PbrARF4 contributes to calyx shedding of fruitlets in ‘Dangshan Suli’ pear by partly regulating the expression of abscission genes

Fruitlet calyx shedding in pear plants is apparently regulated via numerous pathways that involve both environmental triggers and phytohormones cues such as auxin. In this study, we found at 10 days after full bloom (DAFB) higher levels of indoleacetic acid (IAA) and tryptophan (Trp) in calyx persistence fruitlet (CPF) than calyx shedding fruitlet (CSF) ofDanshan Suli’ pear (Pyrus bretschneideri Rhed.). Consisting with this, the activity of indolealdehyde oxidase (IAAIdO), which promotes IAA synthesis, was remarkably increased, and that of peroxidase(POD), which degrades IAA, dropped markedly in CPF but not in CSF. Further, qRT-PCR results revealed that most of 31 PbrARFs (encoding auxin response factors) in Pyrus bretschneideri were highly expressed in CPF, whereas PbrARF4, PbrARF24 and PbrARF26 were significantly downregulated in CPF vis-a-vis CSF. Phylogenetic analysis revealed that 6 PbrARFs clustered in the group III, where PbrARF4 showed the closest affinity with AtARF1 that promotes organ abscission, indicating a putative role of PbrARF4 in mediating the process of calyx shedding in pear. In fact, the ectopic overexpression of PbrARF4 in Solanum lycopersicum resulted in an earlier-formed and deeper abscission layer (AL) in the transgenic plants, whose calyxes were more prone to wilt at the mature red stage (MR) compared with the control plants (wild-type). More importantly, expression levels of the abscission genes SILS and Sl Cel2 in transgenic plants overexpressing PbrARF4 were significantly upregulated in comparation with the WT, whereas those of Sl BI and Sl TAPG2 were considerably inhibited. Further, PbrJOINTLESS and PbrIDA,the two genes related to calyx shedding in pear, were up-regulated more in CSF than CPF. The findings contribute to a better understanding of PbrARFs involved in fruitlet calyx shedding of pear, which could prove beneficial to improving the quality of pear fruit.

Genomic selection of eight fruit traits in pear

Genomic selection (GS) has the potential to improve selection efficiency and shorten the breeding cycle in fruit tree breeding. In this study,we evaluated the effect of prediction methods, marker density and the training population (TP) size on pear GS for improving its performance and reducing cost. We evaluated GS under two scenarios:(1) five-fold cross-validation in an interspecific pear family;(2) independent validation. Based on the cross-validation scheme, the prediction accuracy (PA) of eight fruit traits varied between 0.33 (fruit core vertical diameter)and 0.65 (stone cell content). Except for single fruit weight, a slightly better prediction accuracy (PA) was observed for the five parametrical methods compared with the two non-parametrical methods. In our TP of 310 individuals, 2 000 single nucleotide polymorphism (SNP) markers were sufficient to make reasonably accurate predictions. PAs for different traits increased by 18.21%-46.98%when the TP size increased from 50to 100, but the increment was smaller (-4.13%-33.91%) when the TP size increased from 200 to 250. For independent validation, the PAs ranged from 0.11 to 0.45 using rrBLUP method. In summary, our results showed that the TP size and SNP numbers had a greater impact on the PA than prediction methods. Furthermore, relatedness among the training and validation sets, and the complexity of traits should be considered when designing a TP to predict the test panel.

PbGIF1 promoting cell-proliferation in pear fruit is transcriptionally activated by Pb RR1

