Freezing Tolerance Research Articles

Registration of ‘CP 10‐1620’ sugarcane

Abstract‘CP 10‐1620’ (Reg. no. CV‐216, PI 693837) sugarcane (a complex hybrid of Saccharum spp.) was developed through cooperative research conducted by the USDA–ARS, the University of Florida, and the Florida Sugar Cane League, Inc. At the June 7, 2018, Florida Sugarcane Variety Committee Meeting, CP 10‐1620 was released to growers for mineral (sand) soils in Florida, and seed was available in September 2018. CP 10‐1620 was selected from a polycross of ‘CP 00‐1100’ × ‘CP 05‐1740’ (Cross no. X07‐1225) made at Canal Point, FL, in December 2007. Neither the female parent, CP 00‐1100, nor the male parent, CP 05‐1740, is a commercial cultivar. Cane yield of CP 10‐1620 on sand soils, averaged across 12 harvests through three crop cycles (plant cane, first ratoon, and second ratoon), was 11.0% higher (P < 0.05) than the combined mean of three checks, ‘CL 88‐4730’, ‘CP 96‐1252’, and ‘CPCL 97‐2730’, which are commercial checks for sand soils. The commercial recoverable sucrose for CP 10‐1620 was not significantly lower than the combined mean of the checks but the higher cane yield resulted in 8.4% higher (P < 0.05) sucrose yield than the combined mean of the checks. CP 10‐1620 was released because of its high cane yields on sand soils, and its acceptable levels of resistance to brown rust (caused by Puccinia melanocephala H. & P. Sydow), leaf scald (caused by Xanthomonas albilineans Ashby, Dowson), Sugarcane mosaic virus strain E (mosaic), smut (caused by Sporisorium scitamineum), Sugarcane yellow leaf virus, and ratoon stunt [caused by Leifsonia xyli subsp. xyli (Davis et al.) Evtushenko]. CP 10‐1620 was moderately susceptible to orange rust (caused by Puccinia kuehnii E. J. Butler), and it has high freeze tolerance. CP 10‐1620 tested positive for the Bru1 locus, which is an indicator of brown rust resistance.

Enhancing Freeze Tolerance of Field-grown Lettuce with Salicylic Acid, Ascorbic Acid, and Calcium Chloride

With the climate continuing to change, specialty crop growers are particularly at risk for economic loss caused by erratic weather conditions, excess heat and rain, and frost damage. Although tolerant to cold, lettuce (Lactuca sativa) is a major horticultural crop at risk for exposure to freezing temperatures in late fall or early spring in the Midwest. Exogenous applications of salicylic acid, ascorbic acid, and calcium chloride have been shown to improve abiotic stress in plants, particularly with freezing tolerance. This research investigated the effects of the exogenous application of salicylic acid, ascorbic acid, and calcium chloride, in varying concentrations, on field-grown lettuce. The study was conducted at the Iowa State University Horticulture Research Station, Ames, IA, USA. Weekly applications of salicylic acid (0.5 and 1.0 mM), ascorbic acid (0.5 and 1.0 mM), and calcium chloride (10 and 20 mM) were applied until plants were a marketable size. A control treatment (no spray) was also included. Data regarding plant size, yield, leaf area, leaf number, plant dry weight, plant nutrient analysis, and freeze tolerance were collected. Freeze tolerance assessments were conducted through laboratory-simulated freeze events and the evaluation of natural in-field freeze events. Freeze injury was quantified through the electrolyte leakage method. Marketable weights in 2020 and 2021 were statistically similar, except for 1.0 mM salicylic acid in 2020, which showed a significantly lower marketable weight and head diameter. Trends during both years showed that stress protectant applications had the most effective freeze protection at −12 °C in laboratory-simulated freeze events. Calcium chloride at 20 mM had the highest protection at −12 °C, with 13.3% and 24.0% less injury compared with the control in 2020 and 2021, respectively. Salicylic acid 1.0 mM at −12 °C had 4.7% and 23.7% less injury compared with control during these two years, respectively. Ascorbic acid 1.0 mM at −12 °C also showed 6.8% and 9.5% less injury than the control in 2020 and 2021, respectively. Similar trends were observed after in-field freezing events, with 20 mM calcium chloride providing the highest protection against frost in 2020 and 2021. These findings advance the understanding of the capabilities of stress protectants on lettuce as chemical primers after freezing events. This research highlights the benefits of ascorbic acid, salicylic acid, and calcium chloride as stress protectants and encourages future research and further exploration of concentrations, methods of application, and timing of application of products.

