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Asparagus (Asparagus officinalis L.) is an important vegetable crop in southern Ontario, Canada, where winter air and soil temperatures below 0°C are common. Consequently, cultivars growing in this area must possess winterhardiness and freezing tolerance for survival. Asparagus acquires freezing tolerance in the fall through cold acclimation and loses freezing tolerance in the spring through deacclimation. To understand the molecular bases of these processes, transcriptomic analysis (RNA-Seq) was conducted on two cultivars, one adapted, 'Guelph Millennium' (GM), and one unadapted, 'UC157' (UC), to the winter conditions of southern Ontario. RNA extracted from bud and rhizome tissues, sampled on three dates during early spring and late fall, was subjected to sequencing. In the fall, the numbers of differentially expressed (DE) genes at the second and third harvests increased, relative to the first harvest, in dormant buds and rhizomes as freezing tolerance of cultivars increased, and the majority of DE genes were downregulated. In spring, freezing tolerance decreased as plants deacclimated and most genes DE at second and third harvests were upregulated in both cultivars. GM had lower LT50 (lethal temperature at which 50% of plants die) values and hence higher freezing tolerance than UC on specific sampling dates during both spring and fall, and expression patterns of specific genes were correlated with LT50 differences. Functional analysis revealed that these genes were involved in carbohydrate metabolic process, plant hormone signal transduction (auxin and gibberellin), proline metabolism, biosynthesis of secondary metabolites, circadian rhythm, and late embryogenesis abundant proteins and could be associated with cold acclimation and deacclimation processes. These findings will help researchers understand the molecular mechanisms of freezing tolerance in asparagus, leading to breeding and genetic strategies to improve the trait. [ABSTRACT FROM AUTHOR]

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

Three wild yeasts isolated at Kagoshima Prefectural College were identified through ITS4/ITS5 sequencing and characterized for their potential application to fermented foods and beverages. Carbohydrate utilization tests and fermentation experiments in dough were conducted for the isolated yeasts. Specifically, Lachancea fermentati (Lf-KPC1) underwent an additional fermentation test in high-sugar broth and bread evaluation. Lf- KPC1 demonstrated remarkable performance, fermenting equivalently or faster than commercial yeasts in three types of dough with various sugar contents, while the others exhibited low fermentation rates in high-sugar dough. Notably, the three wild yeasts exhibited higher freezing tolerance in dough. In high-sugar broth, Lf-KPC1 exhibited a unique capacity for simultaneous lactic acid and alcohol fermentation, suggesting its potential application in producing sour and low-alcohol beverages. The Lf-KPC1 bread contained more sugar and was more moist. This study concluded that Lf-KPC1 has promising fermentative characteristics, making it highly valuable for fermented products. [ABSTRACT FROM AUTHOR]

Three wild yeasts isolated at Kagoshima Prefectural College were identified through ITS4/ITS5 sequencing and characterized for their potential application to fermented foods and beverages. Carbohydrate utilization tests and fermentation experiments in dough were conducted for the isolated yeasts. Specifically, Lachancea fermentati (Lf-KPC1) underwent an additional fermentation test in high-sugar broth and bread evaluation. Lf-KPC1 demonstrated remarkable performance, fermenting equivalently or faster than commercial yeasts in three types of dough with various sugar contents, while the others exhibited low fermentation rates in high-sugar dough. Notably, the three wild yeasts exhibited higher freezing tolerance in dough. In high-sugar broth, Lf-KPC1 exhibited a unique capacity for simultaneous lactic acid and alcohol fermentation, suggesting its potential application in producing sour and low-alcohol beverages. The Lf-KPC1 bread contained more sugar and was more moist. This study concluded that Lf-KPC1 has promising fermentative characteristics, making it highly valuable for fermented products. [ABSTRACT FROM AUTHOR]

Plant antifreeze proteins (AFPs) are special proteins that can protect plant cells from ice crystal damage in low-temperature environments, and they play a crucial role in the process of plants adapting to cold environments. Proteins with these characteristics have been found in fish living in cold regions, as well as many plants and insects. Although research on plant AFPs started relatively late, their application prospects are broad, leading to the attention of many researchers to the isolation, cloning, and genetic improvement of plant AFP genes. Studies have found that the distribution of AFPs in different species seems to be the result of independent evolutionary events. Unlike the AFPs found in fish and insects, plant AFPs have multiple hydrophilic ice-binding domains, and their recrystallization inhibition activity is about 10-100 times that of fish and insect AFPs. Although different plant AFPs have the characteristics of low TH and high RI, their DNA and amino acid sequences are completely different, with small homology. With in-depth research and analysis of the characteristics and mechanisms of plant AFPs, not only has our understanding of plant antifreeze mechanisms been enriched, but it can also be used to improve crop varieties and enhance their freezing tolerance, yield, and quality through genetic engineering. In addition, the study of plant AFPs also contributes to our understanding of freezing resistance mechanisms in other organisms and provides new research directions for the field of biotechnology. Therefore, based on the analysis of relevant literature, this article will delve into the concepts, characteristics, research methods, and mechanisms of plant AFPs, summarize the latest research progress and application prospects of AFPs in plant, and provide prospects for the future development of AFP gene research. [ABSTRACT FROM AUTHOR]

Mitogen-activated protein kinases (MAPKs) play important roles in plant stress response. As a major member of the MAPK family, MPK3 has been reported to participate in the regulation of chilling stress. However, the regulatory function of wheat (Triticum aestivum) mitogen-activated protein kinase TaMPK3 in freezing tolerance remains unknown. Dongnongdongmai No.1 (Dn1) is a winter wheat variety with strong freezing tolerance; therefore, it is important to explore the mechanisms underlying this tolerance. In this study, the expression of TaMPK3 in Dn1 was detected under low temperature and hormone treatment. Gene cloning, bioinformatics and subcellular localisation analyses of TaMPK3 in Dn1 were performed. Overexpressed TaMPK3 in Arabidopsis thaliana was obtained, and freezing tolerance phenotype observations, physiological indices and expression levels of ICE-C-repeat binding factor (CBF)-COR-related genes were determined. In addition, the interaction between TaMPK3 and TaICE41 proteins was detected. We found that TaMPK3 expression responds to low temperatures and hormones, and the TaMPK3 protein is localised in the cytoplasm and nucleus. Overexpression of TaMPK3 in Arabidopsis significantly improves freezing tolerance. TaMPK3 interacts with the TaICE41 protein. In conclusion, TaMPK3 is involved in regulating the ICE-CBF-COR cold resistance module through its interaction with TaICE41, thereby improving freezing tolerance in Dn1 wheat. [ABSTRACT FROM AUTHOR]

Camellia oleifera Abel. is the dominant woody oil crop under significant development in China. Wild C. oleifera in Lu Mountain is a valuable genetic resource with strong freezing tolerance. With high-throughput sequencing, the genome of wild C. oleifera in Lu Mountain was analysed and 700.3 Gb clean reads were obtained. The genome of wild C. oleifera was estimated as allohexaploid, and its haplotype genome size was about 2.69 Gb-2.79 Gb, with repeat content of 63.01%-73.02% and heterozygosity of 6.30%-7.43%, belonging to a very complex genome. The genomic draft was assembled that contained a total of 6,952,303 scaffolds with N50 length of 1.23 kb, and the overall length was 2.39 Gb with GC content of 40.87%. In the genomic draft, 1,104,618 SSRs were identified; scaffold1096012 and scaffold1779458 were identified as key genes associated with freezing tolerance combined with the transcriptome data of field plus lab experiments. In this study, the genomic background of hexaploid wild C. oleifera in Lu Mountain was revealed. This lays the foundation for obtaining the high-quality chromosome-level reference genome of wild C. oleifera. The identification of SSRs and key genes associated with freezing tolerance may contribute to the efficient exploration and utilisation of this genetic resource. [ABSTRACT FROM AUTHOR]