As a cell proliferation regulator involved in wide biological processes in plants,GRF-INTERACTING FACTOR(GIF)controls different tissues development.However,whether GIF participates in fruit development remains unclear.According to transcriptome data,we identified PbGIF1was highly expressed during fruit development in cytokinins induced parthenocarpy pear.In the present study,the biofunction of PbGIF1 was initially verified.Overexpression of PbGIF1 promoted fruit size of transgenic tomato.The size of flesh fruit was not affected by cell expansion but the cell proliferation was promoted by overexpressing Pb GIF1.The accelerated cell proliferation process was also observed in PbGIF1-overexpressed transgenic pear fruit calli.The transcriptional regulation of cytokinins on PbGIF1 was further confirmed by exogenous CPPU treatments in pear fruitlets.To investigate the underlying mechanism,the cytokinins-responded factor,PbRR1,was further focused on.The results of Yeast-one-hybrid assay suggested that PbRR1 can bind to the promoter sequence of PbGIF1.The transcriptional activation of PbRR1 on PbGIF1 was also confirmed by Dual-Luciferase assays.Taken together,the results showed that cytokinins control pear fruit development via the transcriptional activation of PbGIF1 by PbRR1.

E3 ubiquitin ligase PbrATL18 is a positive factor in pear resistance to drought and Colletotrichum fructicola infection

The Arabidopsis Toxicos en Levadura(ATL)protein is a subfamily of the E3 ubiquitin ligases,which exists widely in plants and is extensively involved in plant growth and development.Although the ATL family has been identified in other species,such as Arabidopsis,Oryza sativa,and grapevine,few reports on pear ATL gene families have been reported.In this study,92 PbrATL genes were identified and analyzed from the Pyrus breschneideri genome.Motif analysis and phylogenetic tree generation divided them into nine subgroups,and chromosome localization analysis showed that the 92 PbrATL genes were distributed in 16 of 17 pear chromosomes.Transcriptome data and quantitative real-time polymerase chain reaction(qRT-PCR)experiments demonstrated that PbrATL18,PbrATL41,and PbrATL88 were involved in both pear drought resistance and Colletotrichum fructicola infection.In addition,Arabidopsis thaliana overexpressing PbrATL18 showed greater resistance to drought stress than the wild type(WT),and PbrATL18-silenced pear seedlings showed greater sensitivity to drought and C.fructicola infection than the controls.PbrATL18 regulated plant resistance by regulating chitinase(CHI),phenylalanine ammonia-lyase(PAL),polyphenol oxidase(PPO),catalase(CAT),peroxidase(POD),and superoxide dismutase(SOD)activities.This study provided a reference for further exploring the functions of the PbrATL gene in drought resistance and C.fructicola infection.

A Light and Simplified Branch Bending Method for Young Pear Trees

Aiming at high cost and low efficiency of conventional branch bending method in the modern intensive planting and labor-saving cultivation mode of young pear trees,this paper provides a new branch bending method with wide source of raw materials,cheap price and simple operation,which is also suitable for the management of low-age branches in the process of high grafting and upgrading of traditional big trees.

Study on the Optimal Composition of Chitosan/Nano-TiO_2 Composite Film and Its Application in the Preservation of Jinqiu Pear

Water permeability can be used as a parameter to evaluate the effects of chitosan / nano-TiO2 composite film on the preservation of fruits and vegetables. In this study, using water permeability as the objective function, an L9 （3）3 orthogonal experiment was conducted with addition amounts of chitosan, nano-TiO2 and glycerin as three factors at three levels. According to the experimental results, when the percentages of chitosan, nano-TiO2 and glycerin in chitosan / nano-TiO2 composite film was 2%, 0.03% and 2.5%, respectively （ i. e. , there were 2.0 g of chitosan, 0.03 g of nano-TiO2 and 2.5 g of glycerin per 1130 g composite film solution）, the water permeability of chitesan / nano-TiO2 composite fdm reached the minimum. Chitosan / nano-TiO2 composite film prepared with the optimal composition was used for preservation of Jinqiu pear. The water loss rate, respiratory intensity and good fruit rate of preserved Jinqiu pear were determined, which indicated that the prepared chitosan / nano-TiO2 composite film exhibited good preservative effects.

