Antifreeze Proteins Research Articles

Improving Soluble Expression of SARS-CoV-2 Spike Priming Protease TMPRSS2 with an Artificial Fusing Protein.

SARS-CoV-2 relies on the recognition of the spike protein by the host cell receptor ACE2 for cellular entry. In this process, transmembrane serine protease 2 (TMPRSS2) plays a pivotal role, as it acts as the principal priming agent catalyzing spike protein cleavage to initiate the fusion of the cell membrane with the virus. Thus, TMPRSS2 is an ideal pharmacological target for COVID-19 therapy development, and the effective production of high-quality TMPRSS2 protein is essential for basic and pharmacological research. Unfortunately, as a mammalian-originated protein, TMPRSS2 could not be solubly expressed in the prokaryotic system. In this study, we applied different protein engineering methods and found that an artificial protein XXA derived from an antifreeze protein can effectively promote the proper folding of TMPRSS2, leading to a significant improvement in the yield of its soluble form. Our study also showed that the fused XXA protein did not influence the enzymatic catalytic activity; instead, it greatly enhanced TMPRSS2's thermostability. Therefore, our strategy for increasing TMPRSS2 expression would be beneficial for the large-scale production of this stable enzyme, which would accelerate aniti-SARS-CoV-2 therapeutics development.

Engulfment Avalanches and Thermal Hysteresis for Antifreeze Proteins on Supercooled Ice.

Antifreeze proteins (AFPs) bind to the ice-water surface and prevent ice growth at temperatures below 0 °C through a Gibbs-Thomson effect. Each adsorbed AFP creates a metastable depression on the surface that locally resists ice growth, until ice engulfs the AFP. We recently predicted the susceptibility to engulfment as a function of AFP size, distance between AFPs, and supercooling [ J. Chem. Phys. 2023, 158, 094501]. For an ensemble of AFPs adsorbed on the ice surface, the most isolated AFPs are the most susceptible, and when an isolated AFP gets engulfed, its former neighbors become more isolated and more susceptible to engulfment. Thus, an initial engulfment event can trigger an avalanche of subsequent engulfment events, leading to a sudden surge of unrestrained ice growth. This work develops a model to predict the supercooling at which the first engulfment event will occur for an ensemble of randomly distributed AFP pinning sites on an ice surface. Specifically, we formulate an inhomogeneous survival probability that accounts for the AFP coverage, the distribution of AFP neighbor distances, the resulting ensemble of engulfment rates, the ice surface area, and the cooling rate. We use the model to predict thermal hysteresis trends and compare with experimental data.

Genomics of cold adaptations in the Antarctic notothenioid fish radiation

Numerous novel adaptations characterise the radiation of notothenioids, the dominant fish group in the freezing seas of the Southern Ocean. To improve understanding of the evolution of this iconic fish group, here we generate and analyse new genome assemblies for 24 species covering all major subgroups of the radiation, including five long-read assemblies. We present a new estimate for the onset of the radiation at 10.7 million years ago, based on a time-calibrated phylogeny derived from genome-wide sequence data. We identify a two-fold variation in genome size, driven by expansion of multiple transposable element families, and use the long-read data to reconstruct two evolutionarily important, highly repetitive gene family loci. First, we present the most complete reconstruction to date of the antifreeze glycoprotein gene family, whose emergence enabled survival in sub-zero temperatures, showing the expansion of the antifreeze gene locus from the ancestral to the derived state. Second, we trace the loss of haemoglobin genes in icefishes, the only vertebrates lacking functional haemoglobins, through complete reconstruction of the two haemoglobin gene clusters across notothenioid families. Both the haemoglobin and antifreeze genomic loci are characterised by multiple transposon expansions that may have driven the evolutionary history of these genes.

Adsorption Kinetics of Type-III Antifreeze Protein on Ice Crystal Surfaces.

The spatio-temporal distribution of type-III antifreeze protein (AFP-III) molecules labeled with fluorescent isocyanate (FITC) was visualized at the interfaces between ice and solutions with an FITC-labeled AFP-III (F-AFP-III) concentration of 20-800 μg/mL by fluorescence microscopy. The number density of F-AFP-III on the surface of ice microcrystals was calculated from the calibrated fluorescence intensity. The adsorption of F-AFP-III molecules on the ice crystal surfaces proceeded at a finite rate and then reached the saturation level. The time course of the number density of adsorbed F-AFP-III molecules could be well represented by Langmuir's model. The characteristic adsorption time of F-AFP-III, the adsorption coefficient k1 = (0.5 ± 0.05) × 10-4 (μg/mL)-1 s-1, and the desorption coefficient k2 = 0.005 ± 0.002 s-1 were determined using the Langmuir's model and obtained experimental data. We found that the adsorption of F-AFP-III could have different kinetics depending on the solution conditions and the type of fluorescence molecules conjugated with AFP-III.