Intrinsically disordered late embryogenesis abundant (LEA) proteins play a central role in the tolerance of plants and other organisms to dehydration brought upon, for example, by freezing temperatures, high salt concentration, drought or desiccation, and many LEA proteins have been found to stabilize dehydration‐sensitive cellular structures. Their conformational ensembles are highly sensitive to the environment, allowing them to undergo conformational changes and adopt ordered secondary and quaternary structures and to participate in formation of membraneless organelles. In an interdisciplinary approach, we discovered how the functional diversity of the Arabidopsis thaliana LEA protein COR15A found in vitro is encoded in its structural repertoire, with the stabilization of membranes being achieved at the level of secondary structure and the stabilization of enzymes accomplished by the formation of oligomeric complexes. We provide molecular details on intra‐ and inter‐monomeric helix–helix interactions, demonstrate how oligomerization is driven by an α‐helical molecular recognition feature (α‐MoRF) and provide a rationale that the formation of noncanonical, loosely packed, right‐handed coiled‐coils might be a recurring theme for homo‐ and hetero‐oligomerization of LEA proteins. [ABSTRACT FROM AUTHOR]

While the position of alpine and arctic treelines can be predicted by climatic data, the underlying biological mechanisms are still unclear. In a recent paper in this journal (Körner C, Lenz A, Hoch G (2023) Chronic in situ tissue cooling does not reduce lignification at the Swiss treeline but enhances the risk of 'blue' frost rings. Alpine Botany https://doi.org/10.1007/s00035-023-00293-6) we presented results of an in situ stem-cooling experiment at a Swiss treeline site. The experiment provided answers to two entirely different questions, related to xylogenesis at treeline: (a) the absence of chronic effects of low temperature on lignification, and (b) a high time resolution insight into the rare occurrence of damages in young, still undifferentiated, and thus, non-lignified cells at the occasion of an exceptional early season frost event. In the last issue of Alpine Botany (August 7, 2023), our data had been re-interpreted by (Büntgen, Alpine Botany, 2023) by confusing (b) with (a). Cell death before secondary wall formation interrupts all metabolism, and thus, cannot exert a specific limitation of lignification. For the xylem to lignify, it requires a secondary cell wall in the first place. A frost damage in young tracheid cells is unsuitable for a dendrological treeline hypothesis based on a low-temperature threshold for lignification. Generally, the global pattern of treeline position is not associated with local freezing conditions. [ABSTRACT FROM AUTHOR]

Wearable electronics based on conductive hydrogels (CHs) easily suffer from prolonged response times, reduced wearing comfort, shortened service lives, and impaired signal accuracy in cold environments, because conventional CHs tend to freeze at subzero temperatures and lose their flexibility, adhesion, transparency, and conductivity, which will limit their applications in extreme environments. Inspired by the way psychrotolerant creatures and superabsorbent materials interfere with the hydrogen bonding networks of water, a freeze‐resistant conductive organohydrogel (COH) is facilely fabricated. The synergy effect between charged polar terminal groups and a binary solvent system of water–ethylene glycol weakens the hydrogen bonding between water molecules and endows the COH with remarkable freezing tolerance (−78 °C). Additionally, the obtained COH is ultra‐stretchable (≈6185%), tough (9.2 MJ m−3), highly transparent (≈99%), self‐adhesive (10.2–27.8 kPa), and biocompatible. This versatile COH is assembled into a strain sensor and a well‐designed bracelet electrocardiogram sensor. Benefiting from the exceptional low‐temperature tolerance of the prepared COH, these devices exhibit fast response with delay‐free signals even at −40 °C. Overall, this work proposes a strategy to develop multifunctional COHs for supporting human health in cold environments. [ABSTRACT FROM AUTHOR]

Asparagus (Asparagus officinalis L.) is an important vegetable crop in southern Ontario, Canada, where winter air and soil temperatures below 0°C are common. Consequently, cultivars growing in this area must possess winterhardiness and freezing tolerance for survival. Asparagus acquires freezing tolerance in the fall through cold acclimation and loses freezing tolerance in the spring through deacclimation. To understand the molecular bases of these processes, transcriptomic analysis (RNA-Seq) was conducted on two cultivars, one adapted, ‘Guelph Millennium’ (GM), and one unadapted, ‘UC157’ (UC), to the winter conditions of southern Ontario. RNA extracted from bud and rhizome tissues, sampled on three dates during early spring and late fall, was subjected to sequencing. In the fall, the numbers of differentially expressed (DE) genes at the second and third harvests increased, relative to the first harvest, in dormant buds and rhizomes as freezing tolerance of cultivars increased, and the majority of DE genes were downregulated. In spring, freezing tolerance decreased as plants deacclimated and most genes DE at second and third harvests were upregulated in both cultivars. GM had lower LT50 (lethal temperature at which 50% of plants die) values and hence higher freezing tolerance than UC on specific sampling dates during both spring and fall, and expression patterns of specific genes were correlated with LT50 differences. Functional analysis revealed that these genes were involved in carbohydrate metabolic process, plant hormone signal transduction (auxin and gibberellin), proline metabolism, biosynthesis of secondary metabolites, circadian rhythm, and late embryogenesis abundant proteins and could be associated with cold acclimation and deacclimation processes. These findings will help researchers understand the molecular mechanisms of freezing tolerance in asparagus, leading to breeding and genetic strategies to improve the trait.

Cold stress is a serious threat to global crop production and food security, but plant cold resistance is accompanied by reductions in growth and yield. In this study, we determined that the novel gene BcGSTF10 in non-heading Chinese cabbage [NHCC; Brassica campestris (syn. Brassica rapa) ssp. chinensis ] is implicated in resistance to cold stress. Biochemical and genetic analyses demonstrated that BcGSTF10 interacts with BcICE1 to induce C-REPEAT BINDING FACTOR (CBF) genes that enhance freezing tolerance in NHCC and in Arabidopsis. However, BcCBF2 represses BcGSTF10 and the latter promotes growth in NHCC and Arabidopsis. This dual function of BcGSTF10 indicates its pivotal role in balancing cold stress and growth, and this important understanding has the potential to inform the future development of strategies to breed crops that are both climate-resilient and high-yielding. [ABSTRACT FROM AUTHOR]

The adaptation and resistance of the vine to low temperatures during the dormancy involves the realization of processes of accumulation and dynamics of some biochemical compounds in the tissues. In this study, between November and March (2022-2023), the dynamics of carbohydrates (soluble sugars and starch) was monitored at 10 days in the annual and multiannual vine wood in the 'Cabernet Sauvignon', 'Fetească neagră' and 'Merlot' from the Banu Mărăcine wine-growing center (plateau-type area, slightly leached reddish-brown soil) and Șimnicu de Sus wine-growing area (slope-type area, medium eroded reddish-brown soil). Regarding the carbohydrate content during the dormancy, significant differences between the two viticultural areas were highlighted only concerning the content of soluble sugars (%) in multiannual wood for the 'Merlot' and 'Cabernet Sauvignon'. As for the starch content (%) in annual woody shoots and in multiannual wood, no statistically significant differences were observed either between the studied cultivars or between the two areas. [ABSTRACT FROM AUTHOR]

Abstract The objective of this work was to evaluate cryoprotectants on the physiological quality of Adenium obesum seeds preserved in liquid nitrogen. The following treatments were used: T1, control, without cryoprotectant and without immersion in liquid nitrogen; T2, with immersion in liquid nitrogen and without cryoprotectant; T3, with 0. 4 mol L-1 sucrose; T4, with 0.8 mol L-1 sucrose; T5, with 1.0 mol L-1 glycerol; T6, with 2.0 mol L-1 glycerol with PVS1; T7, with PVS1; T8, with PVS2; T9, with PVS2 + phloroglucinol (modified PVS2); and T10, with PVS3. Adenium obesum seeds show tolerance to immersion and storage in liquid nitrogen and do not need cryoprotectants to maintain their physiological quality when stored in a water content of 7.5%.

