Genome-wide identification of the CONSTANS-LIKE(COL)family and mechanism of fruit senescence regulation by PpCOL8 in sand pear(Pyrus pyrifolia)

Pyrus pyrifolia Nakai‘Whangkeumbae'is a sand pear fruit with excellent nutritional quality and taste.However,the industrial development of pear fruit is significantly limited by its short shelf life.Salicylic acid(SA),a well-known phytohormone,can delay fruit senescence and improve shelf life.However,the mechanism by which SA regulates CONSTANS-LIKE genes(COLs)during fruit senescence and the role of COL genes in mediating fruit senescence in sand pear are poorly understood.In this study,22 COL genes were identified in sand pear,including four COLs(Pp COL8,Pp COL9a,Pp COL9b,and Pp COL14)identified via transcriptome analysis and 18 COLs through genome-wide analysis.These COL genes were divided into three subgroups according to the structural domains of the COL protein.Pp COL8,with two B-box motifs and one CCT domain,belonged to the first subgroup.In contrast,the other three Pp COLs,Pp COL9a,Pp COL9b,and Pp COL14,with similar conserved protein domains and gene structures,were assigned to the third subgroup.The four COLs showed different expression patterns in pear tissues and were preferentially expressed at the early stage of fruit development.Moreover,the expression of Pp COL8 was inhibited by exogenous SA treatment,while SA up-regulated the expression of Pp COL9a and Pp COL9b.Interestingly,Pp COL8 interacts with Pp MADS,a MADS-box protein preferentially expressed in fruit,and SA up-regulated its expression.While the production of ethylene and the content of malondialdehyde(MDA)were increased in Pp COL8-overexpression sand pear fruit,the antioxidant enzyme(POD and SOD)activity and the expression of Pp POD1 and Pp SOD1 in the sand pear fruits were down-regulated,which showed that Pp COL8 promoted sand pear fruit senescence.In contrast,the corresponding changes were the opposite in Pp MADS-overexpression sand pear fruits,suggesting that Pp MADS delayed sand pear fruit senescence.The co-transformation of Pp COL8 and Pp MADS also delayed sand pear fruit senescence.The results of this study revealed that Pp COL8 can play a key role in pear fruit senescence by interacting with Pp MADS through the SA signaling pathway.

Insights into the dwarfing mechanism of pear(Pyrus betulaefolia) based on anatomical and structural analysis using X-ray scanning

The lack of a suitable rootstock to control scion growth has limited the development of high-density plantations in pear production, which is partly attributed to poor understanding of the dwarfing mechanism. In the present study, the rootstock of the dwarf-type pear (Pyrus betulaefolia)PY-9’ was identified and used as the material for anatomical analysis.PY-9’ grew to half the tree height of the normal cultivar Zhengdu’, along with fewer internodes and shorter length. Significant differences in growth rate betweenPY-9’ andZhengdu’ were detected at approximately 30 days after full bloom, which corresponded with the time of the greatest difference in water potential between the dwarf and normal cultivar.PY-9’ showed a higher photosynthetic rate thanZhengdu’. Anatomical analysis showed thatPY-9’ had higher area ratios of both phloem and xylem and more developed vascular tissues thanZhengdu’. The three-dimensional reconstructed skeleton of the xylem from X-ray computed tomography scanning revealed greater intervessel connectivity inZhengdu’ than inPY-9’, which could contribute to the more vigorous growth ofZhengdu’. This study thus provides the first comparison of the microstructural properties of xylem elements between a dwarfing-type and vigorous-type pear rootstock, providing new insights into the dwarfing mechanism in pear and facilitating breeding of dwarf pear rootstocks to increase crop productivity.