Cryopreservation in the Era of Cell Therapy: Revisiting Fundamental Concepts to Enable Future Technologies

AbstractCryopreservation strives to maximize the viability and biofunctionality of cells and tissues by cooling them to a subzero temperature to facilitate storage and delivery. This technology has enabled clinics and labs to preserve rare and crucial samples and is poised to become more important with rising interest in cell therapy. Here, the biological impact of cooling rates on different cellular components is first described, paying special emphasis on the differences between slow cooling and vitrification with a heat transfer perspective based on the Biot number. This is followed by an overview of various classes of chemical‐based cryoprotective agents including small molecules, antifreeze proteins, hydrogels, and cryoprotective nanomaterials. Most importantly, fundamental concepts of cryopreservation including Mazur's “two‐factor hypothesis” are revisited, gaps in them are highlighted, and experiments to validate reported claims to deepen mechanistic understanding of cryoprotection are proposed. A matric is also introduced to assess the suitability of biomaterials for use in cell therapy to support manufacturers in making strategic choices for storing clinical samples. It is believed that this review would inspire readers to scrutinize fundamental concepts in cryopreservation to facilitate the development of new cryoprotective materials and technologies to support the emerging cell manufacturing and therapy industry.

Polyproline type II helical antifreeze proteins are widespread in Collembola and likely originated over 400 million years ago in the Ordovician Period

Antifreeze proteins (AFPs) bind to ice crystals to prevent organisms from freezing. A diversity of AFP folds has been found in fish and insects, including alpha helices, globular proteins, and several different beta solenoids. But the variety of AFPs in flightless arthropods, like Collembola, has not yet been adequately assessed. Here, antifreeze activity was shown to be present in 18 of the 22 species of Collembola from cold or temperate zones. Several methods were used to characterize these AFPs, including isolation by ice affinity purification, MALDI mass spectrometry, amino acid composition analysis, tandem mass spectrometry sequencing, transcriptome sequencing, and bioinformatic investigations of sequence databases. All of these AFPs had a high glycine content and were predicted to have the same polyproline type II helical bundle fold, a fold unique to Collembola. These Hexapods arose in the Ordovician Period with the two orders known to produce AFPs diverging around 400 million years ago during the Andean-Saharan Ice Age. Therefore, it is likely that the AFP arose then and persisted in many lineages through the following two ice ages and intervening warm periods, unlike the AFPs of fish which arose independently during the Cenozoic Ice Age beginning ~ 30 million years ago.

Ice recrystallization inhibition behavior by wheat flour and its synergy effect with antifreeze protein III

Ice recrystallization (IR) is an undesirable phenomenon in which the size of an ice crystal grows in the frozen medium. Based on the Ostwald ripening theory, the IR kinetics of wheat flour system were investigated under isothermal conditions (−10 °C). The results showed that 30% (w/w) sucrose solution had an IR rate of 3.34 ± 0.29 μm3/min, however adding 5% (w/w) wheat flour reduced the rate to 0.56 ± 0.15 μm3/min. To understand the inhibitory effect of wheat flour, the size distribution was analyzed. The results indicated that wheat flour reduced both the size and size distribution of the ice crystals in the sucrose solution, suggesting that narrowing the size distribution is the candidate mechanism for IR inhibition. Further, combining antifreeze protein III with the wheat flour system decreased IR rate in a synergistic manner. In presence of wheat flour, only 0.1 μg/mL of AFP III was sufficient to lower IR rate significantly, while without it, 0.5 μg/mL of AFP III was required.

Use of Antifreeze Protein from Tenebrio molitor (TmAFP) in Vitrification of In Vitro-Produced Bovine Embryos: An Ultrastructural Study.

