Pyrus Bretschneideri Research Articles

Genome size and chromosome ploidy identification in pear germplasm represented by Asian pears - Local pear varieties

Pear (Pyrus) germplasm resources in Xinjiang were rich, most of which were diploid. Including dozens of varieties such as Pyrus bretschneideri Rehd. (P-br), Pyrus pyrifolia Nakai. (P-py), Pyrus ussuriensis Maxim. (P-us), Pyrus communis L. (P-co), Pyrus betulifolia Bunge. (P-be), Pyrus sinkiangensis Yü. (P-si). The P-si was mainly distributed in Shanshan, Turpan, Korla, Aksu and other places. The genetic background of the main varietieswas not completely clear.The genome size and chromosome ploidy of main pear varieties in Xinjiang were identified by flow cytometry (FCM) and the traditional chromosome pressing method. The results showed that, the genome size of P-xj was 566.54 ± 125.17 Mb, slightly larger than that of the introduced varieties, with a 2C values of 1.16 ± 0.26 pg and a ploidy value (DI) of 1.1 ± 0.24. Twenty-four P-xj were diploid with 34 chromosome numbers. The genome sizes of introduced P-br, P-py, P-us, P-be, and P-co were 485.44 ± 10.05∼567.94 ± 44.40 Mb, and the 2C values was 0.99 ± 0.02∼1.16 ± 0.10 pg. The DI was 0.9 ± 0.06∼1.0 ± 0.09, the FCM detection indicated that they were diploid; the number of chromosomes was 34.There were differences in genome size between pear with the same ploidy. The genome size of all diploid pear was 480.95 ± 16.24∼599.14 ± 38.36 Mb, the 2C values was 0.98 ± 0.03 pg ∼1.23 ± 0.08 pg, and the DI was 0.9 ± 0.00∼1.2 ± 0.06. A total of 5 polyploid materials were found in this study: ‘Yilikamut’, ‘Kotoamut’, ‘Aiwenqieke’, ‘Heisuanli’ and ‘Sha01′, it can provide materials for the innovation and utilization of pear germplasm resources. The results of this study can provide technical support for Pyrus plant on ploidy breeding and omics study, and also a reference for other plants.

Impact of methyl salicylate on storage quality, ethylene action, and protein profiling of ‘Zaosu’ pear (Pyrus bretschneideri)

‘Zaosu’ pear, an early-maturing cultivar harvested in summer in China, readily exhibits quality deterioration and senescence owing to the lack of refrigeration and effective postharvest technology. To explore possible molecular mechanisms of methyl salicylate (MeSA) in delaying senescence, ‘Zaosu’ pear was treated with 0.05 mmol L―1 MeSA and stored at 25 ± 2 °C. The effects on quality, ethylene synthesis and signaling elements, and protein profiling were evaluated. MeSA maintained fruit postharvest quality. In the treated fruit, the yellowing index, weight loss, and color L* value retained lower levels, and the yellowing index was 69 % of that of the control after 20 days, whereas the hue angle and texture properties (fracturability, firmness, and chewiness) had increased levels. The respiration rate and ethylene content, and ethylene biosynthesis and signaling components ACS4, ACO1, ACO4, ETR1, ETR2, ERS2, CTR1, EIN2, and EIL1, were inhibited and downregulated by MeSA, respectively, while ERS1 was upregulated. Thirty-eight different expression proteins were identified by 2-DE and MALDI-TOF/TOF MS analysis in the treated and untreated fruits. Among them, related ripening and senescence proteins 2-methylene-furan-3-one reductase, abscisic stress-ripening protein, annexin RJ4, and mitochondrial outer membrane protein porin were down-accumulated during storage, while electron transfer flavoprotein was up-accumulated later in the storage period. These results indicated that MeSA postponed the ripening and senescence of postharvest ‘Zaosu’ pear.

Pengaruh Penambahan Asam Sitrat Terhadap Proses Non-Enzimatik Browning Jus Buah Pir Yali (Pyrus bretschneideri Rehd.)

The purpose of this study was to find out how effective citric acid is against the nonenzymaticprocess of browning Yali Pear fruit juice (Pyrus bretschneideri Rehd. Theresearch was conducted in a complete randomized design consisting of 5 replications. Thenon-enzymatic browning inhibition process was tested with citric acid treatment as the mainfactor with five concentrations ie 0% w/v, 2,5% w/v, 5% w/v, 7,5% w/v and 10% w/v.Qualitative parameters were dehydrogenase enzyme activity and reducing sugar level.Quantitative parameters were browning index and total soluble carbohydrate content.Levene test, analysis of variance, and Tukey test were performed at 5% significant level. Theresults showed that decrease in dehydrogenase enzyme activity occurs along with increasingthe concentration of citric acid. The level of reducing sugar has increased along with theincrease of citric acid concentration. The 7,5% w/v citric acid concentration effectivelyinhibited non-enzymatic browning of Yali Pear juice with a 21% reduction in the browningindex. Total soluble carbohydrate content increased 7% at 7.5% w/v citric acidconcentration. From the results of the study it was concluded that citric acid at 7,5% w / vconcentration was the inhibitor of non-enzymatic browning and dehydrogenase enzymeactivity, but stimulator of total soluble carbohydrate and reducing sugar level.