Methyltransferase TaSAMT1 mediates wheat freezing tolerance by integrating brassinosteroid and salicylic acid signaling.

Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1 that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops.

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.

Linking temperature sensitivity of mangrove communities, populations and individuals across a tropical‐temperate transitional zone

Abstract Climate change is reshaping coastal wetlands worldwide, driving ecosystem shifts like mangrove poleward expansion into saltmarshes in tropical‐temperate transitional zones. Though warming is recognized as the primary driver, a lack of detailed field studies limits our ability to predict mangrove responses to rapid climate warming. Here, we characterized how mangroves vary across a temperature gradient at 18 sites along Florida's Gulf of Mexico coast (USA). We used minimum air temperature (Tmin) derived from daily data from 1989 to 2021 as the independent variable and applied plot‐based and synoptic approaches to quantify species‐specific mangrove variation at community, population, and individual levels. We then used these results to spatially project future mangrove ecosystem properties under multiple warming scenarios. Across the Tmin gradient from −10.8 to −1.4°C, mangrove canopy height and coverage ranged from 0.4 to 11.5 m and 15% to 98%, respectively, with both exhibiting sigmoidal increases with Tmin. Estimated mangrove aboveground biomass ranged from 0 to 496.7 Mg/ha and showed a positive linear relationship with Tmin due both to the tall tree stratum's increased biomass per tree and higher abundance. While the population abundance and coverage of Rhizophora mangle and Laguncularia racemosa had positive linear relationships with Tmin, Avicennia germinans exhibited a significant quadratic relationship, reflecting the higher freeze tolerance of this species. Such tolerance may stem from A. germinans' higher morphological plasticity observed at the individual level, adapting to cold stress by exhibiting a more shrub‐like architecture at colder sites. Based on these field‐derived quantitative relationships, we projected substantial increases in mangrove coverage and canopy height near current range limits, with tall A. germinans dominating in the north and R. mangle dominating the centre and south of the study region. Synthesis. To better predict the ecological consequences in coastal wetlands under future climate change, it is essential to understand how mangroves respond to winter temperature regimes across a temperature gradient. Collectively, these cross‐level and species‐specific results advance our understanding of mangrove temperature sensitivity and provide information about the future of coastal wetland structure and function in response to a changing climate.

Inferring current and Last Glacial Maximum distributions are improved by physiology‐relevant climatic variables in cold‐adapted ectotherms

AbstractAimEcological niche‐based models (ENM) frequently rely on bioclimatic variables (BioV) to reconstruct biogeographic scenarios for species evolution, ignoring mechanistic relations. We tested if climatic predictors relevant to species hydric and thermal physiology better proximate distribution patterns and support location of Pleistocene refugia derived from phylogeographic studies.LocationThe Western Palaearctic.TaxonVipera berus and Zootoca vivipara, two cold‐adapted species.MethodsWe used two sets of variables, that is physiologically meaningful climatic variables (PMV) and BioV, in a multi‐algorithm ENM approach, to compare their ability to predict current and Last Glacial Maximum (LGM) species ranges. We estimated current and LGM permafrost extent to address spatially the cold hardiness dissimilarity between both species.ResultsPMV explained more accurately the current distribution of these two cold‐adapted species and identified the importance of summer temperature and solar radiation that constrain activity in cold habitats. PMV also provide a better insight than BioV predictors on LGM distribution. By including notably, the permafrost extent, PMV‐based models gave parsimonious putative arrangement and validity of refugia for each clade and subclade in accordance with phylogeographic data. Northern refugia were also identified from 48 to 52° N for V. berus and from 50 to 54° N for Z. vivipara.Main ConclusionsOur hybrid approach based on PMV generated more realistic predictions for both current (biogeographical validation) and past distributions (phylogeographic validation). By combining constraints during the activity period (summer climatic niche) and those inherent to the wintering period (freeze tolerance), we managed to identify glacial refuges in agreement with phylogeographic hypotheses concerning post‐glacial routes and colonization scenarios.

