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Autumn sown small seedlings for later transplanting into large containers have been introduced in Swedish forest tree nurseries. Containerized transplants of Norway spruce (Picea abies (L.) Karst.) from three Swedish nurseries were frozen stored during the autumn of 2014 to find out storability and post-storage vitality. Seedling storability was determined by measuring electrolyte leakage after freezing shoots to −25°C (SELdiff−25), by measurements of dry matter content (DMC) of seedling shoots and by the commercial molecular test ColdNSure™. Vitality of seedlings after storage was determined by measuring the leakage of electrolytes from shoots (SEL), and seedlings were also tested in regrowth tests. All three methods for storability assessment gave similar predictions, except in one case where DMC showed "not storable" for successfully stored seedlings. Our results indicated that young transplants can be successfully short term stored before reaching the target levels for safe long-term storage of conventional seedlings. Early storage of young transplants resulted in low post-storage survival and vitality expressed as root growth capacity and shoot electrolyte leakage (SEL). A prolonged duration in storage generally resulted in lower survival as well as lower root growth capacity and higher levels of SEL, especially for seedlings stored at earlier dates. [ABSTRACT FROM AUTHOR]

In plants, most DREB1s/CBFs proteins with a single AP2/ERF domain participate in responses to abiotic stresses. Here, we cloned a DREB subfamily gene from Lilium longiflorum (designated as LlDREB1G). Sequence analyses revealed that it is composed of 219 amino acids, and contains an AP2/ERF domain, a putative nuclear localization signal (NLS), a DSAW/R and LWSY motif. LlDREB1G, which was localized to the nucleus, showed strongly induced expression under low temperature, and was also stimulated by mechanical agitation, heat shock, ABA and H2O2 stresses. Moreover, the LlDREB1G gene had an obvious circadian rhythm and organ specificity in expression. The LlDREB1G transcription factor was functional in the heterologous system (Arabidopsis). Transgenic Arabidopsis plants with higher transcription of LlDREB1G gene exhibited increased acquired thermotolerance and resistance to freezing and dehydration stresses. Besides, the transgenic seeds were insensitive to exogenous ABA treatment than those of the wild type (WT) in germination experiments. Compared with the WT seedlings, the seedlings of transgenic Arabidopsis overexpressing LlDREB1G showed strongly upregulated expression of stress-related genes after cold, drought or acclimation to heat shock treatments. Furthermore, overexpression of LlDREB1G increased the proline levels and survival rates, but decreased the electrolyte leakage and H2O2 accumulation in the transgenic plants after freezing, dehydration and high temperature treatments. These results imply that LlDREB1G gene may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner. Key message: LlDREB1G gene, a DREB subfamily gene from Lilium longiflorum, may be involved in the responses to multiple abiotic stresses in an ABA-dependent and -independent manner. [ABSTRACT FROM AUTHOR]

Temperate plants are at risk of being exposed to late spring freezes. These freeze events—often called false springs—are one of the strongest factors determining temperate plants species range limits and can impose high ecological and economic damage. As climate change may alter the prevalence and severity of false springs, our ability to forecast such events has become more critical, and it has led to a growing body of research. Many false spring studies largely simplify the myriad complexities involved in assessing false spring risks and damage. While these studies have helped advance the field and may provide useful estimates at large scales, studies at the individual to community levels must integrate more complexity for accurate predictions of plant damage from late spring freezes. Here, we review current metrics of false spring, and how, when, and where plants are most at risk of freeze damage. We highlight how life stage, functional group, species differences in morphology and phenology, and regional climatic differences contribute to the damage potential of false springs. More studies aimed at understanding relationships among species tolerance and avoidance strategies, climatic regimes, and the environmental cues that underlie spring phenology would improve predictions at all biological levels. An integrated approach to assessing past and future spring freeze damage would provide novel insights into fundamental plant biology and offer more robust predictions as climate change progresses, which are essential for mitigating the adverse ecological and economic effects of false springs. [ABSTRACT FROM AUTHOR]

• Low elevation ecotype is more sensitive to the short-day in the 1st stage of cold acclimation. • High elevation ecotype is more leaf-hardy than low elevation ecotype in winter. • Low elevation ecotype employed xanthophyll cycle more robustly from summer to winter. • High elevation ecotype is more photoinhibited than low elevation ecotype in winter. • Violaxanthin de-epoxidation in R. catawbiense may not be transcriptionally regulated. For winter survival, evergreens not only need cold acclimation (CA), but also photoprotection. This study was conducted to examine how two elevational ecotypes of Rhododendron catawbiense respond to cold and light stresses in a common garden. Seasonal changes in leaf freezing tolerance (LFT), photosystem II efficiency, photosynthesis, carotenoids (xanthophyll cycle pigments, lutein, beta-carotene, neoxanthin) were investigated from summer to winter. Parallel changes in the expression of violaxanthin de-epoxidase (VDE) gene were examined to explore its association with violaxanthin de-epoxidation. High elevation (HE) ecotype attained overall greater LFT while low elevation (LE) ecotype was more responsive to short-days for the first stage of CA. In summer, both ecotypes predominantly employed reversible xanthophyll cycle (RXC) to dissipate excess light energy, but LE used it more robustly. In autumn, only HE exhibited slight damage to the reaction centers (RCs), i.e. photosystem II degradation (PSD). In winter, both ecotypes suspended photosynthesis, were photoinhibited, and employed primarily sustained de-epoxidation (SD) for photoprotection; but LE still used RXC more effectively than HE. Overall, LE employed xanthophyll cycle more robustly in summer or winter. Results suggest that microclimates of R. catawbiense ecotypes inform their differential physiological response to cold and light stresses. VDE expression data indicate violaxanthin de-epoxidation in R. catawbiense may not be solely transcriptionally regulated. [ABSTRACT FROM AUTHOR]

