Cold Acclimation Research Articles

PhenoCaB: a new phenological model based on carbon balance in boreal conifers.

Traditional phenological models use chilling and thermal forcing (temperature sum or degree-days) to predict budbreak. Because of the heightening impact of climate and other related biotic or abiotic stressors, a model with greater biological support is needed to better predict budbreak. Here, we present an original mechanistic model based on the physiological processes taking place before and during budbreak of conifers. As a general principle, we assume that phenology is driven by the carbon status of the plant, which is closely related to environmental variables and the annual cycle of dormancy-activity. The carbon balance of a branch was modelled from autumn to winter with cold acclimation and dormancy and from winter to spring when deacclimation and growth resumption occur. After being calibrated in a field experiment, the model was validated across a large area (> 34 000 km2 ), covering multiple conifers stands in Québec (Canada) and across heated plots for the SPRUCE experiment in Minnesota (USA). The model accurately predicted the observed dates of budbreak in both Québec (±3.98 d) and Minnesota (±7.98 d). The site-independent calibration provides interesting insights on the physiological mechanisms underlying the dynamics of dormancy break and the resumption of vegetative growth in spring.

Non-photochemical quenching in natural accessions of Arabidopsis thaliana during cold acclimation

Cold acclimation, initiated by non-freezing low temperatures and light, is a natural strategy for increasing plant survival even at sub-zero temperatures. However, it remains unclear how the non-photochemical quenching processes, which are crucial for excessive light energy dissipation, are modulated during cold acclimation. We compared the effects of two weeks of acclimation to sub-optimal temperatures, at 10 °C (AC10) and 4 °C (AC4), with non-acclimated (NAC) Arabidopsis thaliana natural accessions grown at 21 °C, on their growth (rosette area), biochemistry (chlorophylls and epidermal flavonols), and physiology (CO2 assimilation rate, and quantum yields of photochemical and non-photochemical quenching processes). AC10 reduced rosette area in all (six) accessions, while chlorophylls and CO2 assimilation rate (Asat) decreased only in three accessions and it had no effect on maximum quantum yield (Fv/Fm). However, AC4 significantly decreased rosette area, chlorophylls, and Fv/Fm, in all accessions. Both AC10 and AC4 treatments increased the accumulation of epidermal flavonols in all accessions. In AC4 accessions, we found an increase in additional non-regulatory NPQ, Φf,d, and a decrease in the fraction of excitation energy used by PSII photochemistry, ΦPSII. A similar irradiance resulted in a marginal difference in regulatory NPQ, Φnpq, among NAC and AC10 or AC4 plants; however, AC10 plants have more energy-dependent fastest NPQ, ΦqE, whereas AC4 predominates state transition quenching, ΦqT. These variations in dissipation of absorbed light energy, when combined with reduced chlorophylls and accumulated flavonols, help to reduce the risk of photoinhibition in plants during cold periods. These findings provide new insights into how suboptimal temperature acclimation affects the regulation of NPQ molecular mechanisms in Arabidopsis thaliana natural accessions.

1H, 15N, and 13C backbone and side chain resonance assignments of the cold shock domain of the Arabidopsis thaliana glycine-rich protein AtGRP2.

AtGRP2 (Arabidopsis thaliana glycine-rich protein 2) is a 19-kDa RNA-binding glycine-rich protein that regulates key processes in A. thaliana. AtGRP2 is a nucleo-cytoplasmic protein with preferential expression in developing tissues, such as meristems, carpels, anthers, and embryos. AtGRP2 knockdown leads to an early flowering phenotype. In addition, AtGRP2-silenced plants exhibit a reduced number of stamens and abnormal development of embryos and seeds, suggesting its involvement in plant development. AtGRP2 expression is highly induced by cold and abiotic stresses, such as high salinity. Moreover, AtGRP2 promotes double-stranded DNA/RNA denaturation, indicating its role as an RNA chaperone during cold acclimation. AtGRP2 is composed of an N-terminal cold shock domain (CSD) followed by a C-terminal flexible region containing two CCHC-type zinc fingers interspersed with glycine-rich sequences. Despite its functional relevance in flowering time regulation and cold adaptation, the molecular mechanisms employed by AtGRP2 are largely unknown. To date, there is no structural information regarding AtGRP2 in the literature. Here, we report the 1H, 15N, and 13C backbone and side chain resonance assignments, as well as the chemical shift-derived secondary structure propensities, of the N-terminal cold shock domain of AtGRP2, encompassing residues 1-90. These data provide a framework for AtGRP2-CSD three-dimensional structure, dynamics, and RNA binding specificity investigation, which will shed light on its mechanism of action.