A robust, biodegradable and recyclable all-cellulose ionogel from low-value wood

For thousands of years, wood has been utilized as a natural, abundant, and sustainable material for structural construction and furniture. To enhance its value, we have developed a simple process that can directly convert low-value wood (including cracked wood, chips, shives, and other wood residues) into high-performance all-cellulose ionogels. This process involves delignification, in situ dissolution of cellulose, and self-assembly of molecular cellulose chains. The resulting ionogel exhibits excellent properties such as high ion conductivity (∼60 mS/cm), mechanical strength (∼6 MPa), and self-healing capability (∼10 min). Notably, it also demonstrates exceptional freeze tolerance, withstanding temperatures as low as −50 °C while maintaining high ion conductivity (∼6.4 mS/cm). The ionogel is a versatile substrate for flexible electronic circuits. We have developed a sensor utilizing this ionogel that is breathable, flexible, and highly responsive to temperature, humidity, and strain. Furthermore, we have used it as a gel electrolyte to create supercapacitors with exceptional performance even in low-temperature conditions. The ionic liquid can be reused, with an all-cellulose framework that breaks down naturally in moist soil within 15 days. This sustainable method of producing high-performance ionogels from low-value wood has immense potential in shaping the next generation of soft, intelligent devices.

Identification of genetic loci and candidate genes underlying freezing tolerance in wheat seedlings.

Ten stable loci for freezing tolerance (FT) in wheat were detected by genome-wide association analysis. The putative candidate gene TaRPM1-7BL underlying the major locus QFT.ahau-7B.2 was identified and validated. Frost damage restricts wheat growth, development, and geographical distribution. However, the genetic mechanism of freezing tolerance (FT) remains unclear. Here, we evaluated FT phenotypes of 245 wheat varieties and lines, and genotyped them using a Wheat 90K array. The association analysis showed that ten stable loci were significantly associated with FT (P < 1 × 10-4), and explained 6.45-26.33% of the phenotypic variation. In particular, the major locus QFT.ahau-7B.2 was consistently related to all nine sets of FT phenotypic data. Based on five cleaved amplified polymorphic sequence (CAPS) markers closely linked to QFT.ahau-7B.2, we narrowed down the target region to the 570.67-571.16Mb interval (0.49Mb) on chromosome 7B, in which four candidate genes were annotated. Of these, only TaRPM1-7BL exhibited consistent differential expression after low temperature treatment between freezing-tolerant and freezing-sensitive varieties. The results of cloning and whole-exome capture sequencing indicated that there were two main haplotypes for TaRPM1-7BL, including freezing-tolerant Hap1 and freezing-sensitive Hap2. Based on the representative SNP (+1956, A/G), leading to an amino acid change in the NBS domain, a CAPS marker (CAPS-TaRPM1-7BL) was developed and validated in 431 wheat varieties (including the above 245 materials) and 318 F2 lines derived from the cross of 'Annong 9267' (freezing-tolerant) × 'Yumai 9' (freezing-sensitive). Subsequently, the TaRPM1-7BL gene was silenced in 'Yumai 9' by virus-induced gene silencing (VIGS), and these silenced wheat seedlings exhibited enhanced FT phenotypes, suggesting that TaRPM1-7BL negatively regulates FT. These findings are valuable for understanding the complex genetic basis of FT in wheat.

Establishment and characterization of turtle liver organoids provides a potential model to decode their unique adaptations

Painted turtles are remarkable for their freeze tolerance and supercooling ability along with their associated resilience to hypoxia/anoxia and oxidative stress, rendering them an ideal biomedical model for hypoxia-induced injuries (including strokes), tissue cooling during surgeries, and organ cryopreservation. Yet, such research is hindered by their seasonal reproduction and slow maturation. Here we developed and characterized adult stem cell-derived turtle liver organoids (3D self-assembled in vitro structures) from painted, snapping, and spiny softshell turtles spanning ~175My of evolution, with a subset cryopreserved. This development is, to the best of our knowledge, a first for this vertebrate Order, and complements the only other non-avian reptile organoids from snake venom glands. Preliminary characterization, including morphological, transcriptomic, and proteomic analyses, revealed organoids enriched in cholangiocytes. Deriving organoids from distant turtles and life stages demonstrates that our techniques are broadly applicable to chelonians, permitting the development of functional genomic tools currently lacking in herpetological research. Such platform could potentially support studies including genome-to-phenome mapping, gene function, genome architecture, and adaptive responses to climate change, with implications for ecological, evolutionary, and biomedical research.