PEAR1、GSTP1基因型与脑梗死预后的相关性分析

目的 探讨PEAR1、GSTP1基因型与脑梗死预后的相关性,为脑梗死个体化精准治疗及二级预防提供理论参考.方法 选取首发症状型脑梗死患者,入院后遵循抑制血小板聚集、营养神经、改善循环及对症治疗原则,患者24 h内口服阿司匹林100 mg, 1次/d,并长期维持治疗.收集患者的一般资料、既往史、血小板相关指标、PEAR1及GSTP1基因型、相关待测量表评分及脑梗死复发周期,出院随访13个月.根据脑梗死复发及复发周期进行分组,比较PEAR1、GSTP1基因多态性及其他临床资料与脑梗死预后是否具有关联.结果 入组患者PEAR1基因突变型(AA+AG)占60.1%,未突变型(GG)占39.9%;GSTP1基因突变型(AG+GG)占31.8%,未突变型(AA)占68.2%.对比分析两组患者冠心病病史、出院时NIHSS评分、血小板计数及GSTP1基因型与脑梗死复发有关联.多因素Logistic回归分析显示冠心病病史、出院时低NIHSS评分、低血小板计数及GSTP1基因突变型(AG+GG)为脑梗死复发的独立危险因素(P<0.05).结论 首发症状型脑梗死患者年龄越大、出院时低NIHSS评分、低血小板计数或GSTP1为基因突变型(AG+GG)脑梗死预后较差.

Genome-wide identification of the mitogen-activated protein kinase kinases in pear and their functional analysis in response to black spot

The mitogen-activated protein kinase(MAPK)cascade is crucial to plant growth,development,and stress responses.MAPK kinases(MAPKK)play a vital role in linking upstream MAPKK kinases(MAPKKK)with the downstream MAPK.Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China.The MAPKK genes have been identified in many plants,however,none has been reported in pear(Pyrus bretschneideri).In order to explore whether MAPK gene of pear is related to black spot disease,we designed this experiment.The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome.The phylogenetic analysis revealed that PbrMAPKK genes were divided into A,B,C,and D groups.These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication(WGD)event.The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR(qRT-PCR)identified seven candidate genes associated with resistance.Furthermore,virus-induced gene silencing(VIGS)indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

Isolation,identification and artificial inoculation of Rhizoctonia solani on pear during storage

Pear is a fruit crop of worldwide importance and cold storage is an integral part of the production and distribution of pears.An uncharacterized fungal disease has been observed on‘Huangguan’pear fruit during cold storage in Hebei Province.The fungus was consistently isolated from diseased fruit by routine tissue separation method,and shown to be the causal agent according to Koch postulates.Based on its morphology,molecular characteristics,pathogenicity and ITS sequence,the fungus was identified as Rhizoctonia solani.This study recorded postharvest fruit rot caused by Rhizoctonia solani on pear fruit in China.

Evaluation of the early defoliation trait and identification of resistance genes through a comprehensive transcriptome analysis in pears

Early defoliation,which usually occurs during summer in pear trees,is gradually becoming a major problem that poses a serious threat to the pear industry in southern China.However,there is no system for evaluating the responses of different cultivars to early defoliation,and our knowledge of the potential molecular regulation of the genes underlying this phenomenon is still limited.In this study,we conducted field investigations of 155 pear accessions to assess their resistance or susceptibility to early defoliation.A total of 126 accessions were found to be susceptible to early defoliation,and only 29 accessions were resistant.Among them,19 resistant accessions belong to the sand pear species(Pyrus pyrifolia).To identify the resistance genes related to early defoliation,the healthy and diseased samples of two sand pear accessions,namely,the resistant early defoliation accession‘Whasan’and the susceptible early defoliation accession‘Cuiguan’,were used to perform RNA sequencing.Compared with‘Cuiguan’,a total of 444 genes were uniquely differentially expressed in‘Whasan’.Combined with GO and KEGG enrichment analyses,we found that early defoliation was closely related to the stress response.Furthermore,a weighted gene co-expression network analysis revealed a high correlation of WRKY and ethylene responsive factor(ERF)transcription factors with early defoliation resistance.This study provides useful resistant germplasm resources and new insights into potentially essential genes that respond to early defoliation in pears,which may facilitate a better understanding of the resistance mechanism and molecular breeding of resistant pear cultivars.

