Identification, through transcriptome analysis, of transcription factors that regulate anthocyanin biosynthesis in different parts of red-fleshed apple 'May' fruit

Anthocyanins, a class of flavonoid compounds that provide pigmentation to plant organs, are beneficial components of the human diet and there is an interest in understanding the regulation of their formation in crop plants. The apple cultivar ‘May’ represents an excellent model for the study of anthocyanin metabolism due to the spatial color pattern of its flesh, which transitions sequentially from red to white to red again,from the outside to the inside of the apple fruit. To screen the transcription factors involved in the anthocyanin pathway, transcriptomes from differently colored sections of ‘May’ fruits were analyzed by RNA sequencing. Kyoto Encyclopedia of Genes and Genomes(KEGG) database was used to analyse the gene expression data. The results suggested differentially expressed genes(DEGs) that are related to phytohormones are involved in regulating anthocyanin biosynthesis. K-means clustering analysis revealed 167 common DEGs between different sections of fruit with the same expression pattern as candidates for regulating anthocyanin synthesis. Further analysis showed that nine of the 167 DEGs were annotated as transcription factors and quantitative real time PCR(qRT-PCR) confirmed that their expression was obviously higher in red regions of the fruit, consistent with their roles as hub genes that regulate anthocyanin synthesis. This study provides valuable results for future studies of anthocyanin synthesis in apple fruit.

Overexpression of the transcription factor MdWRKY115 improves drought and osmotic stress tolerance by directly binding to the MdRD22 promoter in apple

Abiotic stress reduces plant yield and quality.WRKY transcription factors play key roles in abiotic stress responses in plants,but the molecular mechanisms by which WRKY transcription factors mediate responses to drought and osmotic stresses in apple(Malus×domestica Borkh.)remain unclear.Here,we functionally characterized the apple GroupⅢWRKY gene MdWRKY115.qRT-PCR analysis showed that MdWRKY115 expression was up-regulated by drought and osmotic stresses.GUS activity analysis revealed that the promoter activity of MdWRKY115 was enhanced under osmotic stress.Subcellular localization and transactivation assays indicated that MdWRKY115 was localized to the nucleus and had a transcriptional activity domain at the N-terminal region.Transgenic analysis revealed that the overexpression of MdWRKY115 in Arabidopsis plants and in apple callus markedly enhanced their tolerance to drought and osmotic stresses.DNA affinity purification sequencing showed that MdWRKY115 binds to the promoter of the stress-related gene MdRD22.This binding was further verified by an electrophoretic mobility shift assay.Collectively,these findings suggest that MdWRKY115 is an important regulator of osmotic and drought stress tolerance in apple.

Comparative transcriptome analysis reveals key genes and pathways in response to Alternaria alternata apple pathotype infection

Apple leaf spot,caused by the Alternaria alternata apple pathotype(AAAP),is an important fungal disease of apple.To understand the molecular basis of resistance and pathogenesis in apple leaf spot,the transcriptomes of two apple cultivars‘Hanfu'(HF)(resistant)and‘Golden Delicious'(GD)(susceptible)were analyzed at 0,6,18,24 and 48 h after AAAP inoculation by RNA-Seq.At each time point,a large number of significantly differentially expressed genes(DEGs)were screened between AAAP-inoculated and uninoculated apple leaves.Analysis of the common DEGs at four time points revealed significant differences in the resistance of‘HF'and‘GD'apple to AAAP infection.RLP,RNL,and JA signal-related genes were upregulated in both cultivars to restrict AAAP development.However,genes encoding CNLs,TNLs,WRKYs,and AP2s were only activated in‘HF'as part of the resistance response,of which,some play major roles in the regulation of ET and SA signal transduction.Further analysis showed that many DEGs with opposite expression trends in the two hosts may play important regulatory roles in response to AAAP infection.Transient expression of one such gene MdERF110 in‘GD'apple leaves improved AAAP resistance.Collectively,this study highlights the reasons for differential resistance to AAAP infection between‘HF'and‘GD'apples which can theoretically assist the molecular breeding of disease-resistant apple crops.