Acquisition of Freezing Tolerance of Resurrection Species from Gesneriaceae, a Comparative Study.

Resurrection plants have the unique ability to restore normal physiological activity after desiccation to an air-dry state. In addition to their desiccation tolerance, some of them, such as Haberlea rhodopensis and Ramonda myconi, are also freezing-tolerant species, as they survive subzero temperatures during winter. Here, we compared the response of the photosynthetic apparatus of two other Gesneriaceae species, Ramonda serbica and Ramonda nathaliae, together with H. rhodopensis, to cold and freezing temperatures. The role of some protective proteins in freezing tolerance was also investigated. The water content of leaves was not affected during cold acclimation but exposure of plants to -10 °C induced dehydration of plants. Freezing stress strongly reduced the quantum yield of PSII photochemistry (Y(II)) and stomatal conductance (gs) on the abaxial leaf side. In addition, the decreased ratio of Fv/Fm suggested photoinhibition or sustained quenching. Freezing-induced desiccation resulted in the inhibition of PSII activity, which was accompanied by increased thermal energy dissipation. In addition, an increase of dehydrins and ELIPs was detected, but the protein pattern differed between species. During recovery, the protein abundance decreased and plants completely recovered their photosynthetic activity. Thus, our results showed that R. serbica, R. nathaliae, and H. rhodopensis survive freezing stress due to some resurrection-linked traits and confirmed their freezing tolerance.

Leaf, Root, and Crown Tissue Physiology of Annual Bluegrass after Cold Acclimation at Varying Soil Moisture Levels and Ice Encasement

Annual bluegrass (Poa annua) is a turfgrass species prone to winterkill-induced damage such as from ice encasement stress. This research aimed to determine whether different levels of soil volumetric water content (SWC) influence cold acclimation and recovery from ice encasement. Annual bluegrass was exposed to 8%, 12%, and 20% SWC treatments during cold acclimation in growth chambers. After cold acclimation, plants were subjected to ice encasement by misting at –3 °C until a 2.5-cm ice layer was formed. On 0 (no ice encasement exposure), 40, and 80 days of treatment, plants were analyzed for recovery (percent green canopy cover over time), and leaf, crown, and root tissues were harvested for lipid peroxidation and total nonstructural carbohydrates (TNC) including storage carbohydrates and water-soluble carbohydrates (WSC). Low SWC during cold acclimation enhanced recovery from cold temperatures and ice encasement. Root carbohydrates were influenced by SWC regimes during cold acclimation since day 0 plant roots exposed to the 8% SWC treatment had 143.9% higher TNC and 137.6% higher WSC compared with day 0 plants exposed to 12% and 20% SWC. Root levels of carbohydrates and lipid peroxidation were least influenced by cold and ice encasement among the organs evaluated. Prolonged freezing conditions and ice encasement reduced leaf and crown tissue carbohydrates and increased lipid peroxidation compared with day 0 plants not exposed to freezing temperatures and ice encasement. After 40 days of ice encasement, plants exposed to the 8% SWC treatment recovered faster than plants cold acclimated at higher soil moisture levels. Average percent canopy cover after 36 days of recovery in the greenhouse was 71.9% higher for 8% SWC treated plants than in 12% and 20% SWC treated plants. Turfgrass managers may benefit from annual bluegrass putting green management strategies to reduce fall soil moisture. Given that soil moisture did not significantly influence carbohydrate or lipid peroxidation results, except for in roots, additional research is needed to understand the mechanism associated with these findings.