Pomegranate freezing tolerance evaluation and its relationship with biochemical parameters

Low temperature is one of the most significant environmental factors that threaten the survival of subtropical and tropical plant species. By conducting a study, which was arranged in a completely randomized design with three replicates, the relative freezing tolerance (FT) of four Iranian pomegranate cultivars, including ‘Alak Torsh’, ‘Tabestaneh Torsh’, ‘Poost Sefid’, and ‘Poost Syah’, as well as its correlation with some biochemical indices, were investigated. From each cultivar, pieces of one-year-old shoot samples were treated with controlled freezing temperatures (−11, −14, and −17 ℃) to determine lethal temperatures (LT50) based on survival percentage, electrolyte leakage, phenolic leakage, and tetrazolium staining test (TST) methods. Results showed that FT was higher in the second year with a lower minimum temperature and a higher concentration of cryoprotectants. The stronger correlation of electrolyte leakage with survival percentage (r = 0.93***) compared to the other three indices explained that this index could be the most reliable injury index in determining the pomegranate FT to investigate freezing effects. Of all four cultivars, ‘Poost Syah’ was the hardest by presenting a higher FT than ~ −14 ℃ in mid-winter. Accordingly, this pomegranate cultivar seems to be promising to grow in regions with a higher risk of freezing and to be involved in breeding programs to develop novel commercial cultivars.

Use of electrolyte leakage to assess floral damage after freezing

AbstractPremiseWith growing interest in the impact of false springs on plant reproduction, there is the need to develop reliable, high‐throughput methods for assessing floral freezing damage. Here we present a method for use with floral tissue that will facilitate more comparative work on floral freezing tolerance in the future.Methods and ResultsWe examined the effectiveness of a modified electrolyte leakage protocol to assess floral freezing damage. By comparing data from temperature response curves to an estimate of visual tissue damage, we optimized the protocol for different floral types and improved the signal‐to‐noise ratio for floral data.ConclusionsOur modified protocol provides a quick and straightforward method for quantifying floral freezing damage that can be standardized across floral types. This method allows for cross‐species comparisons and can be a powerful tool for studying broad patterns in floral freezing tolerance.

Registration of ‘CP 15‐2516’ sugarcane for organic soils in Florida

Abstract‘CP 15‐2516’ (Reg. no. CV‐215, PI 703029) sugarcane cultivar, a complex hybrid of Saccharum spp., was developed jointly by USDA‐ARS, the University of Florida, and the Florida Sugar Cane League. CP 15‐2516 was released in June 2022 for commercial cultivation on the Florida organic (muck) soils due to its high cane and sucrose yields, commercial recoverable sucrose (CRS), and economic index (EI). CP 15‐2516 originated from CPX13‐1630, a cross between a female parent (CP 95‐1039) and a male parent (‘CP 06‐2400’). CP 15‐2516 is resistant to brown rust, orange rust, and Sugarcane mosaic virus strain E. CP 15‐2516 harbors the Bru1 marker associated with brown rust resistance. CP 15‐2516 is moderately resistant to leaf scald and ratoon stunt disease, smut, and yellow leaf. Based on the results of three harvest cycles over five locations in Stage 4 on muck soils, cane and sucrose yields of CP 15‐2516 were 10.48% (181.31 Mg ha−1) and 11.49% (18.62 Mg ha−1) respectively, higher (P &lt; .05) than those of the reference cultivar ‘CP 96‐1252’. Compared with CP 96‐1252, the EI of CP 15‐2516 was significantly (P &lt; .05) higher by 15% (3233 ha−1). The mean cane fiber content of CP 15‐2516 was 138.7 kg Mg−1 and was 1% to 8% higher than the reference cultivars (‘CP 00‐1101’, CP 96‐1252, and ‘CPCL 05‐1201’). CP 15‐2516 flowers intermittently during January in Florida's climate. CP 15‐2516 has a better freeze tolerance than commercial reference cultivars CP 00‐1101 and CP 96‐1252. CP 15‐2516 is expected to positively contribute to the profitability and sustainability of the Florida sugarcane industry.