Biochar alleviates apple replant disease by reducing the growth of Fusarium oxysporum and regulating microbial communities

Apple replant disease(ARD)negatively affects plant growth and reduces yields in replanted orchards.In this study,biochar was applied to apple replant soil with Fusarium oxysporum.Our aim was to investigate whether biochar could promote plant growth and alleviate apple replant disease by reducing the growth of harmful soil microorganisms,changing soil microbial community structure and improving the soil environment.This experiment included five treatments:apple replant soil(CK),methyl bromide fumigation apple replant soil(FM),replant soil with biochar addition(2%),replant soil with F.oxysporum spore solution(8×10^(7)spores·mL^(-1)),and replant soil with biochar and F.oxysporum spore solution addition.Seedling biomass,the activity of antioxidant enzymes in the leaves and roots,and soil environmental variables were measured.Microbial community composition and community structure were analyzed using 16SrDNA and ITS2 gene sequencing.Biochar significantly reduced the abundance of F.oxysporum and increased soil microbial diversity and richness.Biochar also increased the soil enzyme activities(urease,invertase,neutral phosphatase,and catalase),the biomass(plant height,fresh weight,dry weight)and the activity of antioxidant enzymes(superoxide dismutase,peroxidase,and catalase).The root indexes of apple seedlings was also increased in replant soil by biochar.In sum,biochar promoted the growth of plants,improved the replant soil environment,and alleviated apple replant disease.

The role of the auxin-response genes MdGH3.1 and MdSAUR36 in bitter pit formation in apple

Apples often exhibit bitter pits in response to metabolic disorders during ripening and storage;however, the mechanisms underlying the bitter pit(BP) development remain unclear. Here, metabolome and transcriptome analyses were performed to investigate BP pulp of 'Fuji'. Two auxin-response genes, MdGH3.1 and MdSAUR36, were screened. Their expression as well as the auxin content in BP pulp were found to be higher than those in healthy pulp(P < 0.01). In the field, excess CO(NH2)2increased the incidence of BP. Moreover, the auxin content and MdGH3.1 expression increased in apples after nitrogen fertilization. On Day 30 before harvest, the two genes were transiently transferred to the fruit, and 20.69% and 23.21% of BP fruits were harvested. After 10 μmol·L-1auxin was infiltrated at low pressure into postharvest fruit, the increase in MdGH3.1 expression occurred earlier than that in MdSAUR36. MdGH3.1 increased the expression of MdSAUR36, but MdSAUR36 did not increase expression of MdGH3.1. Therefore, we suggest that MdGH3.1 acts upstream of MdSAUR36 during BP formation and that these genes induce BP formation by regulating auxin and phenylpropanoid biosynthesis.

Silencing of early auxin responsive genes MdGH3-2/12 reduces the resistance to Fusarium solani in apple

Apple replant disease(ARD)has led to severe yield and quality reduction in the apple industry.Fusarium solani(F.solani)has been identified as one of the main microbial pathogens responsible for ARD.Auxin(indole-3-acetic acid,IAA),an endogenous hormone in plants,is involved in almost all plant growth and development processes and plays a role in plant immunity against pathogens.Gretchen Hagen3(GH3)is one of the early/primary auxin response genes.The aim of this study was to evaluate the function of MdGH3-2 and MdGH3-12 in the defense response of F.solani by treating MdGH3-2/12 RNAi plants with F.solani.The results show that under F.solani infection,RNAi of MdGH3-2/12 inhibited plant biomass accumulation and exacerbated root damage.After inoculation with F.solani,MdGH3-2/12 RNAi inhibited the biosynthesis of acid-amido synthetase.This led to the inhibition of free IAA combining with amino acids,resulting in excessive free IAA accumulation.This excessive free IAA altered plant tissue structure,accelerated fungal hyphal invasion,reduced the activity of antioxidant enzymes(SOD,POD and CAT),increased the reactive oxygen species(ROS)level,and reduced total chlorophyll content and photosynthetic ability,while regulating the expression of PR-related genes including PR1,PR4,PR5 and PR8.It also changed the contents of plant hormones and amino acids,and ultimately reduced the resistance to F.solani.In conclusion,these results demonstrate that MdGH3-2 and MdGH3-12 play an important role in apple tolerance to F.solani and ARD.