1H-NMR analysis reveals cold acclimation induces homeoviscous adaptation in membranes from liver, but not in blood cells or skeletal muscle in the freeze tolerant Cope's gray treefrog

Cope’s gray treefrog Dryophytes chrysoscelis is a seasonally freeze tolerant anuran capable of repeatedly freezing and thawing as much as two-thirds of body water by accumulating and mobilizing cryoprotectants. In cold-acclimated D. chrysoscelis, RBCs are inherently freeze tolerant in vitro, suggesting that membrane composition may contribute to cellular freeze tolerance. The objective of this study is to compare the effects of cold acclimation on lipid profiles of membranes from liver, muscle, and blood cells, to assess the role of membrane composition in cellular and organismal freeze tolerance. It is hypothesized that membrane biochemical composition is tissue-specific and is sensitive to environmental fluctuation. Membranes were prepared from liver (N=7), skeletal muscle (N=7), and blood cells (WBCs, RBCs, and platelets) (N=5) collected from wild-caught male treefrogs that were either cold-acclimated (5°C, 8 hrs daylight) or maintained in warm conditions (22°C, 12 hrs daylight). Fatty acid characterization and cholesterol and triacylglycerol (TAG) content were evaluated using 1 H-NMR spectra collected in a 400 MHz Bruker Avance spectrometer. Principal component analysis demonstrated that membrane lipid profiles were tissue-specific, but distinct warm and cold profiles were detected only in liver. Fatty acids in liver membranes did not vary with acclimation state in chain length ( P=0.46), but total unsaturation was nearly 2-fold greater in cold compared with warm frogs ( P<0.05). Monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids were elevated by 1.7-fold in cold frogs ( P<0.05). Of the PUFA lipid species assessed, linoleic acid (LA) was more than doubled in cold frogs ( P<0.05), while other Ω-3 and Ω-6 fatty acids did not vary between groups ( P>0.5). TAG, free cholesterol, and esterified cholesterol were elevated by 2.5-fold, 2-fold, and 1.4-fold, respectively, in cold frogs (P<0.05), while free cholesterol did not vary between groups ( P=0.38). In membranes from muscle and blood cells, neither fatty acid characterization nor TAG and cholesterol varied between warm and cold frogs, but trends suggested that MUFA ( P=0.10) and LA ( P=0.06) content in blood cells may be elevated in warm frogs. Overall, liver membranes are remodeled during cold acclimation, supporting the hypothesis that membrane composition is adapted to maintain cell function and integrity (i.e. homeoviscous adaptation). Specifically, elevated fatty acid unsaturation and cholesterol in liver likely contribute to cellular freeze tolerance and may support the central role of liver in mobilizing metabolites and cryoprotectants during cold acclimation. However, the absence of such changes (or even changes in the opposite direction) in muscle and blood cells indicates that additional environmental cues, such as exposure to freezing conditions, may be required to induce remodeling and/or that membrane lipid composition does not contribute to freeze tolerance in some tissues. This study was supported by a UD STEM Catalyst Grant awarded to CMK and JE, a Graduate Student Summer Fellowship and Dittrich-Spotila Research Fellowship awarded to EEY, and an NSF-MRI Grant awarded to JE and CMK that was supported by the Ohio Action Fund. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of 5-HT on the cold resistance of mangrove Kandelia obovata seedlings.