The proteome analysis of winter crops during cold acclimation and overwintering can provide important information for designing breeding processes. The current experiment was carried out to investigate the proteome changes in frost-tolerant winter wheat (cv. Norstar) during different cold acclimation (CA) periods under field conditions in a cold and high-altitude region by two-dimensional gel-based proteomic techniques. The results showed that frost tolerance significantly increased by CA and the lethal freezing temperatures (LT50) 10, 14, and 18 weeks after seed sowing were -28°C, -22°C, and -10°C, respectively. By the beginning of the reproductive stage (double ridge stage), the LT50 values had decreased significantly. Around 1000 protein spots were distinguished by Coomassie staining on the gels. The changes in the proteins during the CA often occurred in those with a functional role in photosynthesis, energy production (glycolysis), transcription, chaperone-like activities, membrane and cytoskeleton reorganisation, transport, redox adjustments, and signalling. The results revealed that changes in chloroplast proteins, certain transcription factors such as MADS-box transcription factor 26, and antioxidant enzymes (ascorbate peroxidase) show a similar trend to freezing tolerance, and their expression decreases with the onset of reproductive growth and the loss of freezing tolerance. During the acclimation period, most of the changes were focused on defence systems and cytoskeleton rearrangement, while, photosynthesis, and energy production became the main priority at the beginning of reproductive growth.

The purpose of this study was to identify proteins associated with differences in the freezing tolerance of sheep sperm and to analyze their functions. Qualified fresh semen from four breeds of rams, the Australian White, white-head Dorper, Black-head Dorper, and Hu sheep breeds, were selected for cryopreservation. The sperm freezing tolerance was investigated by evaluation of the overall vitality, progressive vitality, and rapidly advance vitality of the sperm. A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and the protein functions were analyzed. It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. These two breeds were used for the construction of a model based on differences in freezing tolerance and the identification of sperm proteins expressed differentially before freezing and after thawing. A total of 128 differentially expressed proteins (88 up-regulated and 40 down-regulated) were identified before freezing and after thawing in Hu sheep sperm (fresh/frozen Hu sheep sperm referred to as HL vs. HF), while 219 differentially expressed proteins (106 up-regulated and 113 down-regulated) were identified in white-head Dorper sheep (fresh/frozen white-head Dorper sheep sperm referred to as WL vs. WF). A comparison of these differentially expressed proteins showed that 57 proteins overlapped between the two breeds while 71 were only expressed in Hu sheep and 162 were only expressed in white-head Dorper sheep. Functional annotation and enrichment analyses of differentially expressed proteins down-regulated in Hu sheep involved in phosphorylation of phosphatidylinositol phosphate kinases, regulation of GTPase activity and glycolysis/gluconeogenesis signaling pathway. Up-regulated proteins of Hu sheep participated in oxidoreductase activity and oxidative phosphorylation process of sperm freezing. Furthermore, down-regulated in white-head Dorper sheep involved in the metabolic regulation of carbohydrate and nuclear sugar metabolism. Up-regulated proteins of white-head Dorper sheep involved in the ferroptosis and oxidative phosphorylation pathways. Collectively, These proteins were found to participate mainly in oxidative phosphorylation as well as phosphorylation and metabolic processes in the mitochondria to affect the freezing tolerance of sheep sperm. • The purpose of this study was to identify proteins associated with differences in freezing tolerance of sheep sperm and analyze their functions. • A differential model of sperm freezing tolerance was constructed for sheep breeds showing significant differences. • It was found that sperm freezing tolerance was best in Hu sheep and worst in white-head Dorper sheep. • Differentially expressed proteins associated with sperm freezing tolerance were identified using iTRAQ and proteins functions were analyzed. [ABSTRACT FROM AUTHOR]

Cold acclimation is a crucial biological process that enables conifers to overwinter safely. The late embryogenesis abundant (LEA) protein family plays a pivotal role in enhancing freezing tolerance during this process. Despite its importance, the identification, molecular functions and regulatory networks of the LEA protein family have not been extensively studied in conifers or gymnosperms. Pinus tabuliformis , a conifer with high ecological and economic values and with high-quality genome sequence, is an ideal candidate for such studies. Here, a total of 104 LEA genes were identified from P. tabuliformis , and we renamed them according to their subfamily group: PtLEA1 – PtLEA92 (group LEA1–LEA6), PtSMP1 – PtSMP6 (group seed maturation protein) and PtDHN1 – PtDHN6 (group Dehydrin). While the sequence structure of P. tabuliformis   LEA genes are conserved, their physicochemical properties exhibit unique characteristics within different subfamily groupings. Notably, the abundance of low-temperature responsive elements in PtLEA genes was observed. Using annual rhythm and temperature gradient transcriptome data, PtLEA22 was identified as a key gene that responds to low-temperature induction while conforming to the annual cycle of cold acclimation. Overexpression of PtLEA22 enhanced Arabidopsis freezing tolerance. Furthermore, several transcription factors potentially co-expressed with PtLEA22 were validated using yeast one-hybrid and dual-luciferase assays, revealing that PtDREB1 could directly bind PtLEA22 promoter to positively regulate its expression. These findings reveal the genome-wide characterization of P. tabuliformis   LEA genes and their importance in the cold acclimation, while providing a theoretical basis for studying the molecular mechanisms of cold acclimation in conifers. [ABSTRACT FROM AUTHOR]

Antarctic macroalgal communities of the upper intertidal zone are particularly poor compared with other coastal regions. Exposure to desiccation and freezing combined with the abrasive effect of ice threatens the life of sessile organisms. One of the few species able to colonize this environment is the rhodophyte Pyropia endiviifolia. It belongs to the Bangiales, one of the oldest extant clades of photosynthetic eukaryotes, which has occurred for more than one billion years with basically the same morphological structure. Considering that the extent of Antarctic glaciation is a geologically recent event, we hypothesized that pre-adaptations to desiccation in bangiophytes may have contributed to the success of P. endiviifolia in Antarctica. To test this, we compared its photosynthetic performance and tolerance to desiccation and freezing with those from a related intertidal species, the temperate Atlantic species Porphyra linearis. As evidenced by gas exchange and chlorophyll fluorescence, P. endiviifolia is more adapted to high irradiances than P. linearis. The former species was also more desiccation-tolerant, and showed a higher glass transition temperature. Both species did not differ in chlorophyll content per dry weight, and tolerance to −20°C, even though the ice-nucleation temperature was much higher in P. endiviifolia. Membrane integrity may depend on fatty acid composition in P. endiviifolia, while on enhanced tocopherol in P. linearis. Overall, both species show different strategies to deal with freezing temperatures: supercooling in P. linearis vs. freezing-tolerance in P. endiviifolia. This matches with the probability of being subjected to sub-zero temperatures in their natural environments (lower in the case of P. linearis). Surprisingly, the higher risk of dehydration in the natural habitat of P. linearis is not matched by a higher desiccation tolerance. This observation does not support the initial hypothesis of the study but suggests the opposite: the acclimation to a cold environment results in higher desiccation tolerance. HIGHLIGHTS ● Porphyra linearis and Pyropia endiviifolia are remarkably tolerant to desiccation and freezing.● Antarctic P. endiviifolia is remarkably tolerant to desiccation and freezing.● Mechanisms of freezing tolerance could induce a higher tolerance to desiccation. [ABSTRACT FROM AUTHOR]