Premise: Physiological responses to temperature extremes are considered strong drivers of species' demographic responses to climate variability. Plants are typically classified as either avoiders or tolerators in their freezing‐resistance mechanism, but a gradient of physiological‐threshold freezing responses may exist among individuals of a species. Moreover, adaptive significance of physiological freezing responses is poorly characterized, particularly under warming conditions that relax selection on cold hardiness. Methods: Freezing responses were measured in winter and again for new foliage in spring for 14 populations of Artemisia tridentata collected throughout its range and planted in a warm common garden. The relationships of the freezing responses to survival were evaluated in the warm garden and in two colder gardens. Results: Winter and spring freezing resistance were not correlated and appeared to be under differing selection regimes, as evident in correlations with different population climate of origin variables. All populations resisted considerably lower temperatures in winter than in spring, with populations from more continental climates showing narrower freezing safety margins (difference in temperatures at which ice‐nucleation occurs and 50% reduction in chlorophyll fluorescence occurs) in spring. Populations with greater winter freezing resistance had lower survivorship in the warmest garden, while populations with greater spring freezing resistance had lower survivorship in a colder garden. Conclusions: These survivorship patterns relative to physiological thresholds suggest excess freezing resistance may incur a survival cost that likely relates to a trade‐off between carbon gain and freezing resistance during critical periods of moisture availability. This cost has implications for seed moved from cooler to warmer environments and for plants growing in warming environments. [ABSTRACT FROM AUTHOR]

Winter wheat (Triticum aestivum L.) is seeded in the autumn and harvested the following summer, and therefore, must survive multiple episodes of subfreezing temperatures throughout the winter months. Cellular membrane stability following exposure to subfreezing temperatures contributes to the ability to survive these episodes. This study investigated the inheritance of the ability to tolerate subfreezing temperatures with a seven‐parent diallel cross analysis of cellular membrane integrity as measured by electrolyte leakage after exposure to −10 or −14 °C. Significant differences in membrane stability were found among the parent lines. The inheritance of the freezing tolerance as measured by electrolyte leakage was complex and characterized by significant additive, dominant and cytoplasmic effects. General combining ability, indicative of additive genetic effects, were significant at both test temperatures, but accounted for 25.5% of the variance at the −10° C test temperature, and only 4% of the variance at the −14 °C test temperature. Specific combining ability, indicative of genetic dominance effects, were significant at both test temperatures, but accounted for only 14.6% of the variance at the −10 °C test temperature, and 38% of the variance at the −14 °C test temperature. Reciprocal (cytoplasmic) effects were significant and accounted for about 20% of the variance at both test temperatures. Cytoplasmic effects contributing to greater membrane stability were especially apparent in the cultivar Tiber when crossed to Masami, Lewjain, or Hatton. These results suggest that efforts to improve freezing tolerance are complicated by differing gene action at different test temperatures and also may benefit from identifying specific combinations of nuclear and cytoplasm sources that are most conducive to membrane stability following freezing. [ABSTRACT FROM AUTHOR]

Plant tolerance to freezing temperatures is governed by endogenous components and environmental factors. Exposure to low non-freezing temperatures is a key factor in the induction of freezing tolerance in the process called cold acclimation. The role of nitric oxide (NO) in cold acclimation was explored in Arabidopsis using triple nia1nia2noa1-2 mutants that are impaired in the nitrate-dependent and nitrate-independent pathways of NO production, and are thus NO deficient. Here, we demonstrate that cold-induced NO accumulation is required to promote the full cold acclimation response through C-repeat Binding Factor (CBF)-dependent gene expression, as well as the CBF-independent expression of other cold-responsive genes such as Oxidation-Related Zinc Finger 2 (ZF/OZF2). NO deficiency also altered abscisic acid perception and signaling and the cold-induced production of anthocyanins, which are additional factors involved in cold acclimation. [ABSTRACT FROM AUTHOR]

Abstract Freezing stress is one of the most important limiting factors of plant survival. Plants have developed a freezing adaptation mechanism upon sensing low temperatures (cold acclimation). Compositional changes in the plasma membrane, one of the initial sites of freezing injury, is prerequisite of achieving cold acclimation and have been investigated in several plant species. Conversely, the cold dehardening process at elevated temperatures (de-acclimation) has not yet been fully characterized and few studies have addressed the importance of the plasma membrane in the de-acclimation process. In the present study, we conducted shotgun proteomics with label-free semiquantification on plasma membrane fractions of Arabidopsis leaves during cold acclimation and de-acclimation. We consequently obtained a list of 873 proteins with significantly changed proteins in response to the two processes. Although the cold-acclimation-responsive proteins were globally returned to non-acclimated levels by de-acclimation, several representative cold-acclimation-responsive proteins tended to remain at higher abundance during de-acclimation process. Taken together, our results suggest plants deharden right after cold acclimation to restart growth and development but some cold-acclimation-induced changes of the plasma membrane may be maintained under de-acclimation to cope with the threat of sudden freezing during de-acclimation process. Significance Plant freezing tolerance can be enhanced by low temperature treatment (cold acclimation), while elevated temperatures right after cold acclimation can result in the dehardening of freezing tolerance (de-acclimation). However, the de-acclimation process, particularly its relevance to the plasma membrane as the primary site of freezing injury, has not been elucidated. In the present study, a comprehensive proteomic analysis of the plasma membrane during cold acclimation and de-acclimation was carried out as a first step to elucidating how plants respond to rising temperatures. Cold acclimation induced a number of proteomic changes as reported in previous studies, but most proteins, in general, immediately returned to NA levels during de-acclimation treatment for two days. However, the abundances of stress-related proteins (e.g. LTI29, COR78 and TIL) decreased slower than other functional proteins during de-acclimation. Therefore, plants harden during cold acclimation by aborting growth and development and accumulating stress-responsive proteins but seem to deharden quickly under subsequent elevated temperature to resume these processes while guarding against the threat of sudden temperature drops. Graphical abstract Unlabelled Image Highlights • Freezing tolerance of Arabidopsis is reversible in cold-acclimation (CA) and de-acclimation (DA). • Plasma membrane (PM) proteomics changes in DA is correlated with freezing tolerance loss. • Some stress-responsive PM proteins kept high abundance in DA period. • Most of DA changed PM proteins were affected by CA treatment. [ABSTRACT FROM AUTHOR]