5-hydroxytryptamine (5-HT) participates in plant growth and development, and can also delay senescence and cope with abiotic stress. To explore the role of 5-HT in regulating the abilities of mangrove in cold resis-tance, we examined the effects of cold acclimation and the spraying of p-chlorophenylalanine (p-CPA, 5-HT synthesis inhibitor) on leaf gas exchange parameters and CO2 response curves (A/Ca), as well as the endogenous phytohormone content levels in the mangrove species Kandelia obovata seedlings under low temperature stress. The results showed that low temperature stress significantly reduced the contents of 5-HT, chlorophyll, endogenous auxin (IAA), gibberellin (GA), and abscisic acid (ABA). It weakened the CO2 utilization abilities of plants and reduced net photosynthetic rate, which ultimately reduced carboxylation efficiency (CE). Under low temperature stress, exogenous p-CPA reduced the contents of photosynthetic pigments, endogenous hormones, and 5-HT in the leaves, which aggravated the damages caused by low temperature stress on photosynthesis. By enhancing cold acclimation abilities, the endogenous IAA content in the leaves could was reduced under low temperature stress, promoted the production of 5-HT, improved the contents of photosynthetic pigments, GA, and ABA, as well as enhanced photosynthetic carbon assimilation abilities, which would increase photosynthesis in the K. obovata seedlings. Under cold acclimation conditions, the spraying of p-CPA could significantly inhibit the synthesis of 5-HT, promote the production of IAA, and reduce the contents of photosynthetic pigments, GA, ABA, and CE, which would weaken the effects of cold acclimation by improving the cold resistance of mangroves. In conclusion, cold acclimation could improve the cold resistance abilities of K. obovata seedlings by regulating photosynthetic carbon assimilation capacity and the contents of endogenous phytohormone. 5-HT synthesis is one of the necessary conditions for improving the cold resistance abilities of mangroves.

Effect of Photoperiod Duration on Efficiency of Low-Temperature Hardening of <i>Arabidopsis thaliana</i> Heynh. (L.)

The effect of photoperiod duration on efficiency of low-temperature hardening was investigated in Arabidopsis thaliana (L.) Heynh. plants, ecotype Col-0. Six-week-old plants were exposed to cold acclimation at a temperature of 2С during 1‒5 days at photoperiods of 0, 8, and 16 h (illuminance of 200 mol/(m2 s)). According to survival data and leakage of electrolytes after test freezing (6C, 24 h), the plants exposed to cold acclimation in the dark did not show frost resistance. The plants hardened in the light (irrespective of the length of photoperiod) considerably improved their frost resistance by the end of the cold-acclimation period. Net photosynthesis/dark respiration ratio in these plants was almost two times greater than in control material (without hardening). The plants exposed to a 16-h-long photoperiod surpassed the type of treatment with 8-h-long illumination both in the highest levels of accumulation of sugars (by almost 40%) and in the rate of reaching these levels in daily dynamics of hardening. It was shown that MDA content transiently rose during the first 24 h of hardening in the light and did not change in the dark, which may point to a signal role of lipid peroxidation products upon cold acclimation. It was discovered that the photoperiod duration affected the formation rate of frost resistance in A. thaliana plants. A more prolonged operation of A. thalianas photosynthetic apparatus at 16-h-long photoperiod considerably accelerated the accumulation of sugars upon cold acclimation and, therefore, hastened development of frost resistance as compared with an 8-h-long photoperiod. It was concluded that rapid formation of frost resistance in A. thaliana requires a combination of low above-zero temperature and 16-h-long photoperiod.

Multiple inducible thermogenic mechanisms in the development of cold acclimatization

Abstract Extreme cold environment can threaten human health and life through increasing the risk of myocardial infarction, stroke, frostbite, and hypothermia. Insufficient heat production to maintain core body temperature is a major cause of cold injury. To cope with cold stress, human and other mammals have developed the capacity of cold acclimatization to adapt to such a harsh environment. Adaptive non-shivering thermogenesis is a ubiquitous form of cold acclimatization. This review article systematically summarizes the role of three inducible thermogenic forms, including food intake, circadian rhythms, and cold exposure in mediating non-shivering thermogenesis under cold exposure and presents the potential interventions for minimizing the adverse health consequences of cold temperature.

Karrikin increases tomato cold tolerance via strigolactone and the abscisic acid signaling network

As a class of biostimulants, karrikins (KARs) were first identified from plant-derived smoke to regulate plant growth, development, and stress tolerance. However, the roles of KARs in plant cold tolerance and their crosstalk with strigolactones (SLs) and abscisic acid (ABA) remain elusive. We studied the interaction among KAR, SLs, and ABA in cold acclimatization with KAI2-, MAX1-, SnRK2.5-silenced, or cosilenced plant materials. KAI2 is involved in smoke-water- (SW-) and KAR-mediated cold tolerance. MAX1 acts downstream of KAR in cold acclimation. ABA biosynthesis and sensitivity are regulated by KAR and SLs, which improve cold acclimation through the SnRK2.5 component. The physiological mechanisms of SW and KAR in improving growth, yield, and tolerance under a long-term sublow temperature environment were also studied. SW and KAR were shown to improve tomato growth and yield under sublow temperature conditions by regulating nutritional uptake, leaf temperature control, photosynthetic defense, ROS scavenging, and CBF transcriptional activation. Together, SW, which functions via the KAR-mediated SL and ABA signaling network, has potential application value for increasing cold tolerance in tomato production.

