Research Progress on Plant Anti-Freeze Proteins

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 environ-ments.Proteins with these characteristics have been found infish 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 evolu-tionary events.Unlike the AFPs found infish and insects,plant AFPs have multiple hydrophilic ice-binding domains,and their recrystallization inhibition activity is about 10–100 times that offish 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 character-istics 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 thefield of biotech-nology.Therefore,based on the analysis of relevant literature,this article will delve into the concepts,character-istics,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.

L-proline feeding for augmented freeze tolerance of Camponotus japonicus Mayr

The successful cryopreservation of organs is a strong and widespread demand around the world but faces great challenges. The mechanisms of cold tolerance of organisms in nature inspirit researchers to find new solutions for these challenges. Especially, the thermal, mechanical, biological and biophysical changes during the regulation of freezing tolerance process should be studied and coordinated to improve the cryopreservation technique and quality of complex organs. Here the cold tolerance of the Japanese carpenter ants, Camponotus japonicus Mayr, was greatly improved by using optimal protocols and feeding on L-proline-augmented diets for 5 days. When cooling to -27.66 ℃, the survival rate of frozen ants increased from 37.50% ± 1.73% to 83.88% ± 3.67%. Profiling of metabolites identified the concentration of whole-body L-proline of ants increased from 1.78 to 4.64 ng g-1 after 5-day feeding. High Lproline level, together with a low rate of osmotically active water and osmotically inactive water facilitated the prevention of cryoinjury. More importantly, gene analysis showed that the expression of ribosome genes was significantly up-regulated and played an important role in manipulating freezing tolerance. To the best of our knowledge, this is the first study to link genetic variation to the enhancement of ants’ cold tolerance by feeding exogenous cryoprotective compound. It is worth noting that the findings provide the theoretical and technical foundation for the cryopreservation of more complex tissues,organs, and living organisms.

Overexpression of galactinol synthase 1 from Solanum commersonii(ScGolS1) confers freezing tolerance in transgenic potato

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.

Effect of Sugar on the Process of Cold-Acclimation-Induced Freezing Tolerance of Populus tomentosa

Populus tomentosa seedlings for cold-acclimating were pretreated wit h or without 20% saccharose. Changes in the concentrations of total soluble sugar , the survival rates, and freezing tolerance of seedlings during cold acclimatio n were investigated. The results showed that cold acclimation increased the conc entrations of total soluble sugar, the survival rates and freezing tolerance. Co ld acclimation, combined with the saccharose-pretreatment, enhanced the above- ment ioned effect of cold acclimation, and obviously increased the concentrations of total soluble sugar, the survival rates and freezing tolerance of seedlings. Fur ther analysis found that the concentrations of total soluble sugar in branches i ncreased greater than that in leaves during both cold acclimation with or withou t the pretreatment of saccharose. Moreover, an increase of the concentrations of total soluble sugar in branches and leaves was closely related to the freezing tolerance of seedlings. The results indicate the accumulation of soluble sugar i n seedlings induced by cold acclimation may be involved in the induction of free zing tolerance .

Effects of cold-hardening on compatible solutes and antioxidant enzyme activities related to freezing tolerance in Ammopiptanthus mongolicus seedlings

Cold acclimation is associated with many metabolic changes that lead to an increase of freezing tolerance. In order to investigate the biochemical process of cold acclimation in Ammopiptanthus mongolicus, seedlings were acclimated at 2℃ under 16-h photoperiod （150 μmol·m^-2·s^-1 photosynthetically active radiation） for 14 d. Freezing tolerance in seedlings increased after 14 d of cold-hardening. Contents of protein, proline and solute carbohydrate in cotyledon increased after cold acclimation. Patterns of isozymes of superoxide dismutase （SOD）, peroxidase, catalase and polyphenol oxidase （PPO） were investigated. The activities of SOD, peroxidase and PPO in cold acclimated plants were increased during cold-hardening. We deduced that compatible solutes and antioxidant enzymes play important roles in development of freezing tolerance during cold acclimation in this evergreen woody plant.