Low-temperature related abiotic stress is an important factor affecting winter survival in lowland switchgrass when grown in northern latitudes in the United States. A better understanding of the genetic architecture of freezing tolerance in switchgrass will aid the development of lowland switchgrass cultivars with improved winter survival. The objectives of this study were to conduct a freezing tolerance assessment, generate a genetic map using single nucleotide polymorphism (SNP) markers, and identify QTL (quantitative trait loci) associated with freezing tolerance in a lowland × upland switchgrass population. A pseudo-F2 mapping population was generated from an initial cross between the lowland population Ellsworth and the upland cultivar Summer. The segregating progenies were screened for freezing tolerance in a controlled-environment facility. Two clonal replicates of each genotype were tested at six different treatment temperatures ranging from −15 to −5°C at an interval of 2°C for two time periods. Tiller emergence (days) and tiller number were recorded following the recovery of each genotype with the hypothesis that upland genotype is the source for higher tiller number and early tiller emergence. Survivorship of the pseudo-F2 population ranged from 89% at −5°C to 5% at −15°C with an average LT50 of −9.7°C. Genotype had a significant effect on all traits except tiller number at −15°C. A linkage map was constructed from bi-allelic single nucleotide polymorphism markers generated using exome capture sequencing. The final map consisted of 1618 markers and 2626 cM, with an average inter-marker distance of 1.8 cM. Six significant QTL were identified, one each on chromosomes 1K, 5K, 5N, 6K, 6N, and 9K, for the following traits: tiller number, tiller emergence days and LT50. A comparative genomics study revealed important freezing tolerance genes/proteins, such as COR47, DREB2B, zinc finger-CCCH, WRKY, GIGANTEA, HSP70, and NRT2, among others that reside within the 1.5 LOD confidence interval of the identified QTL. [ABSTRACT FROM AUTHOR]

Alternating temperatures require fast and coordinated adaptation responses of plants. Cold acclimation has been extensively investigated and results in increased freezing tolerance in Arabidopsis thaliana. Here, we show that the two Arabidopsis accessions, Col‐0 and N14, which differ in their freezing tolerance, showed memory of cold acclimation, that is, cold priming. Freezing tolerance was higher in plants exposed to cold priming at 4°C, a lag phase at 20°C, and a second triggering cold stress (4°C) than in plants that were only cold primed. To our knowledge, this is the first report on cold memory improving plant freezing tolerance. The triggering response was distinguishable from the priming response at the levels of gene expression (RNA‐Seq), lipid (ultraperformance liquid chromatography–mass spectrometry), and metabolite composition (gas chromatography–mass spectrometry). Transcriptomic responses pointed to induced lipid, secondary metabolism, and stress in Col‐0 and growth‐related functions in N14. Specific accumulation of lipids included arabidopsides with possible functions as signalling molecules or precursors of jasmonic acid. Whereas cold‐induced metabolites such as raffinose and its precursors were maintained in N14 during the lag phase, they were strongly accumulated in Col‐0 after the cold trigger. This indicates genetic differences in the transcriptomic and metabolic patterns during cold memory. Cold acclimation results in increased freezing tolerance that is lost upon transfer back to warm conditions. Arabidopsis thaliana shows cold memory, leading to higher freezing tolerance during a second cold treatment that is related to specific transcriptomic, lipidomic, and metabolomic responses. [ABSTRACT FROM AUTHOR]

Cold stress is a major abiotic factor plants face during their life cycle. Although plants often exhibit phenotypic variation in cold tolerance, the underlying mechanism remains poorly understood. In the present study, the 50% lethal temperature (LT50) values of 37 Arabidopsis thaliana accessions at latitudes from 15° to 58° ranged from –13.2°C to –4.9°C and were closely correlated with the cold climates of the collection sites. According to a methylation analysis of all C-repeat (CRT)-binding factor (CBF) pathway genes, the coding and promoter regions of AtICE1, a regulator of CBF genes, exhibited the greatest variability in methylation levels among the accessions and included 5–122 methylated cytosine residues. In contrast, unmethylated or only slightly methylated genes in the CBF pathway showed little variation among the accessions. According to a gene expression analysis of four selected A. thaliana populations with distinct methylation patterns, except for the down-regulated gene AtCBF2, the expression levels of all members of the CBF pathway were negatively correlated with AtICE1 gene methylation levels. Treatment of the four A. thaliana populations with the DNA methylation inhibitory reagent 5-azacytidine resulted in a 30.0–78.3% enhancement of freezing tolerance and decreases in LT50 values of approximately 1.9–3.6°C. Similar effects were observed in drm2 mutants, including 30.0–48.3% increases in freezing tolerance and decreases in LT50 values of approximately 0.7–3.4°C. Thus, the AtICE1 methylation-regulated transcription of CBF pathway genes is responsible for the phenotypic variation in the freezing tolerance observed in A. thaliana. [ABSTRACT FROM AUTHOR]