Acute cold stress and supercooling capacity of Mediterranean fruit fly populations across the Northern Hemisphere (Middle East and Europe)

The Mediterranean fruit fly, Ceratitis capitata (Diptera: Tephritidae), holds an impressive record of successful invasion events promoted by globalization in fruit trade and human mobility. In addition, C. capitata is gradually expanding its geographic distribution to cooler temperate areas of the Northern Hemisphere. Cold tolerance of C. capitata seems to be a crucial feature that promotes population establishment and hence invasion success. To elucidate the interplay between the invasion process in the northern hemisphere and cold tolerance of geographically isolated populations of C. capitata, we determined (a) the response to acute cold stress survival of adults, and (b) the supercooling capacity (SCP) of immature stages and adults. To assess the phenotypic plasticity in these populations, the effect of acclimation to low temperatures on acute cold stress survival in adults was also examined. The results revealed that survival after acute cold stress was positively related to low temperature acclimation, except for females originating from Thessaloniki (northern Greece). Adults from the warmer environment of South Arava (Israel) were less tolerant after acute cold stress compared with those from Heraklion (Crete, Greece) and Thessaloniki. Plastic responses to cold acclimation were population specific, with the South Arava population being more plastic compared to the two Greek populations. For SCP, the results revealed that there is little to no correlation between SCP and climate variables of the areas where C. capitata populations originated. SCP was much lower than the lowest temperature individuals are likely to experience in their respective habitats. These results set the stage for asking questions regarding the evolutionary adaptive processes that facilitate range expansions of C. capitata into cooler temperate areas of Europe.

Screening Cold Tolerance in St. Augustinegrass (Stenotaphrum secundatum (Walt.) Kuntze) for USDA Hardiness Zone 7

Among warm-season turfgrasses, St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] generally has poor cold tolerance yet excellent shade tolerance. As mostly hot summers follow cold winters in USDA Hardiness Zone 7, severely damaging tall fescue [Festuca arundineacea Schreb.] and centipedegrass [Eremochloa ophiuroides (Munro) Hack.], a St. Augustinegrass cultivar cold tolerant enough to be grown for shady lawns would greatly benefit both home owners and sod growers in this and potentially other regions. Eight St. Augustinegrass samples were selected from an initial collection of 30, including industry standards ‘Raleigh’ and ‘Palmetto’, for further testing from an established germplasm collection of material collected from lawns grown in USDA Hardiness Zone 7. Percent regrowth was calculated 4 weeks after samples underwent a 2 week cold acclimation process, followed by freezing at 0°C, -4°C, and -6°C separately for 3 h. The germplasm samples designated at ‘A’, ‘G’, and ‘H’ had greater clipping regrowth percentages compared to the industry cold tolerant standard, ‘Raleigh’ at either -4°C and -6°C indicating possible increased cold tolerance for several selections compared to ‘Raleigh’. An increased cold tolerant St. Augustinegrass would provide a wider selection of desirable turfgrasses in the transition zone growing region of the world, especially shady sites.