The PcERF5 promotes anthocyanin biosynthesis in red-fleshed pear(Pyrus communis)through both activating and interacting with PcMYB transcription factors

As there is a strong interest in red-skinned pears,the molecular mechanism of anthocyanin regulation in red-skinned pears has been widely investigated;however,little is known about the molecular mechanism of anthocyanin regulation in red-fleshed pears due to limited availability of such germplasm,primarily found in European pears(Pyrus communis).In this study,based on transcriptomic analysis in red-fleshed and white-fleshed pears,we identified an ethylene response factor(ERF)from P.communis,PcERF5,of which expression level in fruit flesh was significantly correlated with anthocyanin content.We then verified the function of PcERF5 in regulating anthocyanin accumulation by genetic transformation in both pear skin and apple calli.PcERF5 regulated anthocyanin biosynthesis by different regulatory pathways.On the one hand,PcERF5 can activate the transcription of flavonoid biosynthetic genes(PcDFR,PcANS and PcUFGT)and two key transcription factors encoding genes PcMYB10 and PcMYB114.On the other hand,PcERF5 interacted with PcMYB10 to form the ERF5-MYB10 protein complex that enhanced the transcriptional activation of PcERF5 on its target genes.Our results suggested that PcERF5 functioned as a transcriptional activator in regulating anthocyanin biosynthesis,which provides new insights into the regulatory mechanism of anthocyanin biosynthesis.This new knowledge will provide guidance for molecular breeding of red-fleshed pear.

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.

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.

Genome-wide identification and characterization of the PbrATG family in Pyrus bretschneideri and functional analysis of PbrATG1a in response to Botryosphaeria dothidea

The pear is an economic fruit that is widely planted around the world and is loved by people for its rich nutritional value. Autophagy is a self-protection mechanism in eukaryotes, and its occurrence often accompanied by the degradation of damaged substances in cells and the recycling of nutrients. Autophagy is one of the mechanisms through which plants respond to environmental stress and plays an important role in plant development and stress resistance. Functional studies of autophagy-related genes (ATGs) have been performed on a variety of plant species, but little information is available on the ATG family in pear (Pyrus bretschneideri Rehd). Therefore, we analyzed the evolutionary dynamics and performed a genome-wide characterization of the PbrATG gene family. A total of 28 PbrATG members were identified.Phylogenetic analysis showed that PbrATGs were more closely related to ATGs of European pear and apple. Evolutionary analysis revealed that whole-genome duplication (WGD) and dispersed duplication events were the main driving forces of PbrATG family expansion.Expression analysis of different pear tissues showed that all the genes were expressed in different pear tissues, and different PbrATGs are expressed at different times and in different locations. Moreover, all PbrATGs also responded to different abiotic stresses, especially salt and drought stress, which elicited the highest expression levels. Pear seedlings were subsequently infected with Botryosphaeria dothidea (B.dothidea). The results showed that different PbrATGs had different expression patterns at different infection stages. According to the gene expression data, PbrATG1a was selected as a key autophagy gene for further analysis. Silencing of PbrATG1a reduced the resistance of pear to B. dothidea, which resulted in increased lesions, reactive oxygen species (ROS) contents, antioxidant enzyme activity, and gene expression levels in the silenced pear seedlings after B. dothidea inoculation. In this study, a comprehensive bioinformatic analysis of ATGs was conducted, and the functions of PbrATGs in pear development and in response to stress were elucidated, which laid a foundation for further study of the molecular mechanism of autophagy and a new strategy for pear resistance breeding.

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.

Isolation and Characterization of a Cinnamoyl CoA Reductase Gene(CCR) in Pear(Pyrus pyrifolia)

Pear is a popular and commercially important fresh fruit, and its texture is related to the presence of sclereid formatted by parenchyma cell with lignification in vascular plants. Previous studies have demonstrated that content of lignin may be regulated by cinnamoyl CoA reductase(CCR) in various plants. However, the function of CCR in pears remains very limited. In the present study, we isolated a cDNA encoding CCR(PpCCR, GenBank accession No. KF999958) and its promoter(proPpCCR) from Whangkeumbae pear to investigate the function of CCR in lignin biosynthesis. PpCCR-GFP expressed in rice mesophyll protoplast demonstrated that PpCCR-GFP was localized in the cytoplasm, indicating that CCR may function in cytoplasm without localization signals. In transgenic plants carrying PpCCR, we observed higher lignin content compared with that in wild type plants, further suggesting that PpCCR can affect the lignin contents through regulating lignin biosynthesis in Arabidopsis thaliana. More studies in other plants are needed to confirm our conclusion.