Abstract We studied metabolic adaptations to cold stress in roses and identified genes in the carbohydrate pathway during acclimation and deacclimation. A field experiment with four rose cultivars belonging to different USDA plant hardiness zones was set up in Melle, Belgium (51° 0′ N, 3° 48′ E). The more cold-hardy cultivars ('Dagmar Hastrup' and 'John Cabot') reached their lowest LT 50 value in December, indicating a rapid acclimation after the first occurrence of frost. Less cold-hardy cultivars ('Abraham Darby' and 'Chandos Beauty') reached their lowest LT 50 in January/February when exposed to prolonged freezing temperatures. A cell dehydration pattern was found in the less cold-hardy cultivars 'Abraham Darby' and 'Chandos Beauty'. The expression of dehydrins (RhDHN5 and RhDNH6) was up-regulated during November-January. Carbohydrate metabolism is highly involved in cold acclimation in roses. Starch decreased from November towards January in all four cultivars and the hydrolysis of starch by the β-amylolytic pathway (BAM, DPE2) was identified in 'Dagmar Hastrup' from November to January. Oligosaccharides correlated with cold hardiness in three cultivars although no significant upregulation in RhMIPS and RhRS6, key genes in their biosynthesis, was found. Higher sucrose levels were found during acclimation in hardy cultivars, although transcript levels of RhINV2 was more prominent in 'Chandos Beauty'. [ABSTRACT FROM AUTHOR]

Haloxylon ammodendron, a chenopodiaceae shrub species that mainly distributes in northwest of China, is an ecologically important foundation species and exhibits substantial low-temperature tolerance in the desert area. We employed RNA-Seq technologies to identify genes involved in low temperature, and identified 17995, 15511 and 1550 differentially expressed genes from the comparison of chilling treatment versus control, freezing treatment versus control and freezing treatment versus chilling treatment, respectively. By performing BLAST analysis of these unigenes against public databases, "single-organism metabolic process" and "membrane" were strongly affected at low temperature, and "Plant hormone signal transduction" pathway played an important role in resistance of the plants. In addition, we analyzed transcription factors, and found 499, 453, 55 of them were differentially expressed in these three comparison groups, respectively. A large number of transcription factors were identified at low temperature, and six transcription factors were selected for further expression analysis using qRTPCR. This study can understand the molecular mechanisms of Haloxylon ammodendron at low temperature condition, and also can serve as a valuable resource for relevant research on low-temperature tolerance. [ABSTRACT FROM AUTHOR]

Summary: Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing‐tolerant wild species Solanum acaule (Aca) and freezing‐sensitive cultivated S. tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezing‐tolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S. tuberosum cv. E3, indicating its important role(s) in cold‐acclimated freezing tolerance, which was accompanied with the activation of C‐repeat binding factor genes (CBFs). The present research has identified that the ADC1‐associated putrescine pathway plays an important role in cold‐acclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes. Significance Statement: Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying freezing tolerance of potato is largely unknown. The cold‐responsive genes and metabolites of freezing‐tolerant wild species Solanum acaule were identified and the arginine decarboxylase gene ADC1‐associated putrescine pathway was demonstrated to contribute functionally to potato cold‐acclimated freezing tolerance probably through the upregulation of C‐repeat binding factor gene expression. [ABSTRACT FROM AUTHOR]

Heat shock protein DnaK (Hsp70) can prevent irreversible protein denaturation by chaperoning the unfolded polypeptides under extremely cold environment, and enhance cold and freezing tolerance of microorganism. From an Antarctic sea-ice green alga Chlamydomonas sp. ICE-L, eight Hsp70 genes were identified and divided into seven subfamilies: four in the cytoplasmic subfamilies, two in the chloroplast subgroups, and one in the mitochondria or endoplasmic reticulum subfamilies. Phylogenetic analyses showed that except CidnaK, each of the other Cihsp70 genes had their homologs in Chlamydomonas reinhardtii and Volvox carteri. CiDnak is a cytoplasmic protein with highly homologous to DnaK proteins from prokaryotes, and it had 100% amino acid sequence identities with the DnaK of Psychroflexus torquis ATCC 700755, a psychrophilic bacterium isolated from Antarctic sea ice. The transcription of CidnaK was significantly induced upon freezing stress in ICE-L, eminently higher than that of other Cihsp70 genes. In addition, the transformed C. reinhardtii 137c with CidnaK gene showed much higher rates of survival and growth than the wild type under freezing or low temperature conditions. Our results suggested that CidnaK was likely originated from the symbiotic bacterium in the sea-ice brine, and is an important candidate freezing inducible gene that confers low temperature tolerance to Chlamydomonas cells, and plays a crucial role in ICE-L adapting the frigid Antarctic environments. [ABSTRACT FROM AUTHOR]

Freezing temperatures during the winter and unusual temperature fluctuations during the winter and early spring are the most harmful ambient factors threatening tea plant winter survival and may cause marked economic losses in tea production during the spring. In this study, we simulate natural climate change, to established cold acclimation (CA) and rapid cold stress conditions to investigate the transcriptome changes involved in CA and cold stress. Results revealed transcriptional changes occurring during the initial period of CA and the cell wall changes that occur throughout the entire CA process; these changes play crucial roles in increasing freezing tolerance during this process. Comparing cold-acclimated plants without further treatment against cold-acclimated plants under cold-stress, different cold response mechanisms were rapidly activated under cold stress; however, the subsequent freezing-induced accumulation of reactive oxygen species could be the major signal and harmful factor stimulating stress-associated gene expression and impairing tea leaf physiology. Moreover, we investigated 60 differentially expressed genes shared by both processes and highlighted the importance of β-ketoacyl CoA synthases, HXXXD-type acyl-transferase family proteins, NAC domain containing protein 80, sugar SWEET transporters and enolases in the responses to various cold conditions. The results provide useful information for understanding the regulation mechanism in tea plant responding to complex low temperature conditions. [ABSTRACT FROM AUTHOR]