Anatomical Structure Comparison Between Leaves of Two Winter Wheat Cultivars with Different Cold/Freezing Tolerance Under Low Temperature Stress

Winter wheat （Triticum aestivum） cultivars Dongnongdongmai 1 with strong cold/freezing tolerance and Jimai 22 with weak cold/freezing tolerance were used for investigating the difference of microstructure and ultrastructure between leaves of two cultivars under low temperature stress （5℃ and -15 ℃） using optical and electron microscope. The results showed that there was no obvious difference between leaves of Dongnongdongmai 1 and Jimai 22 in microstructure. However, the difference between those leaves was distinct in ultrastructure. The grana lamella and stroma lamella were stacked regularly and arranged parallelly along the long axis of chloroplasts in cv. Dongnongdongmai 1, while the arrangement directions of thylakoids in Jimai 22＇s leaves were so irregular as to form various angles with the long axis of chloroplasts. At -15℃, the mitochondrias were swelled to be round and the structure of cristaes became blurry in both cultivars＇ leaves, while some cristaes of Jimai 22 disappeared. These results would provide theoretical evidence for selecting cold/freezing tolerant winter wheat germplasm resources

The NAC056 transcription factor confers freezing tolerance by positively regulating expression of CBFs and NIA1 in Arabidopsis

Freezing stress can seriously affect plant growth and development,but the mechanisms of these effects and plant responses to freezing stress require further exploration.Here,we identified a NAM,ATAF1/2,and CUC2(NAC)-family transcription factor(TF),NAC056,that can promote freezing tolerance in Arabidopsis.NAC056 mRNA levels are strongly induced by freezing stress in roots,and the nac056 mutant exhibits compromised freezing tolerance.NAC056 acts positively in response to freezing by directly promoting key C-repeat-binding factor(CBF)pathway genes.Interestingly,we found that CBF1 regulates nitrate assimilation by regulating the nitrate reductase gene NIA1 in plants;therefore,NAC056–CBF1–NIA1 form a regulatory module for the assimilation of nitrate and the growth of roots under freezing stress.In addition,35S::NAC056 transgenic plants show enhanced freezing tolerance,which is partially reversed in the cbfs triple mutant.Thus,NAC056 confers freezing tolerance through the CBF pathway,mediating plant responses to balance growth and freezing stress tolerance.

Photoperiod-and temperature-mediated control of the ethylene response and winter dormancy induction in Prunus mume

Plant dormancy is essential for perennial plant survival.Different genotypes of Prunus mume,including Eumume group and Apricot Mei group,undergo leaf senescence and dormancy at different times.In order to verify the cold resistance of P.mume,freeze resistance evaluation was carried out.Our results showed that Apricot Mei group had a stronger freezing tolerance than Eumume group and that leaf senescence and dormancy of Apricot Mei group occurred at an earlier period before winter.Based on phenotypic data in response to seasonal climate change,the significant candidate regions were selected using GWAS.Furthermore,through KEGG pathway and qRT-PCR analyses,we found that the ethylene-related genes,including PmEIL(Pm002057)and PmERF(Pm004265),were significantly upregulated in‘Songchun’Mei(Apricot Mei group)and downregulated in‘Zaohua Lve’Mei(Eumume group).Ethylene-related genes expression models showed that ethylene may be indirectly involved in the induction of dormancy.The PmEIL and PmERF genes were the core genes of the ethylene signal transduction pathway and were regulated by the exogenous ACC or PZA compounds.For non-dormant or weekly dormant perennial plants,application of ACC was able to induce plant dormancy and thus enhance cold/freeze tolerance.Overall,the expression of the major ethylene genes played a significant role in dormancy induction and freezing tolerance in P.mume;accordingly,application of ACC and PZA compounds were an effective approach for enhancing cold/freeze of tolerance of woody plant.

Cloning and Sequence Analysis of a Glucose-6-Phosphate Dehydrogenase Gene PsG6PDH from Freezing-tolerant Populus suaveolens

A 1 207 bp cDNA fragment (PsG6PDH) was amplified by RT-PCR from cold-induced total RNA of the freez- ing-tolerant P. Suaveolens, using primers based on the highly conserved region of published plant glucose-6-phosphate dehydro- genase (G6PDH) genes. The sequence analysis showed that PsG6PDH coding region had 1 101 bp and encoded 367 predicted amino acid residues. Moreover, the nucleotide sequence of PsG6PDH showed 83%, 82%, 79%, 79% and 78% identity, and the derived amino acid sequence shared 44.2%, 44.7%, 42.0%, 40.5% and 43.9% identity with those of the Solanum tuberosum, Nicotiana ta- bacum, Triticum aestivum, Oryza sativa and Arabidopsis thaliana, respectively. The results show that PsG6PDH is a new member of G6PDH gene family and belongs to the cytosolic G6PDH gene. This is the first report on cloning of the G6PDH gene from woody plants.