Identification of Self-Incompatibility Genotypes in Some Sand Pears (Pyrus pyrifolia Nakai) by PCR-RFLP Analysis

The identification of self-incompatibility genotype （S-genotype） will be useful for selection of pollinizers and design of crossing in cultivar improvement of sand pear. This paper reported the identification of self-incompatibility genotypes of seven Chinese and two Japanese sand pear cultivars using PCR-RFLP analysis and S-RNase sequencing. The Sgenotypes of these cultivars were determined as follows： Huali 1 S1S3, Shounan S1S3, Xizilti S1S4, Qingxiang S3S7, Sanhua S2S7, Huangmi （Imamuranatsu） S1S6, Huali 2 S3S4, Baozhuli S7S33, Cangxixueli S5S15. S-RNase alleles （S1 to S9） in sand pear could be identified effectively by PCR-RFLP analysis.

An assessment of the genetic diversity of pear(Pyrus L.)germplasm resources based on the fruit phenotypic traits

Germplasm resources are an important basis for genetic breeding and analysis of complex traits,and research on genetic diversity is conducive to the exploration and creation of new types of germplasm.In this study,the distribution frequency,coefficient of variation,Shannon-Wiener index,and variance and cluster analyses were used to analyze the diversity and trait differences of 39 fruit phenotypic traits from 570 pear accessions,which included 456 pear accessions from 11 species and 114 interspecific hybrid cultivars that had been stored in the National Germplasm Repository of Apple and Pear(Xingcheng,China).The comprehensive evaluation indices were screened by correlation,principal component and regression analyses.A total of 132 variant types were detected in 28 categorical traits of pear germplasm fruit,which indicate a rich diversity.The diversity indices in decreasing order were:fruit shape(1.949),attitude of calyx(1.908),flesh texture type(1.700),persistency of calyx(1.681),russet location(1.658),relief of area around eye basin(1.644),flavor(1.610)and ground color(1.592).The coefficient of variation of titratable acidity in the 11 numerical traits of pear germplasm fruit was as high as 128.43%,which could more effectively reflect the differences between pear accessions.The phenotypic differentiation coefficient V_(st)(66.4%)among the five cultivated pear species,including Pyrus bretschneideri(White Pear),P.pyrifolia(Sand Pear),P.ussuriensis(Ussurian Pear),P.sinkiangensis(Xinjiang Pear),and P.communis(European Pear),was higher than the within population phenotypic differentiation coefficient V_(st)(33.6%).The variation among populations was the main source of variation in pear fruit traits.A hierarchical cluster analysis divided the 389 accessions of six cultivated pear species,including P.pashia(Himalayan Pear),into six categories.There were certain characteristics within the populations,and the differences between populations were not completely clustered by region.For example,Sand Pear cultivars from Japan and the Korean Peninsula clustered together with those from China.Most of the White Pear cultivars clustered with the Sand Pear,and a few clustered with the Ussurian Pear cultivars.The Ussurian Pear and European Pear cultivars clustered separately.The Xinjiang Pear and Himalayan Pear did not cluster together,and neither did the cultivars.Seventeen traits,three describing fruit weight and edible rate(fruit diameter,fruit length and fruit core size),five describing outer quality and morphological characteristics(over color,amount of russeting,dot obviousness,fruit shape,and stalk length),and nine describing inner quality(flesh color,juiciness of flesh,aroma,flavor,flesh texture,flesh texture type,soluble solid contents,titratable acidity,and eating quality)were selected from the 39 traits by principal component and stepwise regression analyses.These 17 traits could reflect 99.3%of the total variation and can be used as a comprehensive evaluation index for pear germplasm resources.