Transcriptional regulation of MdPIN7 by MdARF19 during gravityinduced formation of adventitious root GSA in self-rooted apple stock

Self-rooted apple stock is widely used for apple production.However,the shallowness of the adventitious roots in self-rooted apple stock leads to poor performance in the barren orchards of China.This is because of the considerable difference in the development of a gravitropic set-point angle(GSA)between self-rooted apple stock and seedling rootstock.Therefore,it is crucial to study the molecular mechanism of adventitious root GSA in self-rooted apple stock for breeding self-rooted and deep-rooted apple rootstock cultivars.An apple auxin response factor MdARF19 functioned to establish the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.MdARF19 bound directly to the MdPIN7 promoter,activating its transcriptional expression and thus regulating the formation of the adventitious root GSA in 12-2 self-rooted apple stock.However,MdARF19 influenced the expression of auxin efflux carriers(MdPIN3 and MdPIN10)and the establishment of adventitious root GSA of self-rooted apple stock in response to gravity signals by direct activation of MdFLP.Our findings provide new information on the transcriptional regulation of MdPIN7 by auxin response factor MdARF19 in the regulation of the adventitious root GSA of self-rooted apple stock in response to gravity and auxin signals.

Improved multi-scale inverse bottleneck residual network based on triplet parallel attention for apple leaf disease identification

Accurate diagnosis of apple leaf diseases is crucial for improving the quality of apple production and promoting the development of the apple industry. However, apple leaf diseases do not differ significantly from image texture and structural information. The difficulties in disease feature extraction in complex backgrounds slow the related research progress. To address the problems, this paper proposes an improved multi-scale inverse bottleneck residual network model based on a triplet parallel attention mechanism, which is built upon ResNet-50, while improving and combining the inception module and ResNext inverse bottleneck blocks, to recognize seven types of apple leaf(including six diseases of alternaria leaf spot, brown spot, grey spot, mosaic, rust, scab, and one healthy). First, the 3×3 convolutions in some of the residual modules are replaced by multi-scale residual convolutions, the convolution kernels of different sizes contained in each branch of the multi-scale convolution are applied to extract feature maps of different sizes, and the outputs of these branches are multi-scale fused by summing to enrich the output features of the images. Second, the global layer-wise dynamic coordinated inverse bottleneck structure is used to reduce the network feature loss. The inverse bottleneck structure makes the image information less lossy when transforming from different dimensional feature spaces. The fusion of multi-scale and layer-wise dynamic coordinated inverse bottlenecks makes the model effectively balances computational efficiency and feature representation capability, and more robust with a combination of horizontal and vertical features in the fine identification of apple leaf diseases. Finally, after each improved module, a triplet parallel attention module is integrated with cross-dimensional interactions among channels through rotations and residual transformations, which improves the parallel search efficiency of important features and the recognition rate of the network with relatively small computational costs while the dimensional dependencies are improved. To verify the validity of the model in this paper, we uniformly enhance apple leaf disease images screened from the public data sets of Plant Village, Baidu Flying Paddle, and the Internet. The final processed image count is 14,000. The ablation study, pre-processing comparison, and method comparison are conducted on the processed datasets. The experimental results demonstrate that the proposed method reaches 98.73% accuracy on the adopted datasets, which is 1.82% higher than the classical ResNet-50 model, and 0.29% better than the apple leaf disease datasets before preprocessing. It also achieves competitive results in apple leaf disease identification compared to some state-ofthe-art methods.