CsFAD2 and CsFAD5 are key genes for C18:2 fatty acid pathway-mediated cold tolerance in tea (Camellia sinensis)

Low temperature is a limiting environmental factor that impairs the growth, yield and quality of tea plants. Fatty acids desaturations (FADs) play a key role in mediating membrane fluidity in response to various abiotic stresses; however, the intrinsic mechanisms underlying FAD-mediated alleviation of cold stress in tea plants are poorly understood. In this study, cold stress exposure significantly increased the content of C18:2 (linoleic acid) in two tea varieties contrasting in cold tolerance (cold-tolerant Longjin43 and cold-sensitive Huangjinye). Three different cDNA sequences, designated CsFAD2, CsFAD5 and CsFAD6, encoding ω-6 FADs that are involved in the response to cold stress were isolated from tea. Yeast experiments further demonstrated that CsFAD2, CsFAD5 and CsFAD6 can convert C18:1 to C18:2. Ectopic expression of CsFAD2, CsFAD5 and CsFAD6 in Arabidopsis seedlings and plants enhanced cold tolerance in pot experiments. Interestingly, CsFAD2-or CsFAD5-silenced tea leaves were more sensitive to cold acclimation than CsFAD6-silenced leaves, and CsFAD2/5-silenced leaves affected cold tolerance more than CsFAD2/6- or CsFAD5/6 -silenced leaves, suggesting that CsFAD2 and CsFAD5 were more important than CsFAD6 in cold acclimation-dependent cold tolerance. Taken together, our results suggest that C18:2 is an important component associated with cold stress and CsFAD2 and CsFAD5 contribute more than CsFAD6 to cold tolerance.

Peruvian Amaranth (kiwicha) Accumulates Higher Levels of the Unsaturated Linoleic Acid

Grain amaranth (Amaranthus spp.) is an emerging crop rich in proteins and other valuable nutrients. It was domesticated twice, in Mexico and Peru. Although global trade is dominated by Mexican species of amaranth, Peruvian amaranth (A. caudatus, kiwicha) has remained neglected, although it harbours valuable traits. In the current study, we investigate the accumulation of polyunsaturated fatty acids, comparing four genotypes of A. caudatus with K432, a commercial variety deriving from the Mexican species A. hypochondriacus under the temperate environment of Southwest Germany. We show that the A. caudatus genotypes flowered later (only in late autumn), such that they were taller as compared to the Mexican hybrid but yielded fewer grains. The oil of kiwicha showed a significantly higher content of unsaturated fatty acids, especially of linoleic acid and α-linolenic acid compared to early flowering genotype K432. To gain insight into the molecular mechanisms behind these differences, we sequenced the genomes of the A. hypochondriacus × hybridus variety K432 and the Peruvian kiwicha genotype 8300 and identified the homologues for genes involved in the ω3 fatty-acid pathway and concurrent oxylipin metabolism, as well as of key factors for jasmonate signalling and cold acclimation. We followed the expression of these transcripts over three stages of seed development in all five genotypes. We find that transcripts for Δ6 desaturases are elevated in kiwicha, whereas in the Mexican hybrid, the concurrent lipoxygenase is more active, which is followed by the activation of jasmonate biosynthesis and signalling. The early accumulation of transcripts involved in cold-stress signalling reports that the Mexican hybrid experiences cold stress already early in autumn, whereas the kiwicha genotypes do not display indications for cold stress, except for the very final phase, when there were already freezing temperatures. We interpret the higher content of unsaturated fatty acids in the context of the different climatic conditions shaping domestication (tropical conditions in the case of Mexican amaranth, sharp cold snaps in the case of kiwicha) and suggest that kiwicha oil has high potential as functional food which can be developed further by tailoring genetic backgrounds, agricultural practice, and processing.

Arabidopsis LFR, a SWI/SNF complex component, interacts with ICE1 and activates ICE1 and CBF3 expression in cold acclimation.

Low temperatures restrict the growth and geographic distribution of plants, as well as crop yields. Appropriate transcriptional regulation is critical for cold acclimation in plants. In this study, we found that the mutation of Leaf and flower related (LFR), a component of SWI/SNF chromatin remodeling complex (CRC) important for transcriptional regulation in Arabidopsis (Arabidopsis thaliana), resulted in hypersensitivity to freezing stress in plants with or without cold acclimation, and this defect was successfully complemented by LFR. The expression levels of CBFs and COR genes in cold-treated lfr-1 mutant plants were lower than those in wild-type plants. Furthermore, LFR was found to interact directly with ICE1 in yeast and plants. Consistent with this, LFR was able to directly bind to the promoter region of CBF3, a direct target of ICE1. LFR was also able to bind to ICE1 chromatin and was required for ICE1 transcription. Together, these results demonstrate that LFR interacts directly with ICE1 and activates ICE1 and CBF3 gene expression in response to cold stress. Our work enhances our understanding of the epigenetic regulation of cold responses in plants.