The Variation of Stone Cell Content in 236 Germplasms of Sand Pear(Pyrus pyrifolia)and Identification of Related Candidate Genes

Stone cells have been described to substantially influence pear fruit quality,as lignin and cellulose are the main components of stone cells.However,there are limited studies on the relationship between the variation and molecular basis of stone cells,lignin and cellulose content among different pear varieties.Here,to reveal the variation of stone cell content within different cultivated species,we collected 236 germplasms of sand pear(Pyrus pyrifolia)at 50 days after flower blooming(DAFB),the key stage of stone cell formation.In our results,we measured the content of stone cells,lignin and cellulose and found that these contents ranged from2.82%to 29.00%,8.84%to 55.30%and 11.52%to 30.55%,respectively.Further analysis showed that the variation coefficient of stone cell,lignin and cellulose content was 39.10%,28.03%and 16.71%,respectively.Additionally,a significant correlation between stone cell,lignin and cellulose content were detected,and the correlation coefficient between the contents of stone cell and lignin(0.912)was higher than between the contents of stone cell and cellulose(0.796).Moreover,the average lignin content(29.73%)was higher than the average cellulose content(18.03%)in stone cells in pear fruits,indicating that lignin is the main component of stone cell in pears.Finally,on the basic of the transcriptome data,we identified 10 transcription factors belonging to bHLH,ERF,MYB,and NAC transcript families,which might be involved in lignin formation in stone cells.qRT-PCR experiments verified coincident trends between expression of candidate genes and stone cell content.This research laid foundation for future studies on genetic variation of stone cells in pear fruits and provided important gene resources for stone cell regulation.

Transcriptional profiles underlying the effects of salicylic acid on fruit ripening and senescence in pear(Pyrus pyrifolia Nakai)

Salicylic acid(SA) plays a pivotal role in delaying fruit ripening and senescence. However, little is known about its underlying mechanism of action. In this study, RNA sequencing was conducted to analyze and compare the transcriptome profiles of SA-treated and control pear fruits. We found a total of 159 and 419 genes differentially expressed between the SA-treated and control pear fruits after 12 and 24 h of treatment, respectively. Among these differentially expressed genes(DEGs), 125 genes were continuously differentially expressed at both treatment times, and they were identified as candidate genes that might be associated with SA-regulated fruit ripening and senescence. Bioinformatics analysis results showed that 125 DEGs were mainly associated with plant hormone biosynthesis and metabolism, cell wall metabolism and modification, antioxidant systems, and senescence-associated transcription factors. Additionally, the expression of several candidate DEGs in ripening and senescent pear fruits after SA treatments were further validated by quantitative real-time PCR(qRT-PCR). This study provides valuable information and enhances the understanding of the comprehensive mechanisms of SA-meditated pear fruit ripening and senescence.

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.

The Storability and Its Regulatory Mechanism of HuanghuaPear(Pyrus pyrifolia Nakai.)Fruit as Influencedby Postharvest Treatments

Different temperatures and PEF packing treatments were carried out on postharvest Huanghua pear fruit to investigate their effects on fruit storability and the regulatory mechanism. LOX activity, O2- content, AOS activity, ACC synthase activity, ACC content, ACC oxidase activity and ethylene production changed with peaks in the ripening fruit at 20℃ and were inhibited by cold storage, incidence of fruit woolness and fruit decay were lightened as well. Low temperature combined with PEF packing (PEF1 and PEF2) treatments could further improve the fruit storability, maintain preferable quality. There was no significant difference between PEF1 and PEF2 both during cold storage at 1℃ and shelf life at 20℃. The recommended storage period of Huanghua fruit was two months at It and could be extended one month longer with PEF packing treatments.