Effect of Different Raw Material Processing Methods on the Quality of Apple Distilled Spirits

In this study,the effect of yeast strains(X16 and RMS2),fruit seed,and pectinase on the quality of apple distilled spirits were investigated with apple as material.The results showed that pectinase shortened the fermentation period.The strain X16,fruit seed remaining,and pectinase addition groups had higher yields of crude distilled spirits than the strain RMS2,fruit seed removal,and without pectinase groups,respectively.Regarding the first-grade distilled spirits quality,the X16 group had higher content of total acids,total esters,higher alcohols,and alcohol content than the RMS2 group;the group with fruit seeds had higher total acids but lower alcohol content and total esters than the group with fruit seed removal;the group with pectinase addition had higher total acids and alcohol content than the group without pectinase addition.Regarding the second-grade distilled spirits quality,the X16 group had higher total acids,total esters,and alcohol content than the RMS2 group;the group without fruit seeds had higher alcohol content than the group with fruit seeds;the group with pectinase addition showcased higher total acids but lower alcohol content than the group without pectinase addition.In summary,yeast strains and pectinase affected the quality of apple distilled spirits,and strain X16 was more suitable for brewing apple distilled spirits.Pectinase affected the quality of apple distilled spirits by affecting fermentation rate and temperature.

Overexpression of auxin/indole-3-acetic acid gene MdIAA24 enhances Glomerella leaf spot resistance in apple(Malus domestica)

Auxin is throughout the entire life process of plants and is involved in the crosstalk with other hormones,yet its role in apple disease resistance remains unclear.In this study,we investigated the function of auxin/indole-3-acetic acid(IAA)gene Md IAA24 overexpression in enhancing apple resistance to Glomerella leaf spot(GLS)caused by Colletotrichum fructicola(Cf).Analysis revealed that,upon Cf infection,35S::Md IAA24 plants exhibited enhanced superoxide dismutase(SOD)and peroxidase(POD)activity,as well as a greater amount of glutathione(reduced form)and ascorbic acid accumulation,resulting in less H_(2)O_(2)and superoxide anion(O_(2)^(-))in apple leaves.Furthermore,35S::Md IAA24 plants produced more protocatechuic acid,proanthocyanidins B1,proanthocyanidins B2 and chlorogenic acid when infected with Cf.Following Cf infection,35S::Md IAA24 plants presented lower levels of IAA and jasmonic acid(JA),but higher levels of salicylic acid(SA),along with the expression of related genes.The overexpression of Md IAA24 was observed to enhance the activity of chitinase andβ-1,3-glucanase in Cfinfected leaves.The results indicated the ability of Md IAA24 to regulate the crosstalk between IAA,JA and SA,and to improve reactive oxygen species(ROS)scavenging and defense-related enzymes activity.This jointly contributed to GLS resistance in apple.

Potassium alleviated high nitrogen-induced apple growth inhibition by regulating photosynthetic nitrogen allocation and enhancing nitrogen utilization capacity

There is a close relationship between potassium(K)and nitrogen(N).However,the roles of K under high N conditions remain unclear.Using a hydroponics approach,we monitored the morphological,physiological,and molecular changes in M9T337 apple(Malus domestica)rootstocks under different nitrate(10 and 30 mmol·L^(-1)NO_(3)^(-))and K supply(0.5,6,10,and 20 mmol·L_(-1)K^(+))conditions.Results revealed that high nitrate inhibited the root growth of M9T337 rootstocks,downregulated the expressions of K transporter genes(MdPT5,MdHKT1,and MdATK1),and reduced the net NO3-and K+influx at the surface of roots,thereby resulting in an N/K imbalance in rootstocks.Further investigation showed that 10 mmol·L^(-1)K increased the activity of N metabolic enzymes(NR,GS,NiR,and GOGAT),upregulated the expressions of genes related to nitrate uptake and transport(MdNRT1.1,MdNRT1.2,MdNRT1.5,and MdNRT2.4),promoted15N transport from the roots to the shoots,optimized leaf N distribution,and improved photosynthetic N utilization efficiency under high nitrate conditions.These results suggest that the negative effects of high nitrate may be related to the N/K imbalance and that reducing N/K in plants by increasing K supply level can effectively alleviate the inhibition of N assimilation by high nitrate stress.