The objective of this study was to evaluate the effects of different concentrations of antifreeze protein (AFP) extracted from the larva of the beetle, Tenebrio molitor (TmAFP), on vitrification of in vitro-produced bovine embryos. In vitro-produced blastocysts were divided into three experimental groups and vitrified using a cryotop. TmAFP was added to the equilibrium solution (ES) and vitrification solution (VS) at a concentration of 0 ng/mL (control), 500 ng/mL (500TmAFP), or 1000 ng/mL (1000TmAFP). Vitrification was carried out by first placing the blastocysts in ES for 2 minutes (7.5% ethylene glycol [EG] and 7.5% dimethyl sulfoxide [DMSO]). The blastocysts were then transferred to VS (15% EG and 15% DMSO) and promptly deposited on a cryotop stem and submerged in liquid nitrogen. Warming was carried out in three steps with decreasing sucrose concentrations. After warming, the blast cells were cultured for 24 hours for subsequent survival analysis and ultrastructural evaluation. There was a significant difference in the survival rate and expansion in the 500TmAFP group compared with the other groups. The ultrastructural analysis revealed intracellular lesions in all vitrified embryos; however, the embryos of the 500TmAFP and 1000TmAFP groups showed fewer cytoplasmic lesions compared with the control group. Taken together, addition of TmAFP can mitigate cellular changes that involve organelles and cellular components essential for proper functioning and improve the viability of warmed and vitrified in vitro-produced bovine embryos.

Chilling injury in human kidney tubule cells after subzero storage is not mitigated by antifreeze protein addition

By preventing freezing, antifreeze proteins (AFPs) can permit cells and organs to be stored at subzero temperatures. As metabolic rates decrease with decreasing temperature, subzero static cold storage (SZ-SCS) could provide more time for tissue matching and potentially lead to fewer discarded organs. Human kidneys are generally stored for under 24 h and the tubule epithelium is known to be particularly sensitive to static cold storage (SCS). Here, telomerase-immortalized proximal-tubule epithelial cells from humans, which closely resemble their progenitors, were used as a proxy to assess the potential benefit of SZ-SCS for kidneys. The effects of hyperactive AFPs from a beetle and Cryostasis Storage Solution were compared to University of Wisconsin Solution at standard SCS temperatures (4 °C) and at −6 °C for up to six days. Although the AFPs helped guard against freezing, lower storage temperatures under these conditions were not beneficial. Compared to cells at 4 °C, those stored at −6 °C showed decreased viability as well as increased lactate dehydrogenase release and apoptosis. This suggests that this kidney cell type might be prone to chilling injury and that the addition of AFPs to enable SZ-SCS may not be effective for increasing storage times.

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-preserved Ultrastructure.

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP). Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking. A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

Mechanism of Binding of Polyproline to Ice via Interfacial Water: An Experimental and Theoretical Study.

The molecular mechanism underlying inhibition of ice growth by polyproline (PPro), a minimal antifreeze glycoprotein mimic, remains unclear. In this work, the change in the structure of water during the growth of ice in PPro solutions was investigated using a combination of near-infrared spectroscopy and molecular dynamics (MD) simulations. The results show that only high concentrations of PPro solutions can effectively inhibit ice growth, as indicated by the variation in the spectral intensity of ice with time. When PPro exhibits an antifreeze effect, the spectral intensity of hydrated water associated with PPro in a solution is weakened. The experiments and MD simulations reveal that the quantity of the interfacial water between the ice crystal and the hydrophobic groups of PPro progressively reaches a plateau. Most significantly, we present clear evidence that the stable existence of this interfacial water is critical for the antifreeze activity of PPro.

Flat Solenoidal Ice‐Binding Proteins as Scaffolds for Solid‐Binders

AbstractSolid interfacing biomaterials is a crucial aspect of bionanotechnology and important for applications such as biosensing. Because of their potentially large contact area, flat solenoidal proteins are ideal scaffolds for designing proteins binding to surfaces of man‐made solids such as minerals, metals, and plastics. To explore this opportunity, a naturally occurring flat solenoidal protein: the Rhagium inquistor Antifreeze Protein from the insect Rhagium inquisitor is re‐designed. By mutating 4, 6, and 10 out of its 4 × 5 arrays of threonines into arginines, it have arrived at the silica‐binding proteins RiSiBP‐4, RiSiBP‐6, and RiSiBP‐10. Variants with increasing numbers of arginines bind stronger to silica, but are also less stable and increasingly difficult to produce. It is found that the RiSiBP‐6 variant binds strongly to silica yet still has good stability and easy production. It is shown that sfGFP‐RiSiBP‐6 fusions allow for the functional display of a monolayer of sfGFP cargo on silica surfaces, suggesting the general usefulness of flat solenoidal proteins as scaffolds for designing solid‐binding proteins.