The PcHY5 methylation is associated with anthocyanin biosynthesis and transport in ‘Max Red Bartlett' and ‘Bartlett' pears

The red coloring of pear fruits is mainly caused by anthocyanin accumulation. Red sport, represented by the green pear cultivar ‘Bartlett’(BL) and the red-skinned derivative ‘Max Red Bartlett’(MRB), is an ideal material for studying the molecular mechanism of anthocyanin accumulation in pear. Genetic analysis has previously revealed a quantitative trait locus(QTL) associated with red skin color in MRB. However, the key gene in the QTL and the associated regulatory mechanism remain unknown. In the present study, transcriptomic and methylomic analyses were performed using pear skin for comparisons between BL and MRB. These analyses revealed differential PcHY5 DNA methylation levels between the two cultivars;MRB had lower PcHY5 methylation than BL during fruit development, and PcHY5 was more highly expressed in MRB than in BL. These results indicated that PcHY5 is involved in the variations in skin color between BL and MRB. We further used dual luciferase assays to verify that PcHY5 activates the promoters of the anthocyanin biosynthesis and transport genes PcUFGT, PcGST, PcMYB10 and PcMYB114, confirming that PcHY5 not only regulates anthocyanin biosynthesis but also anthocyanin transport. Furthermore, we analyzed a key differentially methylated site between MRB and BL, and found that it was located in an intronic region of PcHY5. The lower methylation levels in this PcHY5 intron in MRB were associated with red fruit color during development, whereas the higher methylation levels at the same site in BL were associated with green fruit color. Based on the differential expression and methylation patterns in PcHY5 and gene functional verification, we hypothesize that PcHY5, which is regulated by methylation levels, affects anthocyanin biosynthesis and transport to cause the variations in skin color between BL and MRB.

Genome-wide identification of the MATE gene family and functional characterization of PbrMATE9 related to anthocyanin in pear

Plant multidrug and toxic compound extrusion(MATE) genes play an important role in the process of detoxification, plant morphogenesis, and anthocyanin accumulation. However, whether the MATE gene family functions in pear peel coloration is still unknown. To evaluate and identify the MATE gene family members which are involving in anthocyanin accumulation and coloration in pear. In this study, 85 MATE genes were identified in the reference pear genome of ‘Dangshansuli’ through genome-wide identification. Based on gene structure and phylogenetic tree analysis, the MATE family was divided into five subfamilies. RNA sequencing and quantitative real-time polymerase chain reaction(qRTPCR) indicated that the expression patterns of PbrMATEs were tissue-specific. 28.24%(24) of PbrMATE genes were expressed in the fruits, and44.71%(38) of PbrMATE genes were expressed in the leaves. Additionally, we found that the expression levels of PbrMATE9, PbrMATE26,PbrMATE50, and PbrMATE69 in debagged fruits with red peel were significantly higher than those in bagged fruits without red peel, according to our bagging/debagging treatment of ‘Mantianhong’. The expression pattern of PbrMATE9 was consistent with the variation trend in anthocyanin content, suggesting that it might play an important role in anthocyanin accumulation in response to light exposure. Subcellular localization showed that PbrMATE9 was a membrane protein. More strikingly, the transient overexpression of PbrMATE9 promoted anthocyanin accumulation in the peel of pear, and the expression of structural genes(PbrCHI, PbrANS, PbrDFR, and PbrUFGT) in the anthocyanin biosynthesis pathway also increased significantly. Through co-expression network analysis, the transcription factors were identified, such as WRKY, COL,GATA, and BBX, which might be involved in the regulation of PbrMATE9. The study has enriched the genetic resources and improved the understanding of the regulation network of anthocyanin accumulation in pear.