Ultrastructural and Extracellular Protein Changes in Cell Suspension Cultures of Populus euphratica Associated with Low Temperature-induced Cold Acclimation

Populus euphratica Olive is the only tree species that can grow in the saline land and also survive cold winters in northwest China, and it plays a very important role in stabilizing the vulnerable ecosystem there. A cell suspension culture was initiated from callus derived from plantlets of Populus euphratica. Cold acclimation was induced (LT50 of 17.5 ℃) in cell suspension at 45 ℃ in the dark for 30 days and the freezing tolerance increased from LT50 of 12.5 ℃ in nonacclimated cells to LT50 of 17.5 ℃ in cold-acclimated cells. Microvacuolation, cytoplasmic augmentation and accumulation of starch granules were observed in cells that were cold-acclimated by exposure to low temperatures. Several qualitative and quantitative changes in proteins were noted during cold acclimation. Antibodies to carrot extracellular (apoplastic) 36 kD antifreeze protein did not cross react on immunoelectroblots with extracellular proteins in cell suspension culture medium of Populus euphratica, indicating no common epitopes in the carrot 36 kD antifreeze protein and P. euphratica extracellular proteins. The relationship of these changes to cold acclimation in Populus euphratica cell cultures was discussed.

Major physiological adjustments in freezing-tolerant grey tiger longicorn beetle(Xylotrechus rusticus) during overwintering period

1Xylotrechus rusticus （Linnaeus） is one of the most destruc-tive woodborers in northeast China;it damages poplar and is listed as a domestic forestry quarantine pest. Overwintering larvae were collected during October 2012 and March 2013 in Harbin, China, to quantify indi-cators related to the insect’s overwintering strategy and the major cryo-protectants. The supercooling points （SCPs）, which ranged from-14.7＆#176;C to -2.9＆#176;C, were higher than the lethal temperatures of LT50 （-33.64＆#176;C） and LT99 （-40.17＆#176;C） after 24 h exposure. , also the minimum mean daily temperature （-24.5＆#176;C） and mean monthly temperature （-18.0＆#176;C） at the sampling site in January during 2008-2012. Thus, X. rusticus is a typical freezing-tolerant insect. Glycerol serves as a major cryoprotectant for overwintering larvae , because it was the only polyol accumulated during the winter and it also had a significant negative correlation with the SCP （p=0.033, R=0.907）. The glycogen and lipid are major sources of ener-gy and their levels decreased substantially in the middle of overwintering, when glycogen had a significant correlation with the SCP （p= 0.006, R=0.971） whereas the lipid contents did not. Moreover, inter-conversions between glycerol and glycogen, as well as mannose and glycogen, were suggested by their negative correlations. The water content did not change obviously during the winter and was not correlated with the SCP. The free amino acids in the hemolymph and the total protein contents of the bodies of larvae changed significantly during winter, although both had no correlations with the SCP.

Seasonal variation of foliar δ^(13)C values and its indictor significance in Sabina przewalskii and Sabina chinensis

Seasonal variation of foliar δ13C values as well as proline, silicon, MDA and relative water content in Sabina przewalskii Kom. and S. chinensis （Lin.） Ant. were measured. The results show that foliar δ13C values were significantly different at different seasons： lower in winter but higher in summer. δ13C values were positively correlated with air temperature and soil temperature. This demonstrates that foliar δ13C of Sabina is a successful empirical indictor of temperature. Furthermore, foliar δ13C values were positively related to proline and silicon content, and negatively related to relative water content and MDA content. Compared with S. chinensis, S. przewalskii has higher δ13C values, proline and silicon content as well as lower MDA and relative water content. All these results provided strong evidence that it is feasible for δ13C to be regarded as another index to evaluate freezing tolerance of Sabina.