Calmodulin (CaM), a multifunctional Ca2+ sensor, mediates multiple reactions involved in regulation of plant growth and responses to environmental stress. In this study, we found that AtCaM4 plays a negative role in freezing tolerance in Arabidopsis. The deletion of AtCaM4 resulted in enhanced freezing tolerance in cam4 mutant plants. Although AtCaM4 and AtCaM1 were cold-induced isoforms, cam4/cam1Ri double-mutant and cam4 single-mutant plants exhibited similar improvements in freezing tolerance, indicating that AtCaM4 plays major role. Furthermore, we found that AtCaM4 may influence freezing tolerance in a C-repeat binding factor (CBF)-independent manner as cold-induced expression patterns of CBF s did not change in the cam4/cam1Ri mutant. In addition, among the cold-responsive (COR) genes detected, KIN1, COR15b, and COR8.6 exhibited clearly enhanced expression over the long term in cam4/cam1Ri mutant plants exposed to cold stress. Using immunoprecipitation and mass spectrometry, we identified multiple candidate AtCaM4-interacting proteins. Co-immunoprecipitation assays confirmed the interaction of AtCaM4 with PATL1 in vivo and a phenotype analysis showed that patl1 mutant plants exhibited enhanced freezing tolerance. Thus, we conclude that AtCaM4 negatively regulates freezing tolerance in Arabidopsis by interacting with the novel CaM-binding protein PATL1. [ABSTRACT FROM AUTHOR]

Abstract: In northern countries, Lolium perenne L. generally survives poorly when grown inland and north of 60°N because of extensive winter damage. With the projected future climate change, it could become a promising option for improving production efficiency of the agricultural sector in these regions. Here, we compare the biomass production potential of cultivars of diverse origin across five locations stretching from Estonia to Iceland over a period of three harvest years, and their freezing tolerance under artificial conditions. The aim was to relate the observed pattern of adaptation to the geographic origin of the cultivars and their response to prevailing agroclimatic conditions. Significant interactions were observed between cultivars and test environments (locations × years), and significant interactions between cultivars and years were detected at four of the five locations. Models of joint regression, additive main effects and multiplicative interaction (AMMI) and factorial regression using several agroclimatic indices showed that cultivars developed in northern countries showed greater yield potential across the test environments and were, thus, generally better adapted than cultivars from Central Europe. Diploid cultivars were more frost tolerant than tetraploid cultivars giving them an advantage in locations which were characterized by low temperatures during the hardening period in autumn and mild and rainy winters, such as at the Icelandic location. Only a few cultivars showed general adaptability to the environmental conditions at the test sites, the most stable cultivar being an admixture of diploids and tetraploids. In future breeding, the best strategy would be to hybridize cultivars developed in northern countries with more exotic materials that combine high yield potential, adequate winter survival and superior disease resistance under northern conditions. [ABSTRACT FROM AUTHOR]

Mini-plug transplant seedlings of Norway spruce have been cultivated in closed growth systems, so-called plant factories, for few years. The aim of the experiment was to define a short-day treatment (SD) that harden seedlings to sustain 3 months of cold storage, but does not have adverse effects on growth, morphology, and vitality. The seedlings were subjected to one of the following treatments: (1) 12 h photoperiod + 3 weeks duration; (2) 8 h photoperiod + 3 wk duration; (3) 12 h photoperiod + 5 wk duration; and (4) 8 h photoperiod + 5 wk SD. All the SD treatments yielded healthy seedlings that grew well after the cold storage. The frost hardiness of the seedlings improved when the photoperiod was reduced from 12 to 8 h, and when the SD duration was increased from 3 weeks to 5 weeks, but reducing the photoperiod from 12 h to 8 h caused growth reductions. The root and shoot regrowth after cold storage was highest in seedlings that had received 12 h photoperiod and 5 wk duration. However, 12 h photoperiod and 3 wk duration may be an adequate practice for nurseries that treat multiple crops in their SD facilities. [ABSTRACT FROM AUTHOR]

Main conclusion: Freezing resistance strategies vary in Arabidopsis depending on origin. Southern accessions may avoid or tolerate freezing, while northern ones are always tolerant and reduce the proportion of freezable tissue water during acclimation.Survival of sub-zero temperatures can be achieved by either avoiding or tolerating extracellular ice formation. Conflicting evidence has been presented showing that detached leaves of Arabidopsis thaliana are either freeze avoiding or tolerant. Here, we used three different natural Arabidopsis accessions from different habitats to investigate the frost resistance strategy of whole plants in soil. Plants were cooled to fixed temperatures or just held at their individual ice nucleation temperature for different time intervals. Tissue damage of whole plants was compared to the standard lethal temperature determined for detached leaves with external ice nucleation. While all detached leaves survived freezing when ice nucleation was externally initiated at mild sub-zero temperatures, whole plants of the southern accession behaved as freeze avoiding in the non-acclimated state. The northern accessions and all cold acclimated plants were freezing tolerant, but the duration of the freezing event affected tissue damage. Because this pointed to cell dehydration as mechanism of damage, the proportion of freezable water in leaves and osmolality of cell sap was determined. Indeed, the freezing tolerant accession Rsch had a lower proportion of freezable water and higher cell sap osmolality compared to the sensitive accession C24 in the cold acclimated state. [ABSTRACT FROM AUTHOR]