Genome-wide comparative analysis of the BAHD superfamily in seven Rosaceae species and expression analysis in pear (Pyrus bretschneideri)

BackgroundThe BAHD acyltransferase superfamily exhibits various biological roles in plants, including regulating fruit quality, catalytic synthesizing of terpene, phenolics and esters, and improving stress resistance. However, the copy numbers, expression characteristics and associations with fruit aroma formation of the BAHD genes remain unclear.ResultsIn total, 717 BAHD genes were obtained from the genomes of seven Rosaceae, (Pyrus bretschneideri, Malus domestica, Prunus avium, Prunus persica, Fragaria vesca, Pyrus communis and Rubus occidentalis). Based on the detailed phylogenetic analysis and classifications in model plants, we divided the BAHD family genes into seven groups, I-a, I-b, II-a, II-b, III-a, IV and V. An inter-species synteny analysis revealed the ancient origin of BAHD superfamily with 78 syntenic gene pairs were detected among the seven Rosaceae species. Different types of gene duplication events jointly drive the expansion of BAHD superfamily, and purifying selection dominates the evolution of BAHD genes supported by the small Ka/Ks ratios. Based on the correlation analysis between the ester content and expression levels of BAHD genes at different developmental stages, four candidate genes were selected for verification as assessed by qRT-PCR. The result implied that Pbr020016.1, Pbr019034.1, Pbr014028.1 and Pbr029551.1 are important candidate genes involved in aroma formation during pear fruit development.ConclusionWe have thoroughly identified the BAHD superfamily genes and performed a comprehensive comparative analysis of their phylogenetic relationships, expansion patterns, and expression characteristics in seven Rosaceae species, and we also obtained four candidate genes involved in aroma synthesis in pear fruit. These results provide a theoretical basis for future studies of the specific biological functions of BAHD superfamily members and the improvement of pear fruit quality.

Role of membrane lipid hydrolysis genes in the aroma formalion of Chinese white pear ‘Xiang Mian Li’

Pear is an economically important fruit worldwide. We recently reported a crispy texture pear cultivar ‘Xiang Mian Li’ (XML) with intense aroma as a good candidate for Chinese white pear breeding. Chinese white pears usually have faint odor, thus it's interesting to uncover the molecular mechanisms for the development of aroma in ‘XML’. Freshly plucked faint odor ‘XML’ fruit (XML-FA) and postharvest ripened fruit with intense aroma (XML-IA) were studied through RNA-Seq. A total of 3758 differentially expressed genes (DEGs) were found between XML-FA and XML-IA, which significantly enriched in 288 Gene Ontology (GO) terms and 101 KEGG pathways. Besides the expected “Plant hormone signal transduction” and “ethylene biosynthetic process”, changes in volatile biosynthesis related pathways were also found. Highly expressed LOX and lipase were found to be the most important genes involved in the characteristic aroma formation of ‘XML’, suggesting the great role of membrane lipid hydrolysis in the mechanism of aroma formation in ‘XML’ pear. Additionally, PDC2, ADH, FAD, PLD and 4CL were also closely related to the formation of ‘XML’ aroma. Our data can be applied to improve practically the breeding program for elite Chinese white pear cultivars.

Effects of Metaxenia on Stone Cell Formation in Pear (Pyrus bretschneideri) Based on Transcriptomic Analysis and Functional Characterization of the Lignin-Related Gene PbC4H2

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on stone cell formation has not been fully elucidated to date. This study used P. bretschneideri (Chinese white pear) cv. ‘Yali’ (high-stone cell content) and P. pyrifolia (Sand pear) cv. ‘Cuiguan’ (low-stone cell content) as pollination trees to pollinate P. bretschneideri cv. ‘Lianglizaosu’ separately to fill this gap in the literature. The results of quantitative determination, histochemical staining and electron microscopy indicated that the content of stone cells and lignin in YL fruit (‘Yali’ (pollen parent) × ‘Lianglizaosu’ (seed parent)) was significantly higher than that in CL fruit (‘Cuiguan’ (pollen parent) × ‘Lianglizaosu’ (seed parent)). The transcriptome sequencing results that were obtained from the three developmental stages of the two types of hybrid fruits indicated that a large number of differentially expressed genes (DEGs) related to auxin signal transduction (AUX/IAAs and ARFs), lignin biosynthesis, and lignin metabolism regulation (MYBs, LIMs, and KNOXs) between the CL and YL fruits at the early stage of fruit development. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of transcription factors (TFs) related to lignin metabolism, and ultimately affecting lignin deposition and stone cell development. In addition, we performed functional verification of a differentially expressed gene, PbC4H2 (cinnamate 4-hydroxylase). Heterologous expression of PbC4H2 in the c4h mutant not only restored its collapsed cell wall, but also significantly increased the lignin content in the inflorescence stem. The results of our research help to elucidate the metaxenia-mediated regulation of pear stone cell development and clarify the function of PbC4H2 in cell wall development and lignin synthesis, which establishes a foundation for subsequent molecular breeding.