Structural changes in cell wall pectic polymers contribute to freezing tolerance induced by cold acclimation in plants

Subzero temperatures are often lethal to plants. Many temperate herbaceous plants have a cold acclimation mechanism that allows them to sense a drop in temperature and prepare for freezing stress through accumulation of soluble sugars and cryoprotective proteins. As ice formation primarily occurs in the apoplast (the cell wall space), cell wall functional properties are important for plant freezing tolerance. Although previous studies have shown that the amounts of constituent sugars of the cell wall, in particular those of pectic polysaccharides, are altered by cold acclimation, the significance of this change during cold acclimation has not been clarified. We found that β-1,4-galactan, which forms neutral side chains of the acidic pectic rhamnogalacturonan-I, accumulates in the cell walls of Arabidopsis and various freezing-tolerant vegetables during cold acclimation. The gals1 gals2 gals3 triple mutant, which has reduced β-1,4-galactan in the cell wall, exhibited impaired freezing tolerance compared with wild-type Arabidopsis during initial stages of cold acclimation. Expression of genes involved in the galactan biosynthesis pathway, such as galactan synthases and UDP-glucose 4-epimerases, was induced during cold acclimation in Arabidopsis, explaining the galactan accumulation. Cold acclimation resulted in a decrease in extensibility and an increase in rigidity of the cell wall in the wild type, whereas these changes were not observed in the gals1 gals2 gals3 triple mutant. These results indicate that the accumulation of pectic β-1,4-galactan contributes to acquired freezing tolerance by cold acclimation, likely via changes in cell wall mechanical properties.

Extracellular freezing induces a permeability transition in the inner membrane of muscle mitochondria of freeze-sensitive but not freeze-tolerant Chymomyza costata larvae.

Background: Many insect species have evolved the ability to survive extracellular freezing. The search for the underlying principles of their natural freeze tolerance remains hampered by our poor understanding of the mechanistic nature of freezing damage itself. Objectives: Here, in search of potential primary cellular targets of freezing damage, we compared mitochondrial responses (changes in morphology and physical integrity, respiratory chain protein functionality, and mitochondrial inner membrane (IMM) permeability) in freeze-sensitive vs. freeze-tolerant phenotypes of the larvae of the drosophilid fly, Chymomyza costata. Methods: Larvae were exposed to freezing stress at -30°C for 1h, which is invariably lethal for the freeze-sensitive phenotype but readily survived by the freeze-tolerant phenotype. Immediately after melting, the metabolic activity of muscle cells was assessed by the Alamar Blue assay, the morphology of muscle mitochondria was examined by transmission electron microscopy, and the functionality of the oxidative phosphorylation system was measured by Oxygraph-2K microrespirometry. Results: The muscle mitochondria of freeze-tolerant phenotype larvae remained morphologically and functionally intact after freezing stress. In contrast, most mitochondria of the freeze-sensitive phenotype were swollen, their matrix was diluted and enlarged in volume, and the structure of the IMM cristae was lost. Despite this morphological damage, the electron transfer chain proteins remained partially functional in lethally frozen larvae, still exhibiting strong responses to specific respiratory substrates and transferring electrons to oxygen. However, the coupling of electron transfer to ATP synthesis was severely impaired. Based on these results, we formulated a hypothesis linking the observed mitochondrial swelling to a sudden loss of barrier function of the IMM.

Freezing stress response of wild and cultivated chickpeas.