Cold stress is a major abiotic stress that adversely affects plant growth and crop productivity. The C‐REPEAT BINDING FACTOR/DRE BINDING FACTOR 1 (CBF/DREB1) transcriptional regulatory cascade plays a key role in regulating cold acclimation and freezing tolerance in Arabidopsis (Arabidopsis thaliana). Here, we show that max (more axillary growth) mutants deficient in strigolactone biosynthesis and signaling display hypersensitivity to freezing stress. Exogenous application of GR245DS, a strigolactone analog, enhances freezing tolerance in wild‐type plants and strigolactone‐deficient mutants and promotes the cold‐induced expression of CBF genes. Biochemical analysis showed that the transcription factor WRKY41 serves as a substrate for the F‐box E3 ligase MAX2. WRKY41 directly binds to the W‐box in the promoters of CBF genes and represses their expression, negatively regulating cold acclimation and freezing tolerance. MAX2 ubiquitinates WRKY41, thus marking it for cold‐induced degradation and thereby alleviating the repression of CBF expression. In addition, SL‐mediated degradation of SMXLs also contributes to enhanced plant freezing tolerance by promoting anthocyanin biosynthesis. Taken together, our study reveals the molecular mechanism underlying strigolactones promote the cold stress response in Arabidopsis. Synopsis: Plants respond to extreme cold stress by synthesizing strigolactones; however, how this class of plant hormones promotes freezing tolerance is still poorly understood. Here, cold‐induced strigolactones are shown to promote plant survival in sub‐zero temperatures by enhancing expression of freezing tolerance regulators DREB1/CBF.Strigolactones positively regulate plant freezing tolerance via a CBF‐dependent pathway.The E3 ligase MAX2 interacts with and ubiquitinates the transcription factor WRKY41, mediating its cold‐induced degradation.WRKY41 represses the cold‐induced expression of CBFs by directly binding to their promoters.SLs‐mediated degradation of SMXLs also contributes to enhanced freezing tolerance by accumulating anthocyanin. [ABSTRACT FROM AUTHOR]

Potato (Solanum tuberosum L.) is the fourth largest food crop in the world. Low temperatures cause serious damage to potato plants every year, and freezing tolerance has become a hot spot in potato research. Galactinol synthase (GolS) is a key enzyme in the synthesis of raffinose family oligosaccharides (RFOs), and plays an important role in the response of plants to abiotic stress. In this study, the ScGolS1 gene from Solanum commersonii was cloned and introduced into the S. tuberosum cultivars ‘Atlantic’ and ‘Desiree’ via Agrobacterium-mediated transformation. Phenotyping assays showed that overexpression of ScGolS1 could significantly improve freezing tolerance in transgenic potato plants. Further physiological and biochemical experiments showed that the transgenic lines had lower relative conductivity, malondialdehyde content, and 3,3′-diaminobenzidine staining and a higher plant survival rate compared with wild type (WT) under cold stress. Moreover, the C-repeat binding factors (CBF1, CBF2 and CBF3), the downstream cold-responsive genes COR413 and COR47, and the ethylene-responsive factor (ERF) transcription factor genes ERF3, ERF4 and ERF6, which function in the ethylene signaling pathway, were all induced by freezing treatment and expressed at higher levels in the ScGolS1 overexpression lines compared with WT. Besides, the expression of some genes such as MIPS, STS and RS from the RFO metabolic pathway was up-regulated under cold stress, resulting in changes in the content of some soluble sugars. This indicated that ScGolS1 overexpression altered the sugar composition and enhanced freezing tolerance in transgenic potato by inducing the ethylene and CBF signaling pathways. These results provided theoretical support and genetic resources for freezing tolerance breeding in potato.

The North American genera Ptelea L. and Zanthoxylum L. are distributed farther north than most other genera in the Rutaceae (citrus family). We used 3 methods to characterize cold acclimation and deacclimation of common hoptree (Ptelea trifoliata L.) and common pricklyash (Zanthoxylum americanum Mill.), species with potential to diversify landscapes. Female and male common hoptrees were compared, and we contextualized our results by measuring cold hardiness of western hoptree (Ptelea crenulata Greene) and representatives of other woody genera of the order Sapindales that are hardy in the Upper Midwestern US: maple (Acer L.), ailanthus (Ailanthus Desf.), corktree (Phellodendron Rupr.), and sumac (Rhus L.). A discoloration method was ineffective, so we assessed hardiness based on electrolyte leakage and the reduction of 2,3,5-triphenyltetrazolium chloride (TTC). TTC reduction indicated that Ptelea and Zanthoxylum were more cold-hardy than indicated by electrolyte leakage. Western hoptree, sampled in its native habitat, was the least hardy. Maximal hardiness of common hoptree, common pricklyash, and western hoptree was −42, −33, and −21 °C (−44, −27, and −6 °F), respectively. Hardiness of female and male common hoptrees did not differ consistently. While common hoptree and common pricklyash have been considered hardy to Zone 3, our assessments of plants from a portion of the species' geographic ranges indicate common hoptree is the more cold-hardy species. [ABSTRACT FROM AUTHOR]

Perennial plant species from water-limiting environments (including climates of extreme drought, heat and freezing temperatures) have evolved traits that allow them to tolerate these conditions. As such, traits that are associated with water stress may show evidence of adaptation to climate when compared among closely related species inhabiting contrasting climatic conditions. In this study, we tested whether key hydraulic traits linked to drought stress, including the vulnerability of leaves to embolism (P 50 leaf ) and the minimum diffusive conductance of shoots (g min ), were associated with climatic characteristics of 14 Tasmanian eucalypt species from sites that vary in precipitation and temperature. Across species, greater cavitation resistance (more negative P 50 leaf ) was associated with increasing aridity and decreasing minimum temperature. By contrast, g min showed strong associations with aridity only. Among these Tasmanian eucalypts, evidence suggests that trait variation is influenced by both cold and dry conditions, highlighting the need to consider both aspects when exploring adaptive trait–climate relationships. [ABSTRACT FROM AUTHOR]

Environmental variation is the most crucial problem as it is causing food insecurity and negatively impacts food availability, utilization, assessment, and stability. Wheat is the largest and extensively cultivated staple food crop for fulfilling global food requirements. Abiotic stresses including salinity, heavy metal toxicity, drought, extreme temperatures, and oxidative stresses being the primary cause of productivity loss are a serious threat to agronomy. Cold stress is a foremost ecological constraint that is extremely influencing plant development, and yield. It is extremely hampering the propagative development of plant life. The structure and function of plant cells depend on the cell's immune system. The stresses due to cold, affect fluid in the plasma membrane and change it into crystals or a solid gel phase. Plants being sessile in nature have evolved progressive systems that permit them to acclimatize the cold stress at the physiological as well as molecular levels. The phenomenon of acclimatisation of plants to cold stress has been investigated for the last 10 years. Studying cold tolerance is critical for extending the adaptability zones of perennial grasses. In the present review, we have elaborated the current improvement of cold tolerance in plants from molecular and physiological viewpoints, such as hormones, the role of the posttranscriptional gene, micro RNAs, ICE-CBF-COR signaling route in cold acclimatization and how they are stimulating the expression of underlying genes encoding osmoregulatory elements and strategies to improve cold tolerance in wheat. [ABSTRACT FROM AUTHOR]

