT (anterior cervical), iBAT (interscapular), nBAT (supraclavicular), and sBAT (suprascapular). The levels of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation, were higher in cBAT and iBAT in the cold than in the warm group. The pAMPK/AMPK and pCREB/CREB were increased in cBAT and iBAT during cold acclimation, respectively. These data indicate that these different sites of BAT play the cold-induced thermogenic function for small mammals.

Streamlining Protein Fractional Synthesis Rates Using SP3 Beads and Stable Isotope Mass Spectrometry: A Case Study on the Plant Ribosome.

Ribosomes are an archetypal ribonucleoprotein assembly. Due to ribosomal evolution and function, r-proteins share specific physicochemical similarities, making the riboproteome particularly suited for tailored proteome profiling methods. Moreover, the structural proteome of ribonucleoprotein assemblies reflects context-dependent functional features. Thus, characterizing the state of riboproteomes provides insights to uncover the context-dependent functionality of r-protein rearrangements, as they relate to what has been termed the ribosomal code, a concept that parallels that of the histone code, in which chromatin rearrangements influence gene expression. Compared to high-resolution ribosomal structures, omics methods lag when it comes to offering customized solutions to close the knowledge gap between structure and function that currently exists in riboproteomes. Purifying the riboproteome and subsequent shot-gun proteomics typically involves protein denaturation and digestion with proteases. The results are relative abundances of r-proteins at the ribosome population level. We have previously shown that, to gain insight into the stoichiometry of individual proteins, it is necessary to measure by proteomics bound r-proteins and normalize their intensities by the sum of r-protein abundances per ribosomal complex, i.e., 40S or 60S subunits. These calculations ensure that individual r-protein stoichiometries represent the fraction of each family/paralog relative to the complex, effectively revealing which r-proteins become substoichiometric in specific physiological scenarios. Here, we present an optimized method to profile the riboproteome of any organism as well as the synthesis rates of r-proteins determined by stable isotope-assisted mass spectrometry. Our method purifies the r-proteins in a reversibly denatured state, which offers the possibility for combined top-down and bottom-up proteomics. Our method offers a milder native denaturation of the r-proteome via a chaotropic GuHCl solution as compared with previous studies that use irreversible denaturation under highly acidic conditions to dissociate rRNA and r-proteins. As such, our method is better suited to conserve post-translational modifications (PTMs). Subsequently, our method carefully considers the amino acid composition of r-proteins to select an appropriate protease for digestion. We avoid non-specific protease cleavage by increasing the pH of our standardized r-proteome dilutions that enter the digestion pipeline and by using a digestion buffer that ensures an optimal pH for a reliable protease digestion process. Finally, we provide the R package ProtSynthesis to study the fractional synthesis rates of r-proteins. The package uses physiological parameters as input to determine peptide or protein fractional synthesis rates. Once the physiological parameters are measured, our equations allow a fair comparison between treatments that alter the biological equilibrium state of the system under study. Our equations correct peptide enrichment using enrichments in soluble amino acids, growth rates, and total protein accumulation. As a means of validation, our pipeline fails to find "false" enrichments in non-labeled samples while also filtering out proteins with multiple unique peptides that have different enrichment values, which are rare in our datasets. These two aspects reflect the accuracy of our tool. Our method offers the possibility of elucidating individual r-protein family/paralog abundances, PTM status, fractional synthesis rates, and dynamic assembly into ribosomal complexes if top-down and bottom-up proteomic approaches are used concomitantly, taking one step further into mapping the native and dynamic status of the r-proteome onto high-resolution ribosome structures. In addition, our method can be used to study the proteomes of all macromolecular assemblies that can be purified, although purification is the limiting step, and the efficacy and accuracy of the proteases may be limited depending on the digestion requirements. Key features • Efficient purification of the ribosomal proteome: streamlined procedure for the specific purification of the ribosomal proteome or complex Ome. • Accurate calculation of fractional synthesis rates: robust method for calculating fractional protein synthesis rates in macromolecular complexes under different physiological steady states. • Holistic ribosome methodology focused on plants: comprehensive approach that provides insights into the ribosomes and translational control of plants, demonstrated using cold acclimation [1]. • Tailored strategies for stable isotope labeling in plants: methodology focusing on materials and labeling considerations specific to free and proteinogenic amino acid analysis [2].