Chickpea is an economically and nutritionally important grain legume globally, however, cold stress has adverse effects on its growth. In cold countries, like Canada where the growing season is short, having cold stress-tolerant varieties is crucial. Crop wild relatives of chickpea, especially Cicer reticulatum, can survive in suboptimal environments and are an important resource for crop improvement. In this study, we explored the performance of eleven C. reticulatum wild accessions and two chickpea cultivars, CDC Leader and CDC Consul, together with a cold sensitive check ILC533 under freezing stress. Freezing tolerance was scored based on a 1-9 scale. The wild relatives, particularly Kesen_075 and CudiA_152, had higher frost tolerance compared to the cultivars, which all died after frost treatment. We completed transcriptome analysis via mRNA sequencing to assess changes in gene expression in response to freezing stress and identified 6,184 differentially expressed genes (DEGs) in CDC Consul, and 7,842 DEGs in Kesen_075. GO (gene ontology) analysis of the DEGs revealed that those related to stress responses, endogenous and external stimuli responses, secondary metabolite processes, and photosynthesis were significantly over-represented in CDC Consul, while genes related to endogenous stimulus responses and photosynthesis were significantly over-represented in Kesen_075. These results are consistent with Kesen_075 being more tolerant to freezing stress than CDC Consul. Moreover, our data revealed that the expression of CBF pathway-related genes was impacted during freezing conditions in Kesen_075, and expression of these genes is believed to alleviate the damage caused by freezing stress. We identified genomic regions associated with tolerance to freezing stress in an F2 population derived from a cross between CDC Consul and Kesen_075 using QTL-seq analysis. Eight QTLs (P<0.05) on chromosomes Ca3, Ca4, Ca6, Ca7, Ca8, and two QTLs (P<0.01) on chromosomes Ca4 and Ca8, were associated with tolerance to freezing stress. Interestingly, 58 DEGs co-located within these QTLs. To our knowledge, this is the first study to explore the transcriptome and QTLs associated with freezing tolerance in wild relatives of chickpea under controlled conditions. Altogether, these findings provide comprehensive information that aids in understanding the molecular mechanism of chickpea adaptation to freezing stress and further provides functional candidate genes that can assist in breeding of freezing-stress tolerant varieties.

A large-effect fitness trade-off across environments is explained by a single mutation affecting cold acclimation

Identifying the genetic basis of local adaptation and fitness trade-offs across environments is a central goal of evolutionary biology. Cold acclimation is an adaptive plastic response for surviving seasonal freezing, and costs of acclimation may be a general mechanism for fitness trade-offs across environments in temperate zone species. Starting with locally adapted ecotypes of Arabidopsis thaliana from Italy and Sweden, we examined the fitness consequences of a naturally occurring functional polymorphism in CBF2. This gene encodes a transcription factor that is a major regulator of cold-acclimated freezing tolerance and resides within a locus responsible for a genetic trade-off for long-term mean fitness. We estimated the consequences of alternate genotypes of CBF2 on 5-y mean fitness and fitness components at the native field sites by comparing near-isogenic lines with alternate genotypes of CBF2 to their genetic background ecotypes. The effects of CBF2 were validated at the nucleotide level using gene-edited lines in the native genetic backgrounds grown in simulated parental environments. The foreign CBF2 genotype in the local genetic background reduced long-term mean fitness in Sweden by more than 10%, primarily via effects on survival. In Italy, fitness was reduced by more than 20%, primarily via effects on fecundity. At both sites, the effects were temporally variable and much stronger in some years. The gene-edited lines confirmed that CBF2 encodes the causal variant underlying this genetic trade-off. Additionally, we demonstrated a substantial fitness cost of cold acclimation, which has broad implications for potential maladaptive responses to climate change.

Transcriptome, histological, and physiological responses of Amur sleeper (Perccottus glenii) during cold stress, freezing, and recovery

Freeze tolerance is a survival strategy employed by some ectotherms living in extremely cold environments. Some fish in extremely cold areas can recover from their frozen state, but they also have to endure cold stress. Amur sleeper (Perccottus glenii) can recover from a completely frozen state. To explore the response of freeze-resistant fish to low temperatures, we analyzed histological alterations, and antioxidant and carbohydrate-lipid metabolizing enzymes of P. glenii under low temperatures. So far, sensory genes regulating P. glenii during cold stress, freezing, and recovery have not been identified. Ultrastructure results indicated that glycogen content and mitochondrial ridge decreased during cold stress and freezing, whereas the number of endoplasmic reticulum increased during recovery. Plasma glucose and glycerol levels of the three treatment groups significantly increased. Lactate dehydrogenase and pyruvate kinase levels significantly increased during cold stress and freezing, and hexokinase levels significantly increased during cold stress. In total, 30,560 unigenes were found (average length 1724 bp, N50 2843 bp). In addition, 7370 differentially expressed genes (DEGs; including 2938 upregulated genes and 4432 downregulated genes) were identified. KEGG analysis revealed that the DEGs were enriched in carbohydrate and lipid metabolism, lipid synthesis, immune system, and anti-apoptosis. Genes involved in glycolysis and phospholipid metabolism were significantly upregulated during cold stress; genes related to circadian rhythm, oxidative phosphorylation, and lipid synthesis were significantly upregulated during freezing; and genes involved in the immune system and anti-apoptosis were significantly upregulated during recovery. Our results attempt to offer new insights into the physiological mechanisms of complex adaptation in P. glenii and provide useful information for future studies on the mechanism underlying freezing/recovery in animals.