Identification and functional analysis of arabinogalactan protein expressed in pear pollen tubes

Arabinogalactan proteins(AGPs)are widely distributed in the plant kingdom and play a vital role during the process of plant sexual reproduction.In this study,we performed a comprehensive identification of the PbrAGPs expressed in pear pollen and further explored their influences on pollen tube growth.Among the 187 PbrAGPs that were found to be expressed in pear pollen tubes,38 PbrAGPs were specifically expressed in pollen according to the RNA-seq data.The PbrAGPs were divided into two groups of highly expressed and specifically expressed in pear pollen.We further tested their expression patterns using RT-PCR and RT-qPCR.Most of the PbrAGPs were expressed in multiple tissues and their expression levels were consistent with reads per kilobase per million map reads(RPKM)values during pollen tube growth,implying that PbrAGPs might be involved in the regulation of pear pollen tube growth.We also constructed phylogenetic trees to identify the functional genes in pear pollen tube growth.Therefore,19 PbrAGPs(PbrAGP1 to PbrAGP19)were selected to test their influences on pollen tube growth.Recombinant proteins of the 19 PbrAGP-His were purified and used to treat pear pollen,and 11 of the PbrAGP-His recombinant proteins could promote pear pollen tube growth.Additionally,pollen tube growth was inhibited when the expression levels of PbrAGP1 and PbrAGP5 were knocked down using an antisense oligonucleotide assay.PbrAGP1 and PbrAGP5 were localized in the plasma membrane and might not alter the distribution of pectin in the pollen tube.In summary,this study identified the PbrAGPs expressed in pear pollen and lays the foundation for further exploring their functions in pollen tube growth.

The HY5 transcription factor negatively regulates ethylene production by inhibiting ACS1 expression under blue light conditions in pear

Ethylene is the main factor controlling fruit ripening of pear(Pyrus ussuriensis).Ethylene production rate is negatively correlated with fruit shelf life;therefore,it is important to decrease the ethylene levels for optimal fruit storage.Here,we observed that blue light treatment could inhibit ethylene production and promote the expression of ELONGATED HYPOCOTYL 5(PuHY5),a basic leucine zipper domain(bZIP)transcription factor.The following studies showed that PuHY5 could bind to the promoter of ACC synthase 1(PuACS1),a rate-limiting enzyme in ethylene biosynthesis,and inhibit its expression.For pears in which Pu HY5 was silenced,the ethylene production and PuACS1 expression were much higher than those in the control fruit.These results demonstrated that blue light inhibited ethylene production through the induction of Pu HY5 in pear.Our finding provides a new method for prolonging fruit shelf life.

Study on Adaptability of Introduced Pear Varieties

[Objectives]To study the adaptability of introduced pear.[Methods]Five pear varieties,"Aidang"pear,"Taiwan Zaomi"pear,"Cuiguan"pear,"Tianjin Yali"pear and"Zaosheng Xinshui"pear,were introduced.Then,using these five varieties,the phenology of pear trees,various characters of fruit,stress resistance(heat tolerance and cold tolerance)of varieties were studied.[Results]The plants of 5 varieties of pear trees grew fast and were robust;in late March,it went into the flowering period;"Aidang"pear fruit had a certain number of stone cells;"Taiwan Zaomi"pear had the highest sweetness;"Cuiguan"pear had the largest fruit;these five varieties of pear trees had good water resistance,heat resistance and cold resistance.[Conclusions]This study can provide a reference for the introduction of pear trees,and can also provide a practical basis for the large-scale planting of pear trees.

Pollination Effects of Pear Trees by Honeybee and Its Influencing Factors

[Objectives]To improve the yield and quality of different pear varieties by supplementary pollination.[Methods]The effects of bee pollination were compared with other pollination methods,and the effects of different factors on honeybee pollination were studied through experiments.[Results]Honeybee pollination significantly increased the number of fruit per inflorescence.In addition,it could improve the single fruit weight,fruit shape and other quality indicators of fruit.The pollination effect of honeybee was the best in all supplementary pollination measures.Honeybee pollination had good fruit setting effect on different pear varieties,but there were some differences in the effect of quality improvement.Spraying lime sulfur mixture and other measures could significantly affect the effects of honeybee pollination.[Conclusions]The research results prove the effectiveness of honeybee pollination technology in Hebei Province,and provide a certain reference for the establishment of a simple and practical artificial assisted pollination technology system.