Development and Implementation of Microbial Antifreeze Protein Based Coating for Anti‐Icing

AbstractIce formation on a solid surface is a major challenge in industrial applications, it causes higher energy consumption and performance deterioration and may lead to catastrophic results. The preparation of anti‐icing surfaces to prohibit ice accumulation on a surface is crucial to reduce operational costs and to extend the surface's lifetime. The utilization of cryoprotectants to obtain anti‐icing surfaces is an effective method and is applicable in multiple fields. Antifreeze proteins (AFPs) are natural cryoprotectants to obtain anti‐icing surfaces, which have the ability to decrease the freezing point and to prevent ice‐crystal growth via thermal hysteresis (TH) and ice recrystallization inhibition (IRI). This study reports the molecular cloning, expression, and production of AFP protein from Escherichia coli (E. Coli). This wok also demonstrates the activity of coated AFP on aluminum surfaces. The expressed AFP is immobilized on aluminum surfaces treated by oxygen plasma. The coated AFP exhibits promising antifreeze activity with a high anti‐icing ability on aluminum surfaces in the size of evaporator fins (40 × 40 cm). The outcome of this study provides new insights into the biotechnological implementation of AFPs to various industrial applications for energy‐saving and higher performance.

The alteration of proteins and metabolites in leaf apoplast and the related gene expression associated with the adaptation of Ammopiptanthus mongolicus to winter freezing stress

Ammopiptanthus mongolicus, an evergreen broad-leaved plant, can tolerate severe freezing stress (temperatures as low as −20 °C in winter). The apoplast is the space outside the plasma membrane that plays an important role in plant responses to environmental stress. Here, we investigated, using a multi-omics approach, the dynamic alterations in the levels of proteins and metabolites in the apoplast and related gene expression changes involved in the adaptation of A. mongolicus to winter freezing stress. Of the 962 proteins identified in the apoplast, the abundance of several PR proteins, including PR3 and PR5, increased significantly in winter, which may contribute to winter freezing-stress tolerance by functioning as antifreeze proteins. The increased abundance of the cell-wall polysaccharides and cell wall-modifying proteins, including PMEI, XTH32, and EXLA1, may enhance the mechanical properties of the cell wall in A. mongolicus. Accumulation of flavonoids and free amino acids in the apoplast may be beneficial for ROS scavenging and the maintenance of osmotic homeostasis. Integrated analyses revealed gene expression changes associated with alterations in the levels of apoplast proteins and metabolites. Our study improved the current understanding of the roles of apoplast proteins and metabolites in plant adaptation to winter freezing stress.

Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities.In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to −25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions (‘in air’ or ‘in bulk CPA’).For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 (‘scaffold in a bulk CPA’) vs. 7.71 ± 0.43 (‘bulk CPA’, P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 (‘scaffold in a bulk CPA’) vs. 7.68 ± 0.34 (‘bulk CPA’, P < 0.0001)).At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen ‘in air’ were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 μg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively).Moreover, when frozen ‘in air’ in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend.The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

Antifreeze protein type III addition to freezing extender comprehensively improves post-thaw sperm properties in Okinawan native Agu pig

Sperm cryopreservation often leads to physical cell damage through ice crystal formation. This study evaluates the improvements to freezing extender cryoprotective activity due to antifreeze protein (AFP) addition, which primarily acts on ice crystal formation, through investigating the post-thaw sperm properties of Okinawan native Agu pig. Six individual boar sperm samples were diluted with the freezing extender supplemented with 1 μg/mL of AFP I or AFP III and then subjected to cryopreservation. Treatment with AFP I during the freezing procedure had no improvement for any characteristics after thawing compared to untreated sperm. In contrast, the addition of AFP III to the freezing extender strongly increased sperm motility, mitochondria and cell membrane integrity, and the acrosomal proteolytic activity of frozen-thawed sperm in 5 of 6 individuals (P < 0.05). Furthermore, cryoinjury prevention by AFP III significantly enhanced sperm viability (by ATP content), and maintained DNA quality and in vitro sperm penetrability compared with AFP I treatment (P < 0.05). These findings demonstrate that AFP III addition to the freezing extender of boar sperm is more effective in maintaining sperm characteristics than the extender without AFP III or AFP I, despite individual differences in response.