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In order to investigate the effect of foliar application of potassium sulfate (K2SO4; 0%, 1.5%, and 3%) and iron chelate (Fe-EDDHA; 0%, 0.5%, and 1%) on fruit set, fruit yield and quality, leaf mineral nutrient content, raisin yield, and autumn and winter cold tolerance of 'Bidaneh-Sefid' grape, this study has conduced a factorial experiment, based on randomized complete blocks design in a commercial vineyard in Bahareh, Malayer, Iran, during 2016-2017. Vines have been sprayed during three stages, including one week befor flowering, as well as two and five weeks after corolla abscision. According to the results obtained, the combined effects of K2SO4 and Fe-EDDHA have been significant on fruit set, fruit yield and quality, and raisin yield, not to mention vines' cold tolerance, wi th the highest fruit set percentage and yield belonging to 1.5% of K2SO4 in combination with 0.5% iron chelate. The grapes' K, Fe, and Mn concentration has had a positive and significant correlation with fruit yield per vine and the highest ascorbic acid concentration has been observed in vines, treated with 1.5% K2SO4. It is also found that raisin yield has been the highest in combined treatments of 1.5% K2SO4 and 1% Fe-EDDHA and the lowest in control vines (0% for both K2SO4 and Fe-EDDHA). The highest autumn cold hardiness has been recorded in vines, treated with 3% K2SO4 and 1% Fe-EDDHA, whereas the highest winter cold tolerance has been achieved in the ones, treated with 3% K2SO4 and 0.5% Fe-EDDHA. The lowest cold tolerance belongs to control vines. There has been a positive and significant correlation between cold tolerance and K content and a negative one with Mg concentration. Totally, the highest fruit and raisin yields and their desirable qualities have been obtained in K2SO4 and Fe-EDDHA with moderate concentration; however, the highest bud's cold tolerance has been seen in the combination of these fertilizers with higher doses. [ABSTRACT FROM AUTHOR]

Information concerning genes and signals regulating cold acclimation processes in plants is abundant; however, less is known about genes and signals regulating the deacclimation process. A population of primarily winter B. napus varieties was used to conduct a genome-wide association study and to compare the transcriptomes from two winter B. napus varieties showing time-dependent differences in response to cold acclimation and deacclimation treatments. These studies helped to identify loci, candidate genes, and signaling processes impacting deacclimation in B. napus. GWAS identified polymorphisms at five different loci associated with freezing tolerance following deacclimation. Local linkage decay rates near these polymorphisms identified 38 possible candidate genes. Several of these genes have been reported as differentially regulated by cold stress in arabidopsis (Arabidopsis thaliana), including a calcium-binding EF-hand family protein (encoded by BnaCnng10250D) that was also differentially expressed during deacclimation in this study. Thousands of other genes differentially expressed during the acclimation and deacclimation treatments implicated processes involving oxidative stress, photosynthesis, light-regulated diurnal responses, and growth regulation. Generally, responses observed during acclimation were reversed within one week of deacclimation. The primary differences between the two winter B. napus varieties with differential deacclimation responses involved protection from oxidative stress and the ability to maintain photosynthesis.

Winter wheat (Triticum aestivum L.) undergoes a period of cold acclimation in order to survive the ensuing winter, which can bring freezing temperatures and snow mold infection. Tolerance of these stresses is conferred in part by accumulation of carbohydrates in the crown region. This study investigates the contributions of carbohydrate accumulation during a cold treatment among wheat lines that differ in their snow mold tolerance (SMT) or susceptibility (SMS) and freezing tolerance (FrT) or susceptibility (FrS). Two parent varieties and eight recombinant inbred lines (RILs) were analyzed. The selected RILs represent four combinations of tolerance: SMT/FrT, SMT/FrS, SMS/FrT, and SMS/FrS. It is hypothesized that carbohydrate accumulation and transcript expression will differ between sets of RILs. Liquid chromatography with a refractive index detector was used to quantify carbohydrate content at eight time points over the cold treatment period. Polysaccharide and sucrose content differed between SMT and SMS RILs at various time points, although there were no significant differences in glucose or fructose content. Glucose and fructose content differed between FrT and FrS RILs in this study, but no significant differences in polysaccharide or sucrose content. RNAseq was used to investigate differential transcript expression, followed by modular enrichment analysis, to reveal potential candidates for other mechanisms of tolerance, which included expected pathways such as oxidative stress, chitinase activity, and unexpected transcriptional pathways. These differences in carbohydrate accumulation and differential transcript expression begin to give insight into the differences of wheat lines when exposed to cold temperatures.

Sugar beet (Beta vulgaris L.) is an economic crop integrating grain and feed production, and is a major source of sucrose. Freezing stress can cause dehydration and necrosis of seedlings. Therefore, it is important to study the basic physiological and proteomic mechanisms of different sugar beet cultivars under freezing stress. In this study, compared with freezing-sensitive (SME) cultivar, the freezing-tolerant (TBP) cultivar showed higher photosynthetic rate, antioxidant enzyme activity, proline and abscisic acid levels, lower malondialdehyde and gibberellin levels and relative conductivity under freezing stress. Moreover, comparative proteomic analysis revealed that proteins mapped to photosynthesis, tryptophan synthesis, anthocyanin synthesis, tricarboxylic acid cycle, reactive oxygen species scavenging and sucrose metabolism pathways were more abundant in TBP, indicating that these metabolic pathways may be beneficial to improve freezing tolerance. In particular, compared with SME cultivar, proteins involved in tryptophan synthesis were found to be upregulated only in TBP cultivar under freezing stress, including phospho-2-dehydro-3-deoxyheptonate aldolase 1, probable inactive shikimate kinase like 1, and anthranilate synthase alpha subunit 2. In addition, quantitative real-time polymerase chain reaction analysis showed that mRNA expression of these three proteins was significantly upregulated in the freezing stress (−5 ℃) vs. control group in the TBP cultivar. Additionally, tryptophan content in TBP leaves increased significantly under freezing stress, while in SME it showed no significant change. Furthermore, the application of exogenous tryptophan solution significantly reduced the mortality rate of sugar beet seedlings (17-day-old) under freezing stress. This study provides a foundation for further functional analysis to fully understand the molecular mechanisms of adaptation to freezing stress in sugar beet. [Display omitted] • The tolerant variety has unique anti-freezing mechanism. • TBP showed higher photosynthesis, antioxidant enzyme activity and proline than SME. • Proteins mapped to photosynthesis, anthocyanin synthesis were abundant in TBP. • Proteins mapped to ROS scavenging and sucrose metabolism were abundant in TBP. • TBP acquired freezing tolerance by regulating the tryptophan level. [ABSTRACT FROM AUTHOR]