ALL ABOUT APPLES!

Crunch!Apples are a sweet and delicious fruit.There are over 7,500 different kinds!The fruit is usually red,but it can be green or other colors.Applesaren't expensive.They make a great snack.You can eat a whole apple.You can slice it,too.They're also good with other foods.Do you like apples?

Efficient Breeding and Cultivation of Type 2 Red-fleshed Apple Cultivars Using a Search System for Suitable Apple Cultivar Combination

As apple possesses self-incompatibility, knowledge of the S-genotypes is very important for apple breeding and cultivation. We investigated the S-RNase genotypes of 13 type 2 red-fleshed apple cultivars and 8 lineages. All of the investigated type 2 red-fleshed apples contained the S3-RNase allele closely linked to the red-fleshed trait. We made a search system for ovule parents to efficiently produce new type 2 red-fleshed cultivars based on the S-RNase allele information(http://www.agr.nagoya-u.ac.jp/～hort/apple/red/). More than 90% of progenies from crossing a selected white-fleshed ovule parent with a type 2 red-fleshed pollen parent are expected to show the red-fleshed phenotype. We also compiled a database of the S-RNase genotypes of more than 1 000 apple cultivars, including a survey system of cultivar combinations showing those that were fully incompatible, semicompatible and fully compatible, written in Chinese(http://www.agr.nagoya-u.ac.jp/～hort/apple/ch/) and Japanese(http://www.agr.nagoya-u.ac.jp/～hort/apple/ja/).

A MYB-INDUCED MECHANISM FOR ENHANCED ASCORBATE IN RED-FLESHED APPLES

The results presented in"The anthocyanin biosynthetic regulator MdMYBl positively regulates ascorbic acid biosynthesis in apple"(An et al.#this issue)provide evidence for a new mechanism for the elevation of ascorbate concentration in apple.Using a red-fleshed apple breeding population,the authors show how the anthocyanin-regulating MYB transcription factor,MdMYBl,also increases ascorbate concentrations by directly activating transcription of the dehydroascorbate reductase gene MdDHAR.This gene recycles oxidized ascorbate back to ascorbate,leading to elevated concentrations of vitamin C.These red-fleshed apples have enhanced concentrations of both anthocyanins and ascorbate,both of which are appealing traits for the development of healthier apples.

HATCH评分、APPLE评分在心房颤动患者射频消融术后复发的预测价值研究

目的探讨HATCH评分、APPLE评分在心房颤动(房颤)患者射频消融术后复发的预测价值。方法选取2018年5月至2020年6月于秦皇岛市第一医院心血管内科行射频消融术治疗的105例房颤患者的临床资料进行回顾性分析,观察手术时间、X线曝光时间、消融成功率及复发率;且根据术后3个月后是否出现持续时间≥30 s的快速性房性心律失常分为复发组(n=36)和未复发组(n=69),分析影响复发的危险因素,评估HATCH评分、APPLE评分及两项联合评估房颤患者术后复发的AUC值、敏感度、特异度。结果105例房颤患者均顺利完成射频消融术,消融成功率为65.71%,复发率为34.29%。复发组与未复发组在性别、年龄、病程、合并高血压、糖尿病、心力衰竭、估算的肾小球滤过率(eGFR)、持续性房颤、左心室大小、左室射血分数(LVEF)、右室前后径、升主动脉内径、肺动脉主干内径、室内隔厚度、左室后壁厚度、主动脉瓣口血流速度、肌酐等资料比较中,差异均无统计学意义(P>0.05);而在左心房大小、E峰、HATCH评分、APPLE评分等比较中,差异具有统计学意义(P<0.05)。二元Logistic回归分析显示,左心房大小、E峰、HATCH评分、APPLE评分是影响手术治疗后复发的独立危险因素(P<0.001)。ROC曲线分析显示,HATCH评分、APPLE评分及两项联合评估房颤患者术后复发的AUC值分别为(0.747、0.807、0.927,P<0.05);敏感度分别为44.40%、69.40%、94.40;特异度分别为91.30%、85.50%、84.10%。结论HATCH评分、APPLE评分预测房颤患者射频消融术后复发具有一定价值,且两项联合有利于提高敏感度,能为识别术后复发提供指导。