Jasmonate enhances cold acclimation in jojoba by promoting flavonol synthesis

Abstract Jojoba is an industrial oil crop planted in tropical arid areas, and its low-temperature sensitivity prevents its introduction into temperate areas. Studying the molecular mechanisms associated with cold acclimation in jojoba is advantageous for developing breeds with enhanced cold tolerance. In this study, metabolomic analysis revealed that various flavonols accumulate in jojoba during cold acclimation. Time-course transcriptomic analysis and weighted correlation network analysis (WGCNA) demonstrated that flavonol biosynthesis and jasmonates (JAs) signaling pathways played crucial roles in cold acclimation. Combining the biochemical and genetic analyses showed that ScMYB12 directly activated flavonol synthase gene (ScFLS). The interaction between ScMYB12 and transparent testa 8 (ScTT8) promoted the expression of ScFLS, but the negative regulator ScJAZ13 in the JA signaling pathway interacted with ScTT8 to attenuate the transcriptional activity of the ScTT8 and ScMYB12 complex, leading to the downregulation of ScFLS. Cold acclimation stimulated the production of JA in jojoba leaves, promoted the degradation of ScJAZ13, and activated the transcriptional activity of ScTT8 and ScMYB12 complexes, leading to the accumulation of flavonols. Our findings reveal the molecular mechanism of JA-mediated flavonol biosynthesis during cold acclimation in jojoba and highlight the JA pathway as a promising means for enhancing cold tolerance in breeding efforts.

Cold Acclimation and Deacclimation Processes in Grapevine Buds: Insights into the Regulation of Cod Hardiness and ICE-CBF-COR Gene Expression

Cold Acclimation and Deacclimation Processes in Grapevine Buds: Insights into the Regulation of Cod Hardiness and ICE-CBF-COR Gene Expression

Temperature-Caused Changes in Raman Pattern and Protein Profiles of Winter Triticale (x Triticosecale, Wittm.) Field-Grown Seedlings.

Climate change, which causes periods with relatively high temperatures in winter in Poland, can lead to a shortening or interruption of the cold hardening of crops. Previous research indicates that cold acclimation is of key importance in the process of acquiring cereal tolerance to stress factors. The objective of this work was to verify the hypothesis that both natural temperature fluctuations and the plant genotype influence the content of metabolites as well as proteins, including antioxidant enzymes and photosystem proteins. The research material involved four winter triticale genotypes, differing in their tolerance to stress under controlled conditions. The values of chlorophyll a fluorescence parameters and antioxidant activity were measured in their seedlings. Subsequently, the contribution of selected proteins was verified using specific antibodies. In parallel, the profiling of the contents of chlorophylls, carotenoids, phenolic compounds, and proteins was carried out by Raman spectroscopy. The obtained results indicate that a better PSII performance along with a higher photosystem II proteins content and thioredoxin reductase abundance were accompanied by a higher antioxidant activity in the field-grown triticale seedlings. The Raman studies showed that the cold hardening led to a variation in photosynthetic dyes and an increase in the phenolic to carotenoids ratio in all DH lines.