BZR1 Positively Regulates Freezing Tolerance via CBF-Dependent and CBF-Independent Pathways in Arabidopsis

Cold stress is a major environmental factor that adversely affects plant growth and development. The C-repeat binding factor/DRE binding factor 1 （CBF/DREB1） transcriptional regulatory cascade has been shown to play important roles in plant response to cold. Here we demonstrate that two key components of brassinosteroid （BR） signaling modulate freezing tolerance of Arabidopsis plants. The loss-of-function mutant of the GSK3-1ike kinases involved in BR signaling, bin2-3 bill bil2, showed increased freezing tolerance, whereas overexpression of BIN2 resulted in hypersensitivity to freezing stress under both non-acclimated and acclimated conditions. By contrast, gain-of-function mutants of the transcription factors BZR1 and BES1 displayed enhanced freezing tolerance, and consistently cold treatment could induce the accumulation of dephosphorylated BZR1. Biochemical and genetic analyses showed that BZR1 acts upstream of CBF1 and CBF2 to directly regulate their expression. Moreover, we found that BZR1 also regulated other COR genes uncoupled with CBFs, such as WKRY6, PYL6, SOCl, JMT, and SAG21, to modulate plant response to cold stress. Consistently, wrky6 mutants showed decreased freezing tolerance. Taken together, our results indicate that BZR1 positively modulates plant freezing tolerance through CBF-dependent and CBF-independent pathways.

Cold-Induced CBF–PIF3 Interaction Enhances Freezing Tolerance by Stabilizing the phyB Thermosensor in Arabidopsis

Growth inhibition and cold-acclimation strategies help plants withstand cold stress,which adversely affects growth and survival.PHYTOCHROME B(phyB)regulates plant growth through perceiving both light and ambient temperature signals.However,the mechanism by which phyB mediates the plant response to cold stress remains elusive.Here,we show that the key transcription factors mediating cold acclimation,C-REPEAT BINDING FACTORs(CBFs),interact with PHYTOCHROME-INTERACTING FACTOR 3(PIF3)under cold stress,thus attenuating the mutually assured destruction of PIF3–phyB.Cold-stabilized phyB acts downstream of CBFs to positively regulate freezing tolerance by modulating the expression of stress-responsive and growth-related genes.Consistent with this,phyB mutants exhibited a freezing-sensitive phenotype,whereas phyB-overexpression transgenic plants displayed enhanced freezing tolerance.Further analysis showed that the PIF1,PIF4,and PIF5 proteins,all of which negatively regulate plant freezing tolerance,were destabilized by cold stress in a phytochrome-dependent manner.Collectively,our study reveals that CBFs–PIF3–phyB serves as an important regulatory module for modulating plant response to cold stress.

Disruption of the Arabidopsis Defense Regulator Genes SAG101, EDS1, and PAD4 Confers Enhanced Freezing Tolerance

In Arabidopsis, three lipase-like regulators, SAG101, EDS1, and PAD4, act downstream of resistance protein-associated defense signaling. Although the roles of SAG101, EDS1, and PAD4 in biotic stress have been extensively studied, little is known about their functions in plant responses to abiotic stresses. Here, we show that SAG101, EDS1, and PAD4 are involved in the regulation of freezing tolerance in Arabidopsis. With or without cold acclimation, the sag101, edsl, and pad4 single mutants, as well as their double mutants, exhibited similarly enhanced tolerance to freezing temperatures. Upon cold exposure, the sag101, edsl, and pad4 mutants showed increased transcript levels of C-REPEAT/DRE BINDING FACTORs and their regulons compared with the wild type. Moreover, freezing-induced cell death and accumulation of hydrogen peroxide were ameliorated in sag101, edsl, and pad4 mutants. The sag101, edsl, and pad4 mutants had much lower salicylic acid （SA） and diacylglycerol （DAG） contents than the wild type, and exogenous application of SA and DAG compromised the freezing tolerance of the mutants. Furthermore, SA suppressed the cold-induced expression of DGATs and DGKs in the wild-type leaves. These findings indicate that SAG101, EDS1, and PAD4 are involved in the freezing response in Arabidopsis, at least in part, by modulating the homeostasis of SA and DAG.

Farinose fiavonoids are associated with high freezing tolerance in fairy primrose (Primula malacoides) plants

The deposition of surface （farinose） flavonoids on aerial parts of some Primula species is a well-documented but poorly understood phenomenon. Here, we show that flavonoid deposition on the leaves and winter buds may contribute strongly to preventing freezing damage in these plants. The ice nucleation temperature of fairy primrose （Primula malacoides） leaves covered with natural flavone was approximately 6~C lower compared to those that had their flavone artificially removed. Additionally, farinose flavonoids on the leaves reduced subse- quent electrolyte leakage （EL） from the cells exposed to freezing temperatures. Interestingly, exogenous application of flavone at 4 mg/g fresh weight to P. malacoides leaves, which had the original flavone mechanically removed, restored freezing tolerance, and diminished EL from the cells to pretreatment values. Our results suggest that farinose flavonoids may function as mediators of freezing tolerance in P. malacoides, and exogenous application of flavone could be used to reduce freezing damage during sudden but predictable frost events in other plant species.