Low temperature is a primary abiotic stress that significantly affects plant growth and development in the cool regions of Northeast China. It is the primary limiting factor in the development of the global apple industry. Numerous studies have investigated the alterations in transcriptional metabolism under low-temperature stress in various plants. However, research on the role of dormancy in regulating differences in cold tolerance among apple varieties is limited. Therefore, this study conducted a comprehensive analysis of the physiological and biochemical indices, transcriptome, and metabolome of the cold-tolerant variety 'Hanfu' (HF) and cold-sensitive variety 'Naganofuji 2′ (CF) during endodormancy and ecodormancy. Under the low-temperature treatment, the cross-section of a 1-year-old branch of CF exhibited more severe browning than HF did, and the relative electrolyte leakage value of CF was higher than that of HF. Transcriptomics and metabolomics analysis revealed the key pathways of apples in response to low-temperature stress. Functional enrichment analysis showed that hormone signal transduction pathways and amino acid metabolism-related pathways were significantly enriched during endodormancy and ecodormancy, and these pathways were considered an important way for apples to respond to low-temperature freezing injury. The galactose metabolism pathway was significantly enriched only during endodormancy, while the membrane lipid metabolism pathway was significantly enriched only during ecodormancy. A gene co-expression network was constructed using weighted gene correlation network analysis, and six modules with biological significance were selected from the network. Among them, genes encoding transcription factors such as DREB1C/CBF2, JUB1, CCCH, and VIP1 are hub genes in different modules, which can be used as candidate genes to explore the network regulation pathway of apple response to low-temperature stress. Overall, these findings help to elucidate the molecular mechanism underlying freeze-tolerance in apples and provide novel insights into the regulatory network involved in the response of apples to low-temperature stress during dormancy. • Cold response of apple during dormancy was investigated. • Sugar and amino acid metabolism are key response pathways under cold stress. • WGCNA analysis identified the hub genes related to cold resistance. [ABSTRACT FROM AUTHOR]

In northern China, freezing injury is observed frequently in the rare species Magnolia wufengensis but not in the more common species Magnolia denudata. To investigate the role of the phytohormone abscisic acid (ABA) on frost tolerance in these two species, exogenous ABA was applied to the seedlings and then physiological and biochemical responses were measured during cold acclimation. Shoot growth cessation was stimulated by ABA in M. wufengensis but not in M. denudata. Abscisic acid inhibited shoot growth in M. wufengensis but not in M. denudata. Treatment with ABA stimulated leaf senescence in both species, and this effect was greater in M. denudata. For both species, ABA-treated plants exhibited bud dormancy sooner and had an increased tolerance to freezing, decreased water content and increased accumulation of proline, glucose, and fructose in shoots. These effects were generally greater for M. denudata. Freezing tolerance was significantly correlated with content of water, proline, glucose, and fructose for both species, but freezing tolerance was significantly correlated with raffinose content only in M. wufengensis. We conclude that exogenous ABA could increase cold acclimation and improve cold hardiness of both Magnolia species, although M. denudata was more responsive to ABA than M. wufengensis, which might result from a greater dehydration and accumulation of proline and certain soluble sugars.

Winter pea can be grown in rotation with cereal crops in Central Anatolia, Turkey. However, winterkill can occur during harsh winters. The objective of this study was to screen pea accessions for winter survival, and identify genotypes with differential winter hardiness for future crop development. The plant material consisted of 58 accessions including local landraces, elite winter cultivars, selected lines and several checks. Twenty-five of them were evaluated under both field and laboratory conditions. The rest of the genotypes were tested under field conditions. Field trials were planted in Haymana, Ankara, and in Ulaş, Sivas, Turkey during the autumn of 2014. Winter hardiness was evaluated as the percentage of surviving plants. Differential survival of genotypes was observed at both locations. On average, the survival rate was lower in Ulaş (54.8%) than in Haymana (67.8%), and ranged between 1.5 and 100%. Turkish landraces TR 79404 (88.6%), TR 79407 (88.5%) and TR 80194 (84.8%) had survival percentages comparable with the three winter-hardy checks (Turkish cvs. Taşkent (90.0%) and Özkaynak (85.0%), US cv. Melrose (94.7%)). Twelve single plants were selected from these populations for future cultivar development. The European and US accessions, included in the trials for their previously reported winter hardiness, showed high levels of winter hardiness, and could be used in breeding programs. In the laboratory, no plants survived at -12°C and -16°C three weeks following the freezing test. Screening at -8°C generated differential survival among winter genotypes. Significant positive correlations (r = 0.67-0.87, P < 0.001) were found between the test environments for the percent survival.