Adaptive thermogenesis of the brown adipose tissue in Apodemus chevrieri during cold acclimation

Abstract To investigate the effect of low temperature on body mass, thermogenic activity, and Uncoupling protein-1 (UCP1) content of brown adipose tissue (BAT) in Chevrier’s field mouse (Apodemus chevrieri), 50 healthy adult mice with similar body mass were used in our experiment. They were divided into five groups of ten individuals as follows: a control group (0 d), where animals were maintained under 25 ± 1°C and a 12L:12D (light:dark, lights on 08:00) photoperiod; the other groups were maintained under 5 ± 1°C and a 12L:12D photoperiod for 7 d, 14 d, 21 d and 28 d, respectively. At the end of the experiment, the changes in body mass, resting metabolic rate (RMR), nonshivering thermogenesis (NST), BAT mass, mitochondrial protein (MP) content, cytochrome c oxidase (COX) activity, and UCP1 content were measured. The results showed that compared with the control group (0 d), the body mass of the cold acclimation groups decreased significantly. In contrast, RMR, NST, BAT mass, MP content, COX activity, and UCP1 content of the cold acclimation group increased significantly. After cold acclimation for 28 days, RMR increased by 89.4%, NST increased by 50.4%, BAT mass increased by 44.6%, and UCP1 content increased by 36.0%. The ratio of (NST − RMR)/RMR was 1.03 after seven days and then dropped to 0.59 on day 21, and remained at a steady level thereafter. Evidently, NST was significantly positively correlated with BAT mass and UCP1 content. The results indicated that under continuous cold exposure, A. chevrieri could take appropriate measures to reduce body mass, increase RMR, induce BAT tissue proliferation, and unregulated UCP1 expression, thereby enhancing BAT thermogenic activity to cope with a low-temperature environment. In the early stage of cold acclimation, NST was dominant in thermogenesis, but as time continued, it reduced. This may represent a unique energy adaptation strategy of small rodents originating from the north and spreading southward toward the Hengduan Mountain regions.

SERCA uncoupling may facilitate cold acclimation in dark-eyed juncos (Junco hyemalis) without regulation by sarcolipin or phospholamban.

Homeothermic endotherms defend their body temperature in cold environments using a number of behavioral and physiological mechanisms. Maintaining a stable body temperature primarily requires heat production through shivering (ST) or non-shivering thermogenesis (NST). Although the use of NST is well established in mammalian systems, the mechanisms and extent to which NST is used in birds is poorly understood. In mammals, one well-characterized mechanism of NST is through uncoupling of Ca2+ transport from ATP hydrolysis by sarco/endoplasmic reticulum ATPase (SERCA) in the skeletal muscle, which generates heat and may contribute to Ca2+ signaling for fatigue resistance and mitochondrial biogenesis. Two small proteins-sarcolipin (SLN) and phospholamban (PLN)-are known to regulate SERCA in mammals, but recent work shows inconsistent responses of SLN to cold-acclimation in birds. In this study, we measured SERCA uncoupling in the pectoralis flight muscle of control (18°C) and cold-acclimated (-8°C) dark-eyed juncos (Junco hyemalis) that exhibited suppressed SLN transcription in the cold. We measured SERCA activity and Ca2+ uptake rates for the first time in cold-acclimated birds and found greater SERCA uncoupling in the muscle of juncos in the cold. However, SERCA uncoupling was not related to SLN or PLN transcription or measures of mitochondrial biogenesis. Nonetheless, SERCA uncoupling reduced an individual's risk of hypothermia in the cold. Therefore, while SERCA uncoupling in the cold could be indicative of NST, it does not appear to be mediated by known regulatory proteins in these birds. These results prompt interesting questions about the significance of SLN and PLN in birds and the role of SERCA uncoupling in response to environmental conditions.

Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold‐acclimated Arabidopsis

AbstractOverwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short‐term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second‐phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second‐phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C‐repeat binding factor and cold‐regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea during the 2PFA treatment. Compared with wild‐type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.

Cold stress induces rapid gene-specific changes in the levels of H3K4me3 and H3K27me3 in Arabidopsis thaliana.