Effect of the addition of antifreeze protein type I on the quality of post-thawed domestic cat epididymal sperm

Cryopreservation of domestic cat semen is mainly performed as a model for the establishment of endangered wild feline protocols. The supplementation of antifreeze protein type I (AFP I) to cryopreservation medium has shown improvement in frozen-thawed sperm quality in other species, but its effect on cat semen has not yet been tested. This study aimed to assess the addition of AFP I to cryopreservation medium in domestic cats. Sperm was obtained from the cauda epididymis of orchiectomized cats; sperm was then pooled in Tris buffer and allocated into three treatments, according to AFP I final concentration: 0 (control), 0.1, and 0.5 µg/ml. Nine replicates were cryopreserved in a two-step protocol and subsequently thawed at 37°C for 30 s. There was no difference (P > 0.05) among the control, 0.1 and 0.5 µg/ml groups for parameters such as motility, vitality, functional membrane integrity, mature chromatin, normal morphology, and sperm binding to egg perivitelline membrane. In the 0.5 μg/ml group only, percentages of live sperm with intact acrosome and of sperm with most inactive mitochondria (DAB III) showed a significant reduction, along with a tendency (P = 0.053) to an increase in the percentage of sperm with most active mitochondria (DAB II). In conclusion, the supplementation of 0.1 and 0.5 µg/ml of AFP I did not promote consistent beneficial effects on the overall sperm cryotolerance in domestic cats.

Oxidative stress and DNA fragmentation in frozen/thawed common Carp Cyprinus carpio sperm with and without supplemental proteins

Using cryopreservation techniques can increase the effectiveness of reproducing cultured fish species by ensuring a dependable supply of sperm, although the quality of the sperm could be impacted by the procedures involved. The goal of this study was to investigate the effect of purified seminal plasma transferrin (Tf), bovine serum albumin (BSA), and antifreeze protein (AFP) types I and III at 1 µg mL−1 on relevant characteristics of cryopreserved sperm from common carp Cyprinus carpio. We compared oxidative stress indices, antioxidant activity, and DNA fragmentation of fresh sperm to that frozen with extender only or with Tf, BSA, or AFP types I and III. Fresh sperm had significantly lower levels of thiobarbituric acid reactive substances (TBARS) compared to samples that underwent cryopreservation without protein treatment, which resulted in 0.54 ± 0.06 nmol/108 cells of TBARS. Carbonyl derivatives of proteins (CP) decreased significantly (ANOVA; P > 0.05) in carp sperm with addition of Tf, AFPI, and AFPIII. Significant differences in superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity were seen in sperm supplemented with Tf, BSA, AFPI, and AFPIII from those without. Significantly less DNA damage, expressed as percent tail DNA (11.56 ± 1.34) and olive tail moment (0.59 ± 0.13), was recorded in samples cryopreserved with Tf. The findings indicated that addition of Tf, BSA, AFPI, or AFPIII to cryopreservation medium is beneficial to sperm preservation. The mechanisms through which these proteins act positively on sperm need to be further investigated.

Cover Feature: Regulating Antifreeze Activity through Water: Latent Functions of the Sugars of Antifreeze Glycoprotein Revealed by Total Chemical Synthesis (Chem. Eur. J. 21/2023)

The sugar moiety of the antifreeze glycoprotein AFGP can form a unique dynamic water phase that includes disordered water molecules thereby inhibiting the freezing process. The property of this water phase seems to depend on both the structure and the number of sugar moieties. More information can be found in the Research Article by R. Okamoto, Y. Kajihara, and co-workers (DOI: 10.1002/chem.202203553).

The BrAFP1 promoter drives gene-specific expression in leaves and stems of winter rapeseed (Brassica rapa L.) under cold induction

BrAFP1(antifreeze protein in winter turnip rape) effectively limits recrystallization and growth of ice crystals. The BrAFP1 expression level determines whether the freezing-induced damage to winter turnip rape plants is avoided. This study analyzed the activity of the BrAFP1 promoters of several varieties at various cold tolerance levels. We cloned the BrAFP1 promoters from five winter rapeseed cultivars. The multiple sequence alignment revealed the presence of one inDel and eight single-nucleotide mutations (SNMs) in the promoters. One of these SNMs (base mutation from C to T) at the –836 site away from the transcription start site (TSS) enhanced the transcriptional activity of the promoter at low temperature. The promoter activity was specific in cotyledons and hypocotyls during the seedling stage and was referential in stems, leaves, and flowers but not the calyx. This consequently drove the downstream gene to be specifically expressed in leaves and stems, but not in roots at low temperature. The truncated fragment GUS staining assays revealed that the core region of the BrAFP1 promoter was included in the 98 bp fragment from the –933 to –836 site away from the TSS, which was necessary for transcriptional activity. The LTR element of the promoter significantly enhanced expression at low temperatures and suppressed expression at moderate temperatures. Moreover, the BrAFP1 5′-UTR intron bound the scarecrow-like transcription factor and enhanced expression at low temperature.