Tea plants (Camellia sinensis (L.) O. Kuntze) frequently suffer severe damage as a result of freezing temperatures in early spring, which severely affect tea quality and tea production in China. Emerging evidence has demonstrated that the ethylene signaling pathway plays an important role in tea plants' freezing responses. However, how ethylene modulates the response to freezing in sprouting tea shoots is not clear. This study verified that the measurement of relative electrolyte leakage in young shoots after 1 h at −5 °C is a rapid way to evaluate their freezing tolerance in the laboratory. Further exploration of the mechanism involved in increasing tea-shoot freezing tolerance by monitoring changes in the transcription of ethylene-related genes and cold signaling-related genes, and the physiological and biochemical changes after the application of ethephon (2-chloroethylphosphonic acid, an ethylene release reagent), revealed that exogenous ethephon significantly increased the freezing tolerance of tea shoots within 3 days of treatment, while concomitantly altering the expression of the ethylene signaling pathway-related genes (i.e., CsETR1, CsETR2, and CsEBF1). Moreover, antioxidant enzyme activities, including superoxide dismutase, catalase, and peroxidase, were uniformly upregulated, which might constitute a major physiological change induced by ethylene signaling and may be responsible for the observed increase in freezing resistance. Nevertheless, soluble sugars and starch, trehalose metabolism, and cold signaling-related genes did not appear relevant to the freezing tolerance increase following ethephon application. This study demonstrated that the freezing tolerance of sprouting tea shoots can be rapidly increased by the exogenous activation of the ethylene signaling pathway and upregulation of the plant's antioxidant system. [ABSTRACT FROM AUTHOR]

Global warming effects in temperate and polar regions include higher average temperatures and a decrease in snow cover, which together lead to an increase in the number of freeze–thaw cycles (FTC). These changes could affect the fitness of both terrestrial and aquatic species. In this study, we tested how tardigrades, ubiquitous microscopic invertebrates, face FTC. Tardigrades are amongst the most resistant animals to unfavorable conditions, including long and deep freezing periods, and are an emerging model group for invertebrate ecology and evolution. We used 12 populations of tardigrades, representing different families within order Parachela, inhabiting different ecosystems (glaciers, snow, terrestrial, aquatic), found in various substrates (mosses, sediments in lakes, cryoconite on glaciers, and snow), and originating from different latitudes and altitudes. We estimated the number of cycles required to kill 50% of individuals and tested for its association with ecological characteristics of the natural habitat (e.g., number of months with predicted FTC), while accounting for phylogeny. The most resistant tardigrades to FTC were the ones from mountain areas and glaciers. The estimated number of cycles required to kill 50% of individuals was the highest for mountainous species inhabiting rock pools and cryoconite holes on glaciers (30 and 14 FTC, respectively). Tardigrades from lowlands were the most sensitive to changes, with 50% of individuals dying after three FTC, while lacustrine and subtropical tardigrades required only one FTC to reach 50% mortality. Our study shows that the response to recurrent freezing stress is taxon dependent and related to the local environmental conditions. The predicted increase of FTC cycles will negatively impact tardigrade populations. Considering the abundance and various trophic roles of tardigrades, reduction in population sizes or the disappearance of some fragile species could affect the functioning of both aquatic and terrestrial ecosystems. Tardigrades are candidate indicators of how freeze–thaw cycles impact ubiquitous microscopic metazoans with similar physiological capabilities.

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Summary: Previous discovering meticulously illustrates the post‐translational modifications and protein stability regulation of ICE1 and their role in cold stress. However, the studies on the interaction of ICE1 with other transcription factors, and their function in modulation cold stress tolerance, as well as in the transition between cold stress and growth are largely insufficient.In this work, we found that maltose binding protein (MBP) 43 directly binds to the promoters of CBF genes to repress their expression, thereby negatively regulating freezing tolerance.Biochemical and genetic analyses showed that MYB43 interacts and antagonizes with ICE1 to regulate the expression of CBF genes and plant's freezing stress tolerance. PLEIOTROPIC REGULATORY LOCUS 1 (PRL1) accumulates under cold stress and promotes MYB43 protein degradation; however, when cold stress disappears, PRL1 restores normal protein levels, causing MYB43 protein to re‐accumulate to normal levels. Furthermore, PRL1 positively regulates freezing tolerance by promoting degradation of MYB43 to attenuate its repression of CBF genes and antagonism with ICE1.Thus, our study reveals that MYB43 inhibits CBF genes expression under normal growth condition, while PRL1 promotes MYB43 protein degradation to attenuate its repression of CBF genes and antagonism with ICE1, and thereby to the precise modulation of plant cold stress responses. [ABSTRACT FROM AUTHOR]

Wheat production is often impacted by pre-winter freezing damage and cold spells in later spring. To study the influences of cold stress on wheat seedlings, unstressed Jing 841 was sampled once at the seedling stage, followed by 4 °C stress treatment for 30 days and once every 10 days. A total of 12,926 differentially expressed genes (DEGs) were identified from the transcriptome. K-means cluster analysis found a group of genes related to the glutamate metabolism pathway, and many genes belonging to the bHLH, MYB, NAC, WRKY, and ERF transcription factor families were highly expressed. Starch and sucrose metabolism, glutathione metabolism, and plant hormone signal transduction pathways were found. Weighted Gene Co-Expression Network Analysis (WGCNA) identified several key genes involved in the development of seedlings under cold stress. The cluster tree diagram showed seven different modules marked with different colors. The blue module had the highest correlation coefficient for the samples treated with cold stress for 30 days, and most genes in this module were rich in glutathione metabolism (ko00480). A total of eight DEGs were validated using quantitative real-time PCR. Overall, this study provides new insights into the physiological metabolic pathways and gene changes in a cold stress transcriptome, and it has a potential significance for improving freezing tolerance in wheat. [ABSTRACT FROM AUTHOR]

In their 2013 paper, Lenz et al. illustrated how trees growing at the low-temperature limit respond to a chronic in situ warming or cooling by 3 K, by employing Peltier-thermostated branch collars that tracked ambient temperatures. The micro-coring-based analysis of seasonal tree ring formation included double-staining microtome cross sections for lignification, but these data had not been included in the publication. In this short communication, we complement these data, collected in 2009 at the Swiss treeline, and we show that a 3 K cooling that corresponds to a 500–600 m higher elevation, had no influence on lignification. However, when a frost event occurred during the early part of ring formation, the 3 K cooling produced a blue (non-lignified) layer of cells, followed by normally lignified cells for the rest of the season. Hence, the event did not affect the cambium, but interrupted cell wall maturation in cells that were in a critical developmental stage. We conclude, that chronic cooling does not affect lignification at treeline, but it increases the risk of frost damage in premature xylem tissue. [ABSTRACT FROM AUTHOR]

Abstract Background Cytosine methylation, the main type of DNA methylation, regulates gene expression in plant response to environmental stress. The winter rapeseed has high economic and ecological value in China's Northwest, but the DNA methylation pattern of winter rapeseed during freezing stress remains unclear. Result This study integrated the methylome and transcriptome to explore the genome-scale DNA methylation pattern and its regulated pathway of winter rapeseed, using freezing-sensitive (NF) and freezing-resistant (NS) cultivars.The average methylation level decreased under freezing stress, and the decline in NF was stronger than NS after freezing stress. The CG methylation level was the highest among the three contexts of CG, CHG, and CHH. At the same time, the CHH proportion was high, and the methylation levels were highest 2 kb up/downstream, followed by the intron region. The C sub-genomes methylation level was higher than the A sub-genomes. The methylation levels of chloroplast and mitochondrial DNA were much lower than the B. napus nuclear DNA, the SINE methylation level was highest among four types of transposable elements (TEs), and the preferred sequence of DNA methylation did not change after freezing stress. A total of 1732 differentially expressed genes associated with differentially methylated genes (DMEGs) were identified in two cultivars under 12 h and 24 h in three contexts by combining whole-genome bisulfite sequencing( and RNA-Seq data. Function enrichment analysis showed that most DMEGs participated in linoleic acid metabolism, alpha-linolenic acid metabolism, carbon fixation in photosynthetic organisms, flavonoid biosynthesis, and plant hormone signal transduction pathways. Meanwhile, some DMEGs encode core transcription factors in plant response to stress. Conclusion Based on the findings of DNA methylation, the freezing tolerance of winter rapeseed is achieved by enhanced signal transduction, lower lipid peroxidation, stronger cell stability, increased osmolytes, and greater reactive oxygen species (ROS) scavenging. These results provide novel insights into better knowledge of the methylation regulation of tolerance mechanism in winter rapeseed under freezing stress.