When exposed to low temperatures, plants undergo a drastic reprogramming of their transcriptome in order to adapt to their new environmental conditions, which primes them for potential freezing temperatures. While the involvement of transcription factors in this process, termed cold acclimation, has been deeply investigated, the potential contribution of chromatin regulation remains largely unclear. A large proportion of cold-inducible genes carries the repressive mark histone 3 lysine 27 trimethylation (H3K27me3), which has been hypothesized as maintaining them in a silenced state in the absence of stress, but which would need to be removed or counteracted upon stress perception. However, the fate of H3K27me3 during cold exposure has not been studied genome-wide. In this study, we offer an epigenome profiling of H3K27me3 and its antagonistic active mark H3K4me3 during short-term cold exposure. Both chromatin marks undergo rapid redistribution upon cold exposure, however, the gene sets undergoing H3K4me3 or H3K27me3 differential methylation are distinct, refuting the simplistic idea that gene activation relies on a switch from an H3K27me3 repressed chromatin to an active form enriched in H3K4me3. Coupling the ChIP-seq experiments with transcriptome profiling reveals that differential histone methylation only weakly correlates with changes in expression. Interestingly, only a subset of cold-regulated genes lose H3K27me3 during their induction, indicating that H3K27me3 is not an obstacle to transcriptional activation. In the H3K27me3 methyltransferase curly leaf (clf) mutant, many cold regulated genes display reduced H3K27me3 levels but their transcriptional activity is not altered prior or during a cold exposure, suggesting that H3K27me3 may serve a more intricate role in the cold response than simply repressing the cold-inducible genes in naïve conditions.

Metabolomic Profile and Functional State of Oat Plants (Avena sativa L.) Sown under Low-Temperature Conditions in the Cryolithozone.

Oats are one of the most useful and widespread cereal crops in the world. In permafrost conditions (Central Yakutia), based on metabolic changes in late summer-sown oat plants (Avena sativa L.), the key processes involved in the cold acclimation of a valuable cereal species were identified. During the onset of low ambient temperatures, metabolites from leaf samples were profiled using gas chromatography with mass spectrometry (GC-MS) and were analyzed using principal component analysis (PCA). A total of 41 metabolites were identified in oat leaves. It was found that acclimation to suboptimal temperatures during the fall period leads to biochemical (accumulation of mono- and disaccharides and decrease in fatty acids and polyols) as well as physiological and biophysical changes (decrease in leaf PRI reflectance indices and chlorophyll a fluorescence). Therefore, the study contributes to a more holistic understanding of oat metabolism under low-temperature cryolithozone stress. It is believed that the analysis of changes in leaf reflection properties and JIP-test parameters of chlorophyll a fluorescence using leaf metabolomic profiling can be used in the selection of valuable varieties of cereal crops to obtain plant fodders with high nutrient contents under conditions of a sharply continental climate.

Phenotypic constraints at the top of the world: an Arctic songbird faces the cumulative cost of maintaining a winter-like phenotype during breeding

Among birds, several body composition traits typically decrease in size or mass during breeding likely as a result of competing demands during this critical life history stage. However, a recent outdoor captive study in an Arctic-breeding cold-specialist songbird (snow buntings – Plectrophenax nivalis) demonstrated that these birds maintain winter cold acclimatization during the spring and summer, despite facing summer temperatures much warmer than on their Arctic breeding grounds. This suggests that buntings may face a cumulative physiological cost during breeding: having to support a winter phenotype while also upregulating additional traits for reproduction. The current study aimed to test this hypothesis. Between 2016 and 2019, we examined how body composition and metabolic performance (thermogenic capacity and physiological maintenance costs) changed from pre-breeding to chick provisioning in free-living birds captured at the northern limit of their breeding range in the Canadian Arctic (Alert, NU, 82°). While body mass and fat reserves deceased significantly between pre-breeding and territory defense independent of thermal conditions, cold endurance and associated traits remained stable and elevated up to the nestling provisioning period, as long as ambient temperature remained below a threshold level of 0–2°C. These results indicate that snow buntings must maintain a high thermogenic capacity after arrival on the breeding grounds if temperatures remain below freezing, regardless of whether birds are actively breeding or not. In this context, our research suggests that these birds, and possibly other arctic breeding songbirds, may experience cumulative physiological costs during years with a late onset of spring, when breeding activities (i.e., egg production and incubation) begin while temperatures are still below 0–2°C.