Study on Brewing Technology of Pear Wine

[Objectives]The quality of pear wine is directly affected by parameter control in the production process, especially in the fermentation stage. The fermentation parameters of pear wine were controlled by studying the effects of fermentation parameters on the product, in order to optimize the fermentation parameters. [Methods]The fermentation conditions of pear wine were optimized by single factor experiments and an orthogonal experiment. [Results] The optimum fermentation conditions of pear wine were nitrogen source 1.0 g/L, fermentation temperature 20 ℃, and pH of fermentation liquid 4.3. The pear wine brewed was full, mellow and harmonious, and had a unique style. The ethanol content of the pear wine was 12.33 through physical and chemical analysis. [Conclusions] This study provides theoretical basis and technical support for the industrial production and promotion of pear wine.

Cultivation Techniques of Qiuyue Pear in Shenxian County

According to the production experience,the author summarizes the cultivation techniques of Qiuyue pear from orchard construction,shaping and pruning,fruit management,underground management,coping with natural disasters,and pest control,in order to provide a reference for producers.

广东地区汉族人群GPⅢa、PEAR1、PTGS1和GSTP1基因多态性分析及相关性研究

目的研究广东地区汉族人群血小板膜糖蛋白Ⅲa(GPⅢa)、血小板内皮聚集受体1(PEAR1)、前列腺素内过氧化物合酶1(PTGS1)、谷胱甘肽S转移酶P1(GSTP1)基因多态性分布情况并进行相关性分析。方法收集2019年1月至2022年4月于广州达安临床检验中心实验室检测的患者样本,共2985例。对患者的GPⅢa基因rs5918位点、PEAR1基因rs12041331位点、PTGS1基因rs10306114位点、GSTP1基因rs1695位点进行基因检测。应用统计学方法比较四个基因位点变异情况在广东汉族人群中的分布差异,分析其与性别、年龄的相关性。结果rs5918位点、rs12041331位点、rs10306114位点、rs1695位点在广东汉族人群中的人群频率分别为0.39%、42.55%、0.05%、17.52%。rs1695位点的基因型和等位基因频率在不同年龄组间的分布差异有统计学意义(P<0.05),其他三个基因位点在不同年龄组之间的分布差异无统计学意义(P>0.05)。广东地区汉族人群四个基因位点基因型和等位基因频率的分布与我国汉族人群差异无统计学意义(P>0.05),与南亚、非洲、欧洲、拉丁美洲人群差异有统计学意义(P<0.05)。结论广东地区汉族人群rs1695位点(GSTP1)年龄<60岁组携带G等位基因的比例增加,存在地区和年龄的差异性,可为指导广东地区汉族人群阿司匹林个性化用药提供依据。

Changes in Soil Organic Matter,Nitrogen and Phosphorus Contents during Decomposition of Pear Branches

[Objectives]To investigate the changes in soil organic matter,nitrogen and phosphorus content in the decomposition process of Korla fragrant pear branches by indoor mixed culture.[Methods]The branches of Korla fragrant pear in the orchard were collected and returned to the field for a period of 150 d for indoor mixed culture.[Results]Different ages of Korla fragrant pear branches have different effects on soil nutrient content during the simulated return to field decomposition process.Compared with the control in the same period,the treatment of returning to field reached a significant level(P<0.05).Compared with the control,the average values of organic matter,total nitrogen and available phosphorus content in treatment 1 and treatment 2 increased by 2.16 times and 1.93 times,61%and 59%,5.88 times and 6.88 times,respectively;compared with the control,the average increase performance of the alkaline hydrolysis nitrogen content of the two treatments was basically the same,and the treatment 2 was the best;compared with the control,the average total phosphorus content of treatment 1 and treatment 2 increased but not significantly.[Conclusions]The contents of soil organic matter,nitrogen and phosphorus were all increased during the decomposition of pear branches,and the overall improvement effect of 10-year-old trees was better than that of 5-year-old trees.Returning the pruned branches to the field can provide a reliable theoretical basis for solving the problem of organic fertilizer shortage in orchards,and also can ensure technical support for improving soil fertility and improving the rhizosphere micro-environment of pear trees.