Study on tillering stage cold tolerant response in overwintering cultivated rice via comparative transcriptomic

AbstractRice adaptation to low temperatures involves changes in their genetic and physiological architecture, caused by complex and variable transcriptional regulatory networks to regulate between growth and stress resistance. Overwintering rice survives cold winter and germinates the following spring. The perennial and cold‐tolerant properties present great potential regarding rice breeding and food security. In this study, the cold‐tolerant overwintering cultivar rice, “glutinous rice 89‐1” (Gr 89‐1), and an indica cultivar rice, “R07,” were placed at 4°C at the tillering stage and sampled at 0, 12, 24, and 96 h, and recovery 7 days. Physiological indicators of each sample were measured, and transcriptome analysis was performed. The results revealed that the overwintering cultivar rice Gr 89‐1 exhibited a pattern of physiological parameters consistent with the cold‐tolerant phenotype, less membrane damage accumulation and water loss with increasing duration of low temperature, and a stable increase in peroxidase activities, catalase activities, and soluble sugar content. Transcriptome analysis of two cultivars at 0, 12, and 24 h under 4°C revealed different gene expression patterns to low temperatures. Compared with R07, gene ontology terms of nuclear lumen, endomembrane system, organelle membrane, and envelope were enriched in Gr 89‐1, suggesting an alteration of membrane composition during cold acclimation. Specific upregulated transcription factors and cold‐tolerance regulators indicated a coordinated response between signal transduction, energy regulation, cell cycle, and circadian rhythm, which might help maintain cell homeostasis and improve Gr 89‐1 survival under cold stress. Two candidate genes OsETR3 and NAC78 were screened out, they significantly upregulated at 12 and 24 h of cold treatment in Gr 89‐1 and were higher than R07, and they were involved in cell wall remodeling and endoplasmic reticulum homeostasis under stress, respectively, which may relate to the cold‐tolerant trait of Gr 89‐1.

Evolutionary footprints of cold adaptation in arctic-alpine Cochlearia (Brassicaceae) – Evidence from freezing experiments and electrolyte leakage

As global warming progresses, plants may be forced to adapt to drastically changing environmental conditions. Arctic-alpine plants have been among the first to experience the effects of climate change. As a result, cold acclimation and freezing tolerance may become increasingly crucial for the survival as winter warming events and earlier snowmelt will cause increased exposure to occasional frost. The tribe Cochlearieae in the mustard family (Brassicaceae) offers an instructive system for studying cold adaptation in evolutionary terms, as the two sister genera Ionopsidium and Cochlearia are distributed among different ecological habitats throughout the European continent and the far north into circumarctic regions. By applying an electrolyte leakage assay to leaves obtained from plants cultivated under controlled temperature regimes in growth chambers, the freezing tolerance of different Ionopsidium and Cochlearia species was assessed measuring lethal freezing temperature values (LT50 and LT100), thereby allowing for a comparison across different species and accessions in their responses to cold. We hypothesized that, owing to varying selection pressures, geographically distant species would differ in freezing tolerance. Despite Ionopsidium occurring under warm and dry Mediterranean conditions and Cochlearia species distributed often at cold habitats, all accessions exhibited similar cold responses. The results may indicate that physiological adaptations of primary metabolic pathways to different stressors, such as salinity and drought, may confer an additional tolerance to cold; this is because all these stressors induce osmotic challenges.

De Novo Transcriptome Assembly and Comparative Analysis of Differentially Expressed Genes Involved in Cold Acclimation and Freezing Tolerance of the Arctic Moss Aulacomnium turgidum (Wahlenb.) Schwaegr.