Morphological and physiological responses to freezing were evaluated in two ecotypes of the perennial turfgrass Paspalum vaginatum. Leaf extension rate, number of active meristems, leaf water potential and net photosynthesis were measured on plants of both a commercial cultivar, 'Sea Isle 2000', and a wild ecotype from the Flooding Pampa grasslands of Argentina. Plants were propagated by cloning, cultivated in pots, and examined during 18 consecutive days under two treatments: a non-frozen control treatment (15.5±7 ºC) and a frozen treatment with two stages: Stage 1 with four hours of freezing stress for 10 nights (-5ºC), and Stage 2 with 12 hours of freezing stress for eight nights (five nights at -5ºC and three nights at -8ºC). After these treatments, plants were returned to the outside environment to evaluate shoot injury and post-freezing recovery. Leaf water potential, net photosynthesis and leaf extension rate were significantly higher in the wild ecotype than in the commercial cultivar. Meristem density was reduced after freezing in both ecotypes, but was more pronounced in the commercial cultivar (98.5%) than in the wild ecotype (80%). Thus, the two ecotypes coming from different environments, exhibited different morphological and physiological responses to exposure to freezing temperatures.

Though winter-hardiness is a complex trait, freezing tolerance was proved to be its main component. Species from temperate regions acquire tolerance to freezing in a process of cold acclimation, which is associated with the exposure of plants to low but non-freezing temperatures. However, mechanisms of cold acclimation in Lolium-Festuca grasses, important for forage production in Europe, have not been fully recognized. Thus, two L. multiflorum/F. arundinacea introgression forms with distinct freezing tolerance were used herein as models in the comprehensive research to dissect these mechanisms in that group of plants. The work was focused on: (i) analysis of cellular membranes’ integrity; (ii) analysis of plant photosynthetic capacity (chlorophyll fluorescence; gas exchange; gene expression, protein accumulation, and activity of selected enzymes of the Calvin cycle); (iii) analysis of plant antioxidant capacity (reactive oxygen species generation; gene expression, protein accumulation, and activity of selected enzymes); and (iv) analysis of Cor14b accumulation, under cold acclimation. The more freezing tolerant introgression form revealed a higher integrity of membranes, an ability to cold acclimate its photosynthetic apparatus and higher water use efficiency after three weeks of cold acclimation, as well as a higher capacity of the antioxidant system and a lower content of reactive oxygen species in low temperature.

Low temperature causes injuries to plants during winter, thereby it affects kiwi fruit quality and yield. However, the changes in metabolites and gene expression during cold acclimation (CA) and deacclimation (DA) in kiwi fruit remain largely unknown. In this study, freezing tolerance, carbohydrate metabolism, and β-amylase gene expression in two Actinidia arguta cv. “CJ-1” and “RB-3” were detected from CA to DA stages. In all acclimation stages, the “CJ-1” was hardier than “RB-3” and possessed lower semi-lethal temperature (LT50). Furthermore, “CJ-1” had a more rapid acclimation speed than “RB-3”. Changes of starch, β-amylase, and soluble sugars were associated with freezing tolerance in both cultivars. Starch contents continued to follow a declining trend, while soluble sugars contents continuously accumulated in both cultivars during CA stages (from October to January). To investigate the possible molecular mechanism underlying cold response in A. arguta, in total, 16 AcBAMs genes for β-amylase were identified in the kiwi fruit genome. We carried out localization of chromosome, gene structure, the conserved motif, and the analysis of events in the duplication of genes from AcBAMs. Finally, a strong candidate gene named AaBAM3 from AcBAMs was cloned in Actinidia arguta (A. arguta), The real-time qPCR showed that AaBAM3 gene expression in seasonal changes was consistent with changes of soluble sugars. These results reveal that AaBAM3 may enhance the freezing tolerance of A. arguta through increasing soluble sugar content.

Plant tolerance to environmental stress is determined by a very complicated network composed of many intra- and extracellular factors. The aim of this study was to select candidate genes involved in responses to freezing and drought in barley on the basis of previous proteomic studies and to analyze changes in their expression caused by application of both stress factors. Six candidate genes for freezing tolerance (namely the genes encoding elongation factor 1 alpha (EF1A), ferredoxin-NADP reductase, a 14-3-3a protein, β-fructofuranosidase, CBF2A and CBF4B) and six for drought tolerance (encoding transketolase, periplasmic serine protease, triosephosphate isomerase, a protein with a co-chaperon region (GroEs), pfam14200 and actin) were chosen arbitrarily on the basis of in silico bioinformatic analyses. The expression levels of these genes were measured under control and stress conditions in six DH (doubled haploid) lines with differing freezing and drought tolerance. The results of gene expression analysis confirmed the roles of the candidate genes preselected in this study on the basis of previous proteome analysis in contributing to the differences in freezing and drought tolerance observed in the studied population of DH lines of winter barley.

Abrupt-chilling events threaten the survival of alfalfa plants, the ability to cope with such condition should be considered during cultivar selection in the production. To assess biochemical and molecular responses of alfalfa to abrupt chilling, the cultivars “WL440HQ” (WL) and “ZhaoDong” (ZD) were subjected to a five-phase experimental regime that included two abrupt-chilling events. The freezing tolerance of the crown was determined as the semi-lethal temperature (LT50) calculated from electrolyte leakage. Soluble sugar concentrations were quantified by ion chromatography. The mRNA transcript levels of four genes encoding enzymes (β-amylase, sucrose phosphate synthase, galactinol synthase, and stachyose synthase) involved in sugar metabolism and two cold-regulated genes (Cas15A and K3-dehydrin) were quantified using quantitative real-time PCR analysis. During the abrupt-chilling events, the LT50 decreased significantly in ZD but not in WL. The rapid response of ZD to abrupt chilling may have been due to the large increases in raffinose and stachyose concentrations, which were consistent with increased transcript levels of the galactinol synthase and stachyose synthase genes. Transcript levels of the cold-regulated genes Cas15A and K3-dehydrin were correlated with increased freezing tolerance under abrupt chilling. The results provide a reference for selection of appropriate cultivars to reduce the risk of crop damage in production areas where early autumn or late spring frosts are likely.