Low temperature is one of the most important abiotic factors limiting the growth, development and geographical distribution of plants. Prunus mume is an attractive woody ornamental plant that blooms in early spring in Beijing. However, the molecular mechanisms underlying cold hardening to enhance freezing tolerance in Prunus genus remains elusive. This study examined the dynamic physiological responses induced by cold hardening, and identified freezing-tolerance genes by RNA-seq and ATAC-seq analyses. Cold hardening elevated the content of soluble substances and enhanced freezing resistance in P. mume. Transcriptome analysis indicated that the candidate differentially expressed genes (DEGs) were those enriched in Ca2+ signalling, mitogen-activated protein kinase (MAPK) cascade, abscisic acid signalling, and inducer of CBF expression 1 (ICE)-C-repeat binding factor (CBF) signalling pathways. The openness of gene chromatin positively correlated with the expression level of these genes. Thirteen motifs were identified in the open chromatin regions in the treatment group subjected to freezing after cold hardening. The chromatin opening of transcription start site at the proximal –177 region of cold-shock protein CS120-like (PmCSL) was markedly increased, while the expression level of PmCSL was significantly up-regulated. Overexpression of PmCSL in Arabidopsis significantly improved the freezing tolerance of transgenic plants. These findings provide new insights into the regulatory mechanism of freezing tolerance to improve breeding of cold-hardy P. mume plants. [ABSTRACT FROM AUTHOR]

Winter oilseed cash cover crops are gaining popularity in integrated weed management programs for suppressing weeds. A study was conducted at two field sites (Fargo, North Dakota, and Morris, Minnesota) to determine the freezing tolerance and weed-suppressing traits of winter canola/rapeseed (Brassica napus L.) and winter camelina [Camelina sativa (L.) Crantz] in the Upper Midwestern USA. The top 10 freezing tolerant accessions from a phenotyped population of winter canola/rapeseed were bulked and planted at both locations along with winter camelina (cv. Joelle) as a check. To phenotype our entire winter B. napus population (621 accessions) for freezing tolerance, seeds were also bulked and planted at both locations. All B. napus and camelina were no-till seeded at Fargo and Morris at two planting dates, late August (PD1) and mid-September (PD2) 2019. Data for winter survival of oilseed crops (plants m−2) and their corresponding weed suppression (plants m−2 and dry matter m−2) were collected on two sampling dates (SD) in May and June 2020. Crop and SD were significant (p < 0.05) for crop plant density at both locations, and PD in Fargo and crop x PD interaction in Morris were significant for weed dry matter. At Morris and Fargo, PD1 produced greater winter B. napus survival (28% and 5%, respectively) and PD2 produced higher camelina survival (79% and 72%, respectively). Based on coefficient of determination (r2), ~50% of weed density was explained by camelina density, whereas ≤20% was explained by B. napus density at both locations. Camelina from PD2 suppressed weed dry matter by >90% of fallow at both locations, whereas weed dry matter in B. napus was not significantly different from fallow at either PD. Genotyping of overwintering canola/rapeseed under field conditions identified nine accessions that survived at both locations, which also had excellent freezing tolerance under controlled conditions. These accessions are good candidates for improving freezing tolerance in commercial canola cultivars. [ABSTRACT FROM AUTHOR]

Understanding the evolution of plant defenses against herbivores requires identifying the benefits and costs of defense. Here, we tested the hypothesis that the benefits and costs of hydrogen cyanide (HCN) defense against herbivory on white clover (Trifolium repens) are temperature dependent. We first tested how temperature affected HCN production in vitro, and then examined how temperature influenced the efficacy of HCN defense of T. repens against a generalist slug (Deroceras reticulatum) herbivore using no-choice and choice feeding trial assays. To understand how temperature affected the costs of defense, plants were exposed to freezing, and HCN production, photosynthetic activity, and ATP concentration were quantified. HCN production increased linearly from 5 °C to 50 °C, and cyanogenic plants experienced reduced herbivory compared to acyanogenic plants only at warmer temperatures when fed upon by young slugs. Freezing temperatures induced cyanogenesis in T. repens and decreased chlorophyll fluorescence. Cyanogenic plants experienced lower ATP levels than acyanogenic plants due to freezing. Our study provides evidence that the benefits of HCN defense against herbivores are temperature dependent, and freezing may inhibit ATP production in cyanogenic plants, but the physiological performance of all plants recovered quickly following short-term freezing. These results contribute to understanding how varying environments alter the benefits and costs of defense in a model system for the study of plant chemical defenses against herbivores. [ABSTRACT FROM AUTHOR]

Low temperature is an important factor limiting plant growth. Most cultivars of Vitis vinifera L. are sensitive to low temperatures and are at risk of freezing injury or even plant death during winter. In this study, we analyzed the transcriptome of branches of dormant cv. Cabernet Sauvignon exposed to several low-temperature conditions to identify differentially expressed genes and determine their function based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG)enrichment analyses. Our results indicated that exposure to subzero low temperatures resulted in damage to plant cell membranes and extravasation of intracellular electrolytes, and that this damage increased with decreasing temperature or increasing duration. The number of differential genes increased as the duration of stress increased, but most of the common differentially expressed genes reached their highest expression at 6 h of stress, indicating that 6 h may be a turning point for vines to tolerate extreme low temperatures. Several pathways play key roles in the response of Cabernet Sauvignon to low-temperature injury, namely: (1) the role of calcium/calmodulin-mediated signaling; (2) carbohydrate metabolism, including the hydrolysis of cell wall pectin and cellulose, decomposition of sucrose, synthesis of raffinose, and inhibition of glycolytic processes; (3) the synthesis of unsaturated fatty acids and metabolism of linolenic acid; and (4) the synthesis of secondary metabolites, especially flavonoids. In addition, pathogenesis-related protein may also play a role in plant cold resistance, but the mechanism is not yet clear. This study reveals possible pathways for the freezing response and leads to new insights into the molecular basis of the tolerance to low temperature in grapevine. [ABSTRACT FROM AUTHOR]

High mountain ecosystems are subjected to frequent freeze–thaw events all-year round, consequently plants have developed freezing resistance mechanisms to cope with extreme low temperatures. Additionally, these events have also been correlated with the risk of cavitation so that plants need to adapt their water transport system. Information on freezing resistance and xylem vessel characteristics along elevation gradients is scarce for neotropical high elevation species. In this study we aboard two specific questions: 1. Are there intraspecific differences in freezing resistance between low and high elevation populations of Senecio formosus Kunth? 2. Could an increase in freeze–thaw cycle frequency and lower freezing temperatures at higher elevations determine differences in xylem conduit traits between low and high elevation S. formosus populations? We expect greater freezing resistance and a safer water transport system, mainly shaped by narrower tracheary elements in higher elevation populations compared to lower ones. Freezing resistance (avoidance and tolerance) and tracheary elements were studied in S. formosus at its lower (3100 m) and upper (4200 m) distributional limits in the Venezuelan paramo. Freezing resistance was determined through injury and freezing temperature determinations; whereas xylem conduit characteristics dealt with were: vessel element and tracheid diameters, % conducting area and vessel element density. S. formosus increased freezing resistance and presented narrower vessel element diameters under more extreme thermal conditions (4200 m). Increasing evidence of intraspecific plant trait variations under different environmental gradients will aid to determine the outcome of individual species and their effects on ecosystem functioning under a changing climate. [ABSTRACT FROM AUTHOR]