Cold acclimation refers to a phenomenon in which plants become more tolerant to freezing after exposure to non-lethal low temperatures. Aulacomnium turgidum (Wahlenb.) Schwaegr is a moss found in the Arctic that can be used to study the freezing tolerance of bryophytes. To improve our understanding of the cold acclimation effect on the freezing tolerance of A. turgidum, we compared the electrolyte leakage of protonema grown at 25 °C (non-acclimation; NA) and at 4 °C (cold acclimation; CA). Freezing damage was significantly lower in CA plants frozen at -12 °C (CA-12) than in NA plants frozen at -12 °C (NA-12). During recovery at 25 °C, CA-12 demonstrated a more rapid and greater level of the maximum photochemical efficiency of photosystem II than NA-12, indicating a greater recovery capacity for CA-12 compared to NA-12. For the comparative analysis of the transcriptome between NA-12 and CA-12, six cDNA libraries were constructed in triplicate, and RNA-seq reads were assembled into 45,796 unigenes. The differential gene expression analysis showed that a significant number of AP2 transcription factor genes and pentatricopeptide repeat protein-coding genes related to abiotic stress and the sugar metabolism pathway were upregulated in CA-12. Furthermore, starch and maltose concentrations increased in CA-12, suggesting that cold acclimation increases freezing tolerance and protects photosynthetic efficiency through the accumulation of starch and maltose in A. turgidum. A de novo assembled transcriptome can be used to explore genetic sources in non-model organisms.

Homozygosity mapping identified loci and candidate genes responsible for freezing tolerance in Camelina sativa.

Homozygosity mapping is an effective tool for detecting genomic regions responsible for a given trait when the phenotype is controlled by a limited number of dominant or co-dominant loci. Freezing tolerance is a major attribute in agricultural crops such as camelina. Previous studies indicated that freezing tolerance differences between a tolerant (Joelle) and susceptible (CO46) variety of camelina were controlled by a small number of dominant or co-dominant genes. We performed whole genome homozygosity mapping to identify markers and candidate genes responsible for freezing tolerance difference between these two genotypes. A total of 28 F3 RILs were sequenced to ∼30× coverage, and parental lines were sequenced to >30-40× coverage with Pacific Biosciences high fidelity technology and 60× coverage using Illumina whole genome sequencing. Overall, about 126k homozygous single nucleotide polymorphism markers were identified that differentiate both parents. Moreover, 617 markers were also homozygous in F3 families fixed for freezing tolerance/susceptibility. All these markers mapped to two contigs forming a contiguous stretch of chromosome 11. The homozygosity mapping detected 9 homozygous blocks among the selected markers and 22 candidate genes with strong similarity to regions in or near the homozygous blocks. Two such genes were differentially expressed during cold acclimation in camelina. The largest block contained a cold-regulated plant thionin and a putative rotamase cyclophilin 2 gene previously associated with freezing resistance in arabidopsis (Arabidopsis thaliana). The second largest block contains several cysteine-rich RLK genes and a cold-regulated receptor serine/threonine kinase gene. We hypothesize that one or more of these genes may be primarily responsible for freezing tolerance differences in camelina varieties.

Insight into Hormonal Homeostasis and the Accumulation of Selected Heat Shock Proteins in Cold Acclimated and Deacclimated Winter Oilseed Rape (Brassica napus L.)

The aim of the current work was to characterize disturbances in the hormonal balance and changes in the accumulation of the protective heat shock proteins (HSP) as a result of deacclimation in a few cultivars of oilseed rape. Samples for both analyses were collected from plants that had not been acclimated (before cold acclimation—control), cold acclimated (at 4 °C d/n, three weeks) and then deacclimated at 16/9 °C d/n (one week). The tested hormones included abscisic acid, jasmonic acid, salicylic acid, gibberellins, auxins and cytokinins (including their precursors, intermediates and conjugates). Unambiguous results were obtained for a stress hormone, abscisic acid, whose concentration increased in the leaves of all of the tested cultivars during cold acclimation while it strongly decreased during deacclimation. Deacclimation resulted also in an elevated level of the typical growth hormones. As a result of cold acclimation, the accumulation of protective proteins such as cytoplasmic HSP70 and HSP90 increased in three of the four tested cultivars. The HSP content most often decreased in the deacclimated plants compared to the cold-acclimated plants. The hormonal and protein changes are discussed relative to the frost tolerance changes of the tested cultivar.