Low Temperature Tolerance of Peach Flower Buds and Shoot Tissues Is Differentially Influenced by Freezing and High Temperature Exposure

In northern temperate zones, peach trees are vulnerable to cold temperature injury in the fall, particularly as climate change prolongs warm weather in the fall and potentially delays the onset of cold acclimation. Four experiments evaluated how cold acclimation of flower buds and shoot phloem, cambium, and xylem is affected by exposure to varying temperatures in the fall. One-year-old peach shoots from trees grown in Maine, USA, were collected from October through November, exposed to 1, 3, or 6 days of low, high, and freezing temperatures, and subjected to stepwise controlled freezing to about −30 °C. Injury was visually quantified as oxidative browning of flower buds and shoot tissues. High temperature exposure, even of a single day, decreased cold tolerance of flower buds and shoot tissues until late November, when high temperatures only minimally decreased cold hardiness. In mid-November, increasing the duration of high temperature exposure from 1 to 3 days decreased cambium and phloem hardiness, but hardiness in flower buds was not further decreased by the longer duration of 3 days. By late November, hardiness in flower buds, cambium, and phloem was less responsive to high temperature, and was increased by prior exposure to 6 days of freezing. After high temperature, xylem lost hardiness to a small degree in mid-October and late November, but in mid-November this occurred in only one experiment. In this study, deacclimation during high temperature in the fall was greater in cambium and phloem than in flower buds and at times greater than in xylem.

Integrated analysis of ATAC-seq and transcriptomic reveals the ScDof3-ScproC molecular module regulating the cold acclimation capacity of potato

Low temperature severely affects the geographical distribution and production of potato, which may incur cold damage in early spring or winter. Cultivated potatoes, mainly derived from Solanum tuberosum, are sensitive to freezing stress, but wild species of potato such as S. commersonii exhibit both constitutive freezing tolerance and/or cold acclimation tolerance. Hence, such wild species could assist in cold hardiness breeding. Yet the key transcription factors and their downstream functional genes that confer freezing tolerance are far from clear, hindering the breeding process. Here, we used ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) alongside RNA-seq to investigate the variation in chromatin accessibility and patterns of gene expression in freezing-tolerant CMM5 (S. commersonii), before and after its cold treatment. Our results suggest that after exposure to cold, transcription factors including Dof3, ABF2, PIF4, and MYB4 were predicted to further control the genes active in the synthetic/metabolic pathways of plant hormones, namely abscisic acid, polyamine, and reductive glutathione (among others). This suggests these transcription factors could regulate freezing tolerance of CMM5 leaves. In particular, ScDof3 was proven to regulate the expression of ScproC (pyrroline-5-carboxylate reductase, P5CR) according to dual-LUC assays. Overexpressing ScDof3 in Nicotiana benthamiana leaves led to an increase in both the proline content and expression level of NbproC (homolog of ScproC). These results demonstrate the ScDof3-ScproC module regulates the proline content and thus promotes freezing tolerance in potato. Our research provides valuable genetic resources to further study the molecular mechanisms underpinning cold tolerance in potato.

Study on the estradiol degradation gene expression and resistance mechanism of Rhodococcus R-001 under low-temperature stress