Involvement of long non-coding RNAs in pear fruit senescence under high-and low-temperature conditions

Pear fruit senescence under high-and low-temperature conditions has been reported to be mediated by microRNAs.Long non-coding RNAs(lncRNAs),which can function as competing endogenous RNAs that interact with microRNAs,may also be involved in temperature-affected fruit senescence.Based on the transcriptome and microRNA sequencings,in this study,3330 lncRNAs were isolated from Pyrus pyrifolia fruit.Of these lncRNAs,2060 and 537 were responsive to high-and low-temperature conditions,respectively.Of these differentially expressed lncRNAs,82 and 24 correlated to the mRNAs involved in fruit senescence under high-and low-temperature conditions,respectively.Moreover,three lncRNAs were predicted to be competing endogenous RNAs(ceRNAs)that interact with the microRNAs involved in fruit senescence,while one and two ceRNAs were involved in fruit senescence under high-and low-temperature conditions,respectively.A dual-luciferase assay showed that the interaction of an lncRNA with a microRNA disrupts the action of the microRNA on the expression of its target mRNA(s).Furthermore,four alternative splicing-derived lncRNAs interacted with miR172i homologies(Novel_88 and Novel_69)to relieve the repressed expression of their target and produce an miR172i precursor.Correlation analysis of microRNA expression suggested that Novel_69 is likely involved in the cleavage of the pre-miR172i hairpin to generate mature miR172i.Taken together,lncRNAs are involved in pear fruit senescence under high-or low-temperature conditions through ceRNAs and the production of microRNA.

Integrative transcriptomic and metabolomic analyses reveal the flavonoid biosynthesis of Pyrus hopeiensis flowers under cold stress

Low temperature is among the most restrictive factors to limit the yield and distribution of pear. Pyrus hopeiensis is a valuable wild resource.PCA showed that P. hopeiensis had strong cold resistance. In this study, the mRNA and metabolome sequencing of P. hopeiensis flower organs exposed to different low temperatures were performed to identify changes of genes and metabolites in response to low-temperature stress. A total of 4 851 differentially expressed genes(DEGs) were identified. Trend analysis showed that these DEGs were significantly enriched in profiles 19, 18, 7, 14, 1, 4 and 11. And the KEGG enrichment analysis showed that the DEGs in profile 18 were significantly enriched in flavone and flavonol biosynthesis. Besides, the expressed trends as well as GO and KEGG functional enrichment analyses of DEGs under cold and freezing stress showed significantly difference. Analyses of flavonoid-related pathways indicated that flavonoid structural genes had undergone significant changes. Correlation analysis showed that b HLH and MYB TFs may affect flavonoid biosynthesis by regulating structural gene expression. And PhMYB308 and PhMYB330 were likely candidate repressors of flavonoid biosynthesis by binding to a specific site in bHLH proteins. In total, 92 differentially accumulated metabolites(DAMs) were identified in P. hopeiensis flowers including 12 flavonoids. WGCNA results showed that coral 1, pink and brown 4 modules were closely associated with flavonoids and 11 MYBs and 15 bHLHs among the three modules may activate or inhibit the expression of 23 structural genes of flavonoid biosynthesis. Taken together, the results of this study provided a theoretical basis for further exploration of the molecular mechanisms of flavonoid biosynthesis and cold resistance of P. hopeiensis flower organs and our findings laid a foundation for further molecular breeding in cold-resistant pear varieties.

Preharvest application of melatonin induces anthocyanin accumulation and related gene upregulation in red pear (Pyrus ussuriensis)

Anthocyanins are important components in the peel of red pears and contribute to the appearance of the fruit.Melatonin application is known to affect anthocyanin biosynthesis,but the effect of preharvest melatonin application on fruit coloration remains largely unknown.The objective of this study was to determine the effects of preharvest melatonin application on pigmentation,phenolic compounds,and the expression of related genes in Nanhong pear(Pyrus ussuriensis).The applications were performed during the pre-color-change period by spraying 50 or 200μmol L^(-1)of melatonin on fruits.We found that treatment with melatonin had a significant effect on color development.The concentrations of anthocyanins and flavonols were enhanced by melatonin treatment,whereas hydroxycinnamate and flavanol concentrations were reduced.Quantitative real-time PCR analyses indicated that the transcription levels for most anthocyanin biosynthetic genes and anthocyanin-related transcription factors were induced by melatonin.Melatonin application also stimulated the expression of melatonin biosynthesis-related genes and consequently caused an increase in endogenous melatonin concentration.These results provide insights into melatonin-induced fruit coloration and will facilitate the application of exogenous melatonin in agriculture.