MicroRNA Regulation in Extreme Environments: Differential Expression of MicroRNAs in the Intertidal Snail Littorina littorea During Extended Periods of Freezing and Anoxia

Several recent studies of vertebrate adaptation to environmental stress have suggested roles for microRNAs （miRNAs） in regulating glo- bal suppression of protein synthesis and/or restructuring protein expression patterns. The present study is the first to characterize stress- responsive alterations in the expression of miRNAs during natural freezing or anoxia exposures in an invertebrate species, the intertidal gastropod Littorina littorea. These snails are exposed to anoxia and freezing conditions as their environment constantly fluctuates on both a tidal and seasonal basis. The expression of selected miRNAs that are known to influence the cell cycle, cellular signaling pathways, carbohydrate metabolism and apoptosis was evaluated using RT-PCR. Compared to controls, significant changes in expression were observed for miR-la-1, miR-34a and miR-29b in hepatopancreas and for miR-la-1, miR-34a, miR-133a, miR-125b, miR-29b and miR-2a in foot muscle after freezing exposure at -6 ~C for 24 h （P 〈 0.05）. In addition, in response to anoxia stress for 24 h, significant changes in expression were also observed for miR-la-1, miR-210 and miR-29b in hepatopancreas and for miR-1 a-1, miR-34a, miR-133a, miR-29b and miR-2a in foot muscle （P 〈 0.05）. Moreover, protein expression of Dicer, an enzyme responsible for mature microRNA processing, was increased in foot muscle during freezing and anoxia and in hepatopancreas during freezing. Alterations in expression of these miRNAs in L. littorea tissues may contribute to organismal survival under freezing and anoxia.

Proteomics Analysis Reveals Post-Translational Mechanisms for Cold-Induced Metabolic Changes in Arabidopsis

Cold-induced changes of gene expression and metabolism are critical for plants to survive freezing. Largely by changing gene expression, exposure to a period of non-freezing low temperatures increases plant tolerance to freezing--a phenomenon known as cold acclimation. Cold also induces rapid metabolic changes, which provide instant protection before temperature drops below freezing point. The molecular mechanisms for such rapid metabolic responses to cold remain largely unknown. Here, we use two-dimensional difference gel electrophoresis （2-D DIGE） analysis of sub-cellular fractions ofArabidopsis thaliana proteome coupled with spot identification by tandem mass spectrometry to identify early cold-responsive proteins in Arabidopsis. These proteins include four enzymes involved in starch degradation, three HSP100 proteins, several proteins in the tricarboxylic acid cycle, and sucrose metabolism. Upon cold treatment, the Disproportio- nating Enzyme 2 （DPE2）, a cytosolic transglucosidase metabolizing maltose to glucose, increased rapidly in the centrifugation pellet fraction and decreased in the soluble fraction. Consistent with cold-induced inactivation of DPE2 enzymatic activity, the dpe2 mutant showed increased freezing tolerance without affecting the C-repeat binding transcription factor （CBF） transcriptional pathway. These results support a model that cold-induced inactivation of DPE2 leads to rapid accumulation of maltose, which is a cold-induced compatible solute that protects cells from freezing damage. This study provides evidence for a key role of rapid post-translational regulation of carbohydrate metabolic enzymes in plant protection against sudden temperature drop.

Target deletion of the AAA ATPase PpCDC48II in Physcomitrella patens results in freezing sensitivity after cold acclimation

CDC48 is a highly conserved protein in eukaryotes and belongs to the AAA （ATPase associated with a variety of cellular activities） superfamily. It can interact with many different cofactors and form protein complexes that play important roles in various cellular processes. According to the Physcomitrella patens database, one member of the ATPases, the cell cycle gene PpCDC4811, was cloned. PpCDC48II contains two typical ATPase modules and is highly homologous to AtCDC48A. PpCDC4811 was up-regulated in mRNA levels after incubation at 0~C for 36 and 72 h. To further elucidate protein function, we disrupted the PpCDC4811 gene by transforming P. patens with the corresponding linear genomic sequences. When treated to the same freezing stress, it was found that PpCDC4811 knockout plants were less resistant to freezing treatment than wild type after acclimation. This suggested that PpCDC481I was an essential gene for low-temperature-induced freezing tolerance in P. patens cells.