Estradiol (E2), an endocrine disruptor, acts by mimicking or interfering with the normal physiological functions of natural hormones within organisms, leading to issues such as endocrine system disruption. Notably, seasonal fluctuations in environmental temperature may influence the degradation speed of estradiol (E2) in the natural environment , intensifying its potential health and ecological risks. Therefore, this study aims to explore how bacteria can degrade E2 under low-temperature conditions, unveiling their resistance mechanisms, with the goal of developing new strategies to mitigate the threat of E2 to health and ecological safety. In this paper, we found that Rhodococcus equi DSSKP-R-001 (R-001) can efficiently degrade E2 at 30°C and 10°C. Six genes in R-001 were shown to be involved in E2 degradation by heterologous expression at 30°C. Among them, 17β-HSD, KstD2, and KstD3, were also involved in E2 degradation at 10°C; KstD was not previously known to degrade E2. RNA-seq was used to characterize differentially expressed genes (DEGs) to explore the stress response of R-001 to low-temperature environments to elucidate the strain’s adaptation mechanism. At the low temperature, R-001 cells changed from a round spherical shape to a long rod or irregular shape with elevated unsaturated fatty acids and were consistent with the corresponding genetic changes. Many differentially expressed genes linked to the cold stress response were observed. R-001 was found to upregulate genes encoding cold shock proteins, fatty acid metabolism proteins, the ABC transport system, DNA damage repair, energy metabolism and transcriptional regulators. In this study, we demonstrated six E2 degradation genes in R-001 and found for the first time that E2 degradation genes have different expression characteristics at 30°C and 10°C. Linking R-001 to cold acclimation provides new insights and a mechanistic basis for the simultaneous degradation of E2 under cold stress in Rhodococcus adaptation.

Toward new biomarkers of cold tolerance: microRNAs regulating cold adaptation in fish are differentially expressed in cold-tolerant and cold-sensitive Nile tilapia (Oreochromis niloticus)

Overcoming the negative effects of cold stress in Nile tilapia (Oreochromis niloticus) is of great interest for the overall aquaculture economy. Recent evidence suggests that microRNAs may regulate mechanisms of cold adaptation in fish and could be potential biomarkers of cold tolerance in these animals. However, information on how microRNAs regulating cold adaptation in fish are expressed in cold-tolerant and cold-sensitive fish is still scarce. Therefore, the aim of the present study is to investigate the relationship between a panel of previously identified differentially expressed microRNAs in fish exposed to low temperatures and their association with cold tolerance in Nile tilapia. To this end, fish were individualized based on their weight and length, and subjected to a critical thermal minimum (CTmin) test. Blood samples were collected before and after the CTmin test. After the CTmin, brain and liver tissues were collected. Fish were separated into cold-tolerant and cold-sensitive based on their CTmin results. Untreated fish were used as control. The relative expression of miR-9-3p, miR-135c, miR-9-5p, miR-30b, miR-122, miR-92a was assessed through qPCR. Additionally, qPCR was used to measure the mRNA relative expression of IGF-I and SCD in the tissues. Cortisol and biochemical parameters were determined in plasma samples. As a result, miR-92a was up-regulated in the liver of cold-sensitive fish and in the blood of cold-tolerant fish post-CTmin. miR-30b was up-regulated in the blood and liver of cold stressed fish. In the brain, miR-9-5p and miR-9-3p were up-regulated in cold-tolerant fish post-CTmin. Furthermore, miR-122 and miR-135c expression levels remained similar to the control post-CTmin. In addition, miR-122 and miR-92a were differentially expressed in the blood of cold-tolerant and cold-sensitive Nile tilapia pre-CTmin. Therefore, besides regulating fish cold acclimation responses differently in cold-tolerant and cold-sensitive fish, miRNAs may also regulate pre-existing physiological conditions that are determinant to cold tolerance in Nile tilapia. In the transcriptional level, SCD expression increased in both cold-tolerant and cold-sensitive fish post-CTmin. Cold-tolerant Nile tilapia post-CTmin had increased levels of plasma cholesterol and IGF-I mRNA in the liver, which could mean an adaptive advantage. The present findings provide new insights on the role of post-transcriptional regulation of cold tolerance in Nile tilapia. Furthermore, miRNAs differentially expressed in cold-tolerant Nile tilapia may be potential targets for the development of new biomarkers and aid in the breeding of cold-tolerant strains in this species.