Ionic Liquid/Water Binary Solvent Anti-Freezing Hydrogel for Strain and Temperature Sensors.

Hydrogels are widely applied in the flexible wearable electronic devices field owing to their skin-like stretchability, superb biocompatibility, and high conductivity retention under mechanical deformations. Nevertheless, hydrogels are prone to freezing at low temperatures and losing water at high temperatures, which seriously limits their practical applications. Herein, a binary solvent system of ionic liquid (1-ethyl-3-methylimidazolium chloride) and water was prepared to endow the ionic hydrogel high ionic conductivity (0.28 S m-1 at 25 °C), high transparency (94.26%), and superior freezing tolerance (-50 °C). The multiple hydrogen bonds formed among polymer chains, water, and ionic liquids significantly improved the mechanical properties of the ionic hydrogel, enabling excellent tensile properties (strain >1800%) and durability (1000 times at 100% strain). Moreover, the ionic hydrogel was further assembled into a dual-response sensor, which exhibited satisfactory sensitivity to both tension (gauge factor = 2.15 at 200% strain) and temperature (temperature coefficient of resistance = -1.845%/°C) and can be applied for human motion and body temperature monitoring. This study provides a versatile method for preparing multifunctional hydrogels with a wide range of applications and lays the groundwork for human movement detection and smart health care.

Alkaline Invertase 2 positively modulates cold adaptive of Camellia sinensis and enhances freezing and salt tolerance in transgenic Arabidopsis thaliana

Alkaline/neutral invertase (A/N-Inv) is a type of hydrolases that irreversibly hydrolyze sucrose into glucose and fructose, thus participating in the plant morphogenesis and stress responses. In tea plant, a putative A/N-Inv gene, CsINV2, mediated the abiotic stress responses of tea plant, but the detailed molecular mechanism has not been elucidated yet. In this study, CsINV2 was verified as an alkaline invertase that can split sucrose into glucose and fructose in vitro, which showed the optimal catalytic activity at 45 °C and pH 8 conditions. Besides, the high transcription of CsINV2 contributed to improving the cold adaptation of tea plant, while silencing of CsINV2 reduced cold adaptive of tea plant. Overexpression of CsINV2 improved freezing tolerance of transgenic Arabidopsis by improving A/N-Inv activity, glucose content, hexose/sucrose ratio, and also via a feedback regulation pathway. Meanwhile, overexpression of CsINV2 promoted gemination rate and root elongation of transgenic Arabidopsis under salt stress condition. Finally, a cold-induced transcription factor CsAHL17 was confirmed to interact with the promoter of CsINV2, silencing of CsAHL17 reduced the transcription of CsINV2 both under 25 °C and 4 °C conditions. In summary, it can draw a conclusion that CsINV2 regulates the cold resistance of tea plant by influencing the contents of different soluble sugar components under the regulation of CsAHL17. This study will further promote the understanding to the role of invertase in tea plants.

CsLHY positively regulates cold tolerance by activating CsSWEET17 in tea plants

Low temperature is one of the most important environmental factors limiting tea plants' geographic distribution and severely affects spring tea's yield and quality. Circadian components contribute to plant responses to low temperatures; however, comparatively little is known about these components in tea plants. In this study, we identified a core clock component the LATE ELONGATED HYPOCOTYL, CsLHY, which is mainly expressed in tea plants' mature leaves, flowers, and roots. Notably, CsLHY maintained its circadian rhythmicity of expression in summer, but was disrupted in winter and held a high expression level. Meanwhile, we found that CsLHY expression rhythm was not affected by different photoperiods but was quickly broken by cold, and the low temperature induced and kept CsLHY expression at a relatively high level. Yeast one-hybrid and dual-luciferase assays confirmed that CsLHY can bind to the promoter of Sugars Will Eventually be Exported Transporters 17 (CsSWEET17) and function as a transcriptional activator. Furthermore, suppression of CsLHY expression in tea leaves not only reduced CsSWEET17 expression but also impaired the freezing tolerance of leaves compared to the control. Our results demonstrate that CsLHY plays a positive role in the low-temperature response of tea plants by regulating CsSWEET17 when considered together.