Low temperature is one of the most prominent environmental factors affecting plant growth. As a deciduous arboreal tree, Acer palmatum has considerable ornamental and economic value; however, the molecular mechanisms underlying cold stress regulation in this species have yet to be determined. In this study, we performed Illumina high-throughput sequencing of 18 libraries obtained from A. palmatum subjected to cold treatment and subsequently identified a differentially expressed R2R3-MYB family gene, ApMYB77, which was then cloned and functionally characterized. The expression of ApMYB77 was induced by cold and drought treatments, and overexpression in A. thaliana enhanced the freezing tolerance (− 9 °C for 6 h) of transgenic plants compared with that of wild-type plants. The survival rates of transgenic plants (89% and 92%) were significantly higher than the wild-type plants (52%). Moreover, the transcript abundances of CBF-dependent regulatory pathway genes (AtCBF1, AtCBF2, AtCBF3, AtCBF4, AtCOR6.6A, AtCOR15B, AtCOR78, AtCOR414, and AtKIN1) were found to be significantly up-regulated in the transgenic lines. Enhanced abscisic acid (ABA)-dependent drought tolerance in transgenic plants was a further consequence of the overexpression of ApMYB77 following treatment of 15% polyethylene glycol for 4 d, compared with that in wild-type plants. In contrast, the expressions of genes (AtANAC072, AtDREB2A, AtERD1, AtMYB2, AtRD20, and AtRD29A) positively regulated by ABA were activated. Overall, the findings of this study indicate that ApMYB77 confers both freezing and drought tolerances. [ABSTRACT FROM AUTHOR]

Abstract Background Rapid-cycling Brassica napus (B. napus-RC) has potential as a rapid trait testing system for canola (B. napus) because its life cycle is completed within 2 months while canola usually takes 4 months, and it is susceptible to the same range of diseases and abiotic stress as canola. However, a rapid trait testing system for canola requires the development of an efficient transformation and tissue culture system for B. napus-RC. Furthermore, effectiveness of this system needs to be demonstrated by showing that a particular trait can be rapidly introduced into B. napus-RC plants. Results An in-vitro regeneration protocol was developed for B. napus-RC using 4-day-old cotyledons as the explant. High regeneration percentages, exceeding 70%, were achieved when 1-naphthaleneacetic acid (0.10 mg/L), 6-benzylaminopurine (1.0 mg/L), gibberellic acid (0.01 mg/L) and the ethylene antagonist silver nitrate (5 mg/L) were included in the regeneration medium. An average transformation efficiency of 16.4% was obtained using Agrobacterium-mediated transformation of B. napus-RC cotyledons using Agrobacterium strain GV3101 harbouring a plasmid with an NPTII (kanamycin-selectable) marker gene and the Arabidopsis thaliana cDNA encoding ACYL-COA-BINDING PROTEIN6 (AtACBP6). Transgenic B. napus-RC overexpressing AtACBP6 displayed better tolerance to freezing/frost than the wild type, with enhanced recovery from cellular membrane damage at both vegetative and flowering stages. AtACBP6-overexpressing B. napus-RC plants also exhibited lower electrolyte leakage and improved recovery following frost treatment, resulting in higher yields than the wild type. Ovules from transgenic AtACBP6 lines were better protected from frost than those of the wild type, while the developing embryos of frost-treated AtACBP6-overexpressing plants showed less freezing injury than the wild type. Conclusions This study demonstrates that B. napus-RC can be successfully regenerated and transformed from cotyledon explants and has the potential to be an effective trait testing platform for canola. Additionally, AtACBP6 shows potential for enhancing cold tolerance in canola however, larger scale studies will be required to further confirm this outcome.

Exogenous application of salicylic acid (SA) to plant tissues has been shown to confer tolerance against various abiotic stresses. Recently, SA application through sub-irrigation was shown to improve plant freezing tolerance (FT). For SA treatment to be employable as an effective intervention strategy for frost protection under field conditions, it is important to study its effect on FT when applied as a foliar spray to whole plants. It is also important to determine for how long the FT-improvement by SA lasts. Present study was conducted to compare SA-induced FT of spinach (Spinacia oleracea L. 'Reflect') seedlings following SA-application by foliar spray vs. sub-irrigation. Durability of FT-promotive effect of SA was evaluated using three freeze-tests over a 4-d period, i.e., at 10-d, 12-d, and 14-d after the SA application. Freezing stress was applied using a temperature-controlled freeze-thaw protocol, and FT was assessed by visual observations (leaf flaccidness vs. turgidity) as well as ion-leakage assay. Data indicated that both foliar spray and sub-irrigation methods improved FT of the seedlings against a relatively moderate (−5.5 °C) as well as severe stress (−6.5 °C). Moreover, improved FT against moderate stress was sustained over a 4-d period, whereas such benefit waned somewhat against the severe stress. SA-treated leaves' growth performance was similar to the non-treated control based on dry weight, fresh weight, leaf area, and dry weight/leaf area parameters. Our results suggest that SA application as a foliar spray can potentially be used to protect field-grown transplants against episodic frosts. [ABSTRACT FROM AUTHOR]

Changes to precipitation patterns and warming temperatures are predicted to reduce the water available to Australian alpine plants during the growing season. Soil water deficits are likely to co-occur with frost extremes that are common throughout the year and heatwaves which are increasing in severity with ongoing climate change. We aimed to determine whether co-occurring reductions in soil moisture would affect the capacity of alpine plants to tolerate temperature extremes. We used small rainout shelters to impose a drought treatment in situ in the alpine zone, which chronically reduced soil moisture in plots of alpine plant species including evergreen shrubs, graminoids and perennial forbs. We determined photosynthetic freezing tolerance and heat tolerance during the growing season across 2 years, and measured plant growth, in response to the drought treatment. Thermal tolerance was insensitive to chronically reduced soil moisture, and graminoids exhibited overall greater freezing and heat tolerance thresholds than other life forms. The drought treatment improved shrub growth, likely due to the amelioration of wind and the slightly warmer temperatures provided by the rainout shelters. We conclude that Australian alpine plants maintain high tolerances to both high- and low-temperature extremes during the growing season and are relatively robust to combined temperature extremes and reductions in near-surface soil moisture that are likely to occur with ongoing climate warming. [ABSTRACT FROM AUTHOR]

Resurrection plants are able to deal with complete dehydration of their leaves and then recover normal metabolic activity after rehydration. Only a few resurrection species are exposed to freezing temperatures in their natural environments, making them interesting models to study the key metabolic adjustments of freezing tolerances. Here, we investigate the effect of cold and freezing temperatures on physiological and biochemical changes in the leaves of Haberlea rhodopensis under natural and controlled environmental conditions. Our data shows that leaf water content affects its thermodynamical properties during vitrification under low temperatures. The changes in membrane lipid composition, accumulation of sugars, and synthesis of stress-induced proteins were significantly activated during the adaptation of H. rhodopensis to both cold and freezing temperatures. In particular, the freezing tolerance of H. rhodopensis relies on a sucrose/hexoses ratio in favor of hexoses during cold acclimation, while there is a shift in favor of sucrose upon exposure to freezing temperatures, especially evident when leaf desiccation is relevant. This pattern was paralleled by an elevated ratio of unsaturated/saturated fatty acids and significant quantitative and compositional changes in stress-induced proteins, namely dehydrins and early light-induced proteins (ELIPs). Taken together, our data indicate that common responses of H. rhodopensis plants to low temperature and desiccation involve the accumulation of sugars and upregulation of dehydrins/ELIP protein expression. Further studies on the molecular mechanisms underlying freezing tolerance (genes and genetic regulatory mechanisms) may help breeders to improve the resistance of crop plants. [ABSTRACT FROM AUTHOR]