TaMAPK3 phosphorylates TaCBF and TaICE and plays a negative role in wheat freezing tolerance

Freezing temperature during overwintering often kills plants; plants have thus, developed a defense mechanism called ‘cold acclimation’, in which a number of genes are involved in increasing cell protection and gene expression. Mitogen-activated protein kinase (MAPK) controls proteins' activities by phosphorylation and is involved in numerous metabolic pathways. In this study, we identified the protein interaction between TaMAPK3 and the proteins in the cold response pathway, ICE41, ICE87, and CBFIVd-D9. The subcellular localization and bimolecular fluorescence complement (BiFC) assays revealed that these proteins interact in the nucleus or in the plasma membrane. Furthermore, MAPK3-mediated phosphorylation of ICE41, ICE87, and CBFIVd-D9 was verified using an in vitro phosphorylation assay. TaMAPK3-overexpressing transgenic Brachypodium showed a lower survival rate upon freezing stress and lower proline content during cold acclimation, compared to wild-type plants. Furthermore, cold response gene expression analysis revealed that the expression of these genes was suppressed in the transgenic lines under cold treatment. It was further elucidated that MAPK3 mediates the degradation of ICE and CBF proteins, which implies the negative impact of MAPK3 on the freezing tolerance of plants. This study will help to elucidate the molecular mechanisms of cold tolerance and the activity of MAPK3 in wheat.

Effect of tea polyphenol-trehalose complex coating solutions on physiological stress and flesh quality of marine-cultured Turbot Scophthalmus maximus during waterless transport.

The waterless transport of live fish has changed the present situation of live-fish transport. However, the waterless transport environment may cause stress in fish. This research evaluated the effect of tea polyphenol-trehalose (TPT) coating solutions on Turbot Scophthalmus maximus during waterless transport. After cold acclimation, Turbot were coated and subsequently transported in a waterless environment for 18 h. Physiological and biochemical parameters were measured, including lysozyme (LZM) and immunoglobulin M (IgM) activities, serum creatinine (Cr) and uric acid (UA) concentrations, and nutritional flavor. The results showed that the nonspecific immunity of Turbot was inhibited during the waterless transport; the LZM activity first increased and then decreased, and the serum Cr and UA concentrations significantly increased. In addition, the waterless transport promoted the breakdown of Turbot flesh proteins, leading to changes in nucleotides and free amino acids (FAAs). After waterless transport, the LZM and IgM activities in the TPT-treated Turbot were higher than those in the control group (CK), and the changes in FAA content and nucleotides were smaller than those observed in the CK group. This study shows that the use of TPT coating solution can reduce the impact of waterless transportation stress on the immune and metabolic functions of Turbot and can maintain the meat quality and flavor of Turbot.

PI3K/AKT pathway modulation and cold acclimation alleviation concerning apoptosis and necroptosis in broiler thymus

Moderate cold stimulation regulates the thymus's growth and function and facilitates cold acclimatization in broilers. However, the underlying mechanism remains unknown. To explore the possible mechanism of the thymus in cold-acclimated broilers against cold stress, 240 1-day-old Arbor Acres (AA) broilers were assigned to two groups randomly. The control group (C) was housed at conventional temperatures. The temperature during the first week was 33-34°C. Between the ages of 8 and 32 days, the temperature was lowered by 1°C every two days, i.e., gradually from 32°C to 20°C, and then maintained at 20°C until 42 days of age. The cold-acclimated group (C-3) was housed at the same temperature as C from 1-7 days after birth. Between 8 and 42 days, the temperature of C-3 was 3°C colder than C. After 24 h exposure to acute cold stress (ACS) at 42 days, C and C-3 were named as S and S-3. The results showed that ACS was able to induce oxidation stress, modulate PI3K/AKT signal, and cause necroptosis and apoptosis in broiler thymus. By contrast, cold acclimation could alleviate apoptosis and necroptosis induced by cold stress via alleviating oxidative stress, efficiently activating the PI3K/AKT signal, as well as decreasing apoptotic and necrotic genes’ levels. This study offers a novel theoretical basis for cold acclimation to improve the body's cold tolerance.