高龄患者围术期APPLE-MDT系统设计与应用

目的以患者为中心,以“全人、个体化”诊疗为目标,采用多学科协作围术期全程管理模式,保障高龄手术患者的医疗安全。方法建设高龄患者围术期APPLE-MDT系统,通过多学科专家评估量表,自动生成评估报告,指导临床医生对患者术前、术中、术后、远期进行有针对性的干预治疗。结果APPLE-MDT管理模式应用于高龄骨科患者围术期,初步实现人工智能辅助决策,高龄患者例数逐年增加,术前住院日和住院天数减少,术后并发症发生率降低到2.4%,可手术率提高到90%,术后死亡率为0。结论高龄患者围术期APPLE-MDT系统辅助建立了高龄患者围术期全数据链数据库,构建了高龄患者围术期新路径管理,体现了先进的医疗管理理念,推动了高质量科研产出。

Extracellular and intracellular infection of Botryosphaeria dothidea and resistance mechanism in apple cells

Apple ring rot,which is caused by Botryosphaeria dothidea,severely affects apple production.The mechanisms employed in apple cells against B.dothidea remain unknown.In this research,the pathogen infection mode and the relationship between cell death and disease resistance in‘Fuji’/B.dothidea interaction pathosystem were investigated.By using transmission electron microscopy(TEM),our research showed that the pathogen infects apple cells both intracellularly and extracellularly.However,compared with that in immature fruit,the incidence of hyphae in the interior of mature apple fruit cells increased dramatically,suggesting that cell wall-mediated penetration resistance could be important in apple resistance against B.dothidea.TEM ultrastructural characterization identified the nuclear morphology of programmed cell death induction in both apple fruit and callus cells under B.dothidea infection.Overexpression of MdVDAC2(MDP0000271281),which encodes an outer-membrane localized anion channel protein in mitochondria,significantly promoted cell death under B.dothidea infection and simultaneously inhibited pathogen infection,suggesting that cell death represents a disease resistance mechanism in apple against B.dothidea infection.Furthermore,BdCatalase(KAF4307763),a cytochromeP450 family protein BdCYP52A4(KAF4300696),and subtilisin-domain containing proteinswere identified fromB.dothidea-secreted proteins,which suggested the potential involvement of active oxygen species and phytoalexins in combating B.dothidea infection and triggering or dampening apple resistance.Collectively,our research suggested that cell wall-mediated penetration resistance,programmed cell death machinery and microbial effector-interrelated signaling were among strategies recruited in apple to combat B.dothidea.The current research laid the foundation for further investigations into resistance mechanisms in apple.