Identify the freezing tolerant cultivars is a suitable approach for the success of lentil sowing in autumn. In this respect, an augmented experience was carried out to evaluate freezing tolerance of 259 lentil accessions during the 2016-2017 growing seasons in Mashhad. During the growing season, the phenologic growth stages of the plants and the survival percentage affected by winter hardiness were recorded, and at the end of the growing season, morphological traits, seed yield and components were determined. The lowest recorded temperature during the growing season was -13°C. Survival percentage varied between 0 and 100% and the highest survival percentage (100%) and maximum plant height (53cm) were found in MLC13 MLC409, respectively. Six accessions (MLC424, MLC286, MLC409, MLC303 'MLC334 and MLC407) were identified as freezing tolerant. Among freezing tolerant accessions, MLC424, MLC286, MLC409, MLC303, MLC334 and MLC407 had the highest seed yield (154, 192, 220, 144, 225 and 264 g.m²), respectively. There was a high correlation between survival percentage and seed yield and components. Based on the results, more precise assessment of physiological processes involved in freezing tolerance of lentil accessions in the controlled condition and accessing to winter hardness genotyped is necessary. [ABSTRACT FROM AUTHOR]

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

Low temperature during cold acclimation (CA) leads to the accumulation of detrimental reactive oxygen species (ROS) in plant tissues, which are scavenged by antioxidants, such as ascorbate and glutathione. However, there is a lack of studies examining the dynamics of antioxidants throughout CA, deacclimation (DEA), and reacclimation (REA) in winter wheat. Six winter wheat genotypes were selected to assess the effect of CA, DEA, and REA on the concentrations of ascorbate and glutathione in leaf and crown tissues under two CA temperature treatments. Higher levels of total, reduced, and oxidised ascorbate were determined in leaves, whereas crowns accumulated higher concentrations of nicotinamide adenine dinucleotide (NAD+) after CA, DEA, and REA. Constant low temperature (CLT) during CA led to higher contents of ascorbate and glutathione in both tissues at all stages of acclimation, in comparison with prolonged higher low temperature (PHLT). The concentrations of antioxidants increased after CA, tended to decrease after DEA, and returned to CA levels after REA. Significant positive correlations between freezing tolerance (FT) and antioxidants were only determined under the CA at CLT treatment, thus, affirming the negative effect of PHLT during CA on the FT of winter wheat. [ABSTRACT FROM AUTHOR]

Most cultivars of Vitis vinifera L. are very sensitive to cold. As an exogenous protectant, Biodegradable Liquid Film (BLF) is considered to protect winegrapes from low temperatures and dry winds for safe overwintering. This study aimed to reveal the physiological and biochemical mechanisms of BLF regulating the freezing tolerance of wine grapes. Groups of ten-year-old vines (Cabernet Sauvignon) were sprayed with BLF in November 2020 and 2021, or left untreated as a control treatment, and field plant mortality after overwintering were investigated. Branch samples were collected monthly for determination of biochemical indicators. Dormant two-year-old cuttings (Cabernet Sauvignon) were also used for the determination of relative expression levels of key genes. The results showed that the application of BLF reduced the branch semi-lethal temperature in January and February samples compared with control, and reduced the mortality of above-ground parts, branches and buds. The physiological status of shoots was greatly affected by the climatic conditions of the year, but BLF treatment increased the levels of soluble protein and soluble sugar, and also decreased the content of superoxide anion and malondialdehyde at most sampling times. Correlation analysis showed that the differences in freezing tolerance between BLF and no treated overwintering(CK) vines were mainly related to peroxidase activity, soluble sugar, reducing sugar and starch content. Low temperature stress activated the over expression of ICE1, CBF1, and CBF3, especially for 12h. BLF treatment significantly increased the expression levels of CBF1 and CBF3 under low temperature stress. Overall, these results demonstrate that BLF treatment protects vines from freezing damage by upregulating osmo-regulatory substances and alleviating oxidative damage. [ABSTRACT FROM AUTHOR]

Abstract Background Freezing stress inhibits plant development and causes significant damage to plants. Plants therefore have evolved a large amount of sophisticated mechanisms to counteract freezing stress by adjusting their growth and development correspondingly. Plant ontogenetic defense against drought, high salt, and heat stresses, has been extensively studied. However, whether the freezing tolerance is associated with ontogenetic development and how the freezing signals are delivered remain unclear. Results In this study, we found that the freezing tolerance was increased with plant age at the vegetative stage. The expressions of microRNA156 (miR156) and SQUAMOSA PROMOTER BINDING PROTEIN-LIKE 9 (SPL9), playing roles in regulation of ontogenetic development, were induced by cold stress. Overexpression of SPL9 (rSPL9) promoted the expression of C-REPEAT BINDING FACTOR 2 (CBF2) and hereafter enhanced the freezing tolerance. Genetic analysis indicated that the effect of rSPL9 on freezing tolerance is partially restored by cbf2 mutant. Further analysis confirmed that SPL9 directly binds to the promoter of CBF2 to activate the expression of CBF2, and thereafter increased the freezing tolerance. Conclusions Therefore, our study uncovers a new role of SPL9 in fine-tuning CBF2 expression and thus mediating freezing tolerance in plants, and implies a role of miR156-SPL pathway in balancing the vegetative development and freezing response in Arabidopsis.

• Apple trees grown in warmer regions experience shallow dormancy. • Cold hardiness is different between cultivars and between cultivation sites. • The Eva cultivar does not present endodormancy and deep cold hardiness. • The maximum cold hardiness occurs after the peak of endodormancy. • Cold deacclimation is rapid and with high heterogeneity. In temperate fruit plants, climate is the main factor correlating changes in plant physiology, mainly those related to entry and exit from dormancy and cold hardiness. Acclimation to cold and its relationship with dormancy are still unknown in areas with a subtropical climate. Therefore, this study aimed to investigate the dormancy dynamics and their interaction with the cold hardiness of different tissues of apple cultivars with different chilling requirements in two cultivation sites in a mild winter region in southern Brazil. The analyses were conducted from 2018 to 2022. The cultivars Gala, Fuji, and Eva were used in two environments with different altitudes. The dormancy dynamics were analyzed using the single-node cutting test, and the cold hardiness of the different tissues of each genotype was evaluated through electrolyte leakage in laboratory freezing tests. Cultivars Gala and Fuji had a greater depth of dormancy than Eva. Plants grown in orchards with higher chill accumulation had a greater depth of dormancy. Cold hardiness varied among cultivars, with the Eva cultivar having the lowest cold hardiness in mid-winter. The tissues (bud, bark, and wood) had different cold hardiness in early winter, with wood having the greatest cold hardiness, followed by bud and bark. As Eva did not go into deep dormancy, a superficial and short acclimation to cold was observed, in addition to rapid sprouting, even in winter. This condition predisposed it to cold damage to tissues in the pre-sprouting period. In this study, we found that maximum cold hardiness occurred after the end of the deep-endodormancy phase. Deacclimation of all cultivars occurred very quickly. [ABSTRACT FROM AUTHOR]