Effects of water stress on quality and sugar metabolism in'Gala'apple fruit

Sugar plays an important role in apple fruit development,appearance and quality as well as contributing to a plant’s water stress response.Trehalose and the trehalose biosynthetic metabolic pathways are part of the sugar signaling system in plants,which are important regulator of water stress response in apple.The effect of water stress treatments applied to apple trees and the corresponding effects of ABA on developmental fruit quality were examined for indicators of fruit quality during fruit development.The results indicated that the severe water stress treatment(W2)occurring after the last stage of fruit cell division caused a decrease in the color and size of fruit.The moderate water stress(W1)occurring after the last stage of fruit cell enlargement(S2)caused an increase in the content of fructose and sorbitol while the apple fruit shape was not affected.These changes in sugar are related to the activity of sugar metabolic enzymes.While the enzymatic activity of vacuolar acid invertase(vAINV)was higher,that of sucrose-phosphate synthase(SPS)was lower in water stress treated fruit throughout the developmental period.This indicates that enhanced sucrose degradation and reduced sucrose synthesis leads to an overall reduced sucrose content during times of drought.Thus,water stress reduced sucrose content.Whereas the content of endogenous trehalose and ABA were the highest in water stress treated fruit.A moderate water stress(W1)imposed on apple trees via water restriction(60%–65%of field capacity)after the fruit cell enlargement phase of fruit development yielded sweeter fruit of higher economic value.

YOLO-RD-Apple果园异源图像遮挡果实检测模型

为探索在自然苹果园环境中对高度遮挡果实进行机器人自动化采摘,提出基于RGB与Depth图像双输入的YOLO-RD-Apple果园异源图像遮挡果实检测模型。使用轻量化的MobileNetV2并在此基础上设计的更加轻量化的MobileNetV2-Lite分别作为RGB和Depth图像的特征提取器,保证特征提取能力的同时降低网络的计算量;将CSPNet与深度可分离卷积结合SE注意力模块,提出全新的SE-DWCSP3模块对PANet结构进行改进,提升网络对于残缺苹果目标的特征提取能力;引入SoftNMS算法替代一般NMS算法,以减少对密集目标的错误抑制现象,降低被遮挡苹果的漏检率。实验结果表明,YOLO-RD-Apple在自然遮挡苹果数据集上性能优秀,在测试集上的AP值达到93.1%,较YOLOv4提升了1.4%,参数量则降低了70%,在GPU(V100)的检测速度达到40.5FPS,较YOLOv4速度提升了12.5%,在检测精度和速度上均有不同程度地提升,同时降低了网络参数量,更加适用于实际果园苹果采摘场景。

Comparison of transcriptome and metabolome analysis revealed differences in cold resistant metabolic pathways in different apple cultivars under low temperature stress

Freezing injury in winter is an important abiotic stress that seriously affects plant growth and development.Deciduous fruit trees resist freezing injury by inducing dormancy.However,different cultivars of the same species have different cold resistance strategies.Little is known about the molecular mechanism of apple trees in response to freezing injury during winter dormancy.Therefore,in this study,1-year-old branches of the cold-resistant cultivar‘Hanfu’(HF)and the cold-sensitive cultivar‘Changfuji No.2’(CF)were used to explore their cold resistance through physiological,biochemical,transcriptomics,and metabolomics analyses.Combining physiological and biochemical data,we found that HF had a stronger osmotic regulation ability and antioxidant enzyme activity than CF,as well as stronger cold resistance.The functional enrichment analysis showed that both cultivars were significantly enriched in pathways related to signal transduction,hormone regulation,and sugar metabolism under freezing stress.In addition,the differentially expressed genes(DEGs)encoding galactinol synthase,raffinose synthase,and stachyose synthetase in raffinose family oligosaccharides(RFOs)metabolic pathways were upregulated in HF,and raffinose and stachyose were accumulated,while their contents in CF were lower.HF accumulated 4-aminobutyric acid,spermidine,and ascorbic acid to scavenge reactive oxygen species(ROS).While the contents of oxidized glutathione,vitamin C,glutathione,and spermidine in CF decreased under freezing stress,consequently,the ability to scavenge ROS was low.Furthermore,the transcription factors apetala 2/ethylene responsive factor(AP2/ERF)and WRKY were strongly induced under freezing stress.In summary,the difference in key metabolic components of HF and CF under freezing stress is the major factor affecting their difference in cold resistance.The obtained results deepen our understanding of the cold resistance mechanism in apple trees in response to freezing injury during dormancy.