The Pyrus bretschneideri invertase gene family: identification, phylogeny and expression patterns

Plant invertases comprise a family of enzymes that catalyze the irreversible hydrolysis of sucrose into glucose and fructose, which plays a crucial role in carbohydrate partitioning and regulating plant growth and development. However, currently, there is little information on the roles of invertases in the development of pear trees. In this study, we performed genome-wide identification of a total of 23 invertases from the predicted genome data of the pear plant (Pyrus bretschneideri R.). Based on phylogenetic analysis, the pear invertases could be clustered into 13 acid invertase genes and 10 alkaline/neutral invertase genes. Chromosome location showed that tandem duplication and segmental duplication have played an important role in the expansion of acid and neutral/alkaline invertases in pear plants. Evolutionary analysis of invertases from pear and two model plant species (Arabidopsis thaliana and Oryza sativa) indicated that the evolutionary divergence of invertases occurred before the common ancestor of dicots and monocots. Tissue-specific expression analysis showed that the transcripts of all the identified invertase genes were detectable in various tissues. We hypothesize that the identified invertase genes all participate in regulating pear growth and development.

Knockout of the S-acyltransferase Gene, PbPAT14, Confers the Dwarf Yellowing Phenotype in First Generation Pear by ABA Accumulation.

The development of dwarf fruit trees with smaller and compact characteristics leads to significantly increased fruit production, which is a major objective of pear (Pyrus bretschneideri) breeding. We identified the S-acylation activity of PbPAT14, an S-acyltransferase gene related to plant development, using a yeast (Saccharomyces cerevisiae) complementation assay, and also PbPAT14 could rescue the growth defect of the Arabidopsis mutant atpat14. We further studied the function of PbPAT14 by designing three guide RNAs for PbPAT14 to use in the CRISPR/Cas9 system. We obtained 22 positive transgenic pear lines via Agrobacterium-mediated transformation using cotyledons from seeds of Pyrus betulifolia (‘Duli’). Six of these lines exhibited the dwarf yellowing phenotype and were homozygous mutations according to sequencing analysis. Ultrastructure analysis suggested that this dwarfism was manifested by shorter, thinner stems due to a reduction in cell number. A higher level of endogenous abscisic acid (ABA) and a higher transcript level of the ABA pathway genes in the mutant lines revealed that the PbPAT14 function was related to the ABA pathway. Overall, our experimental results increase the understanding of how PATs function in plants and help elucidate the mechanism of plant dwarfism.

Comparative genomic analysis of the PAL genes in five Rosaceae species and functional identification of Chinese white pear.

Phenylalanine ammonia lyase (PAL) plays an important role in the biosynthesis of secondary metabolites regulating plant growth response. To date, the evolutionary history of the PAL family in Rosaceae plants remains unclear. In this study, we identified 16 PAL homologous genes in five Rosaceae plants (Pyrus bretschneideri, Fragaria vesca, Prunus mume, Prunus persica, and Malus × domestica). We classified these PALs into three categories based on phylogenetic analysis, and all PALs were distributed on 13 chromosomes. We tracked gene duplication events and performed sliding window analysis. These results revealed the evolution of PALs in five Rosaceae plants. We predicted the promoter of the PbPALs by PLANT CARE online software, and found that the promoter region of both PbPAL1 and PbPAL3 have at least one AC element. The results of qRT-PCR analysis found that PbPAL1 and PbPAL2 were highly expressed in the stems and roots, while expression level of PbPAL3 was relatively low in different tissues. The expression of PbPAL1 and PbPAL2 increased firstly and then decreased at different developmental periods of pear fruit. Among them, the expression of PbPAL1 reached the highest level 55 days after flowering. Three PbPALs were induced by abiotic stress to varying degrees. We transfected PbPAL1 and PbPAL2 into Arabidopsis thaliana, which resulted in an increase in lignin content and thickening of the cell walls of intervascular fibres and xylem cells. In summary, this research laid a foundation for better understanding the molecular evolution of PALs in five Rosaceae plants. Furthermore, the present study revealed the role of PbPALs in lignin synthesis, and provided basic data for regulating lignin synthesis and stone cells development in pear plants.

PbGA2ox8 induces vascular-related anthocyanin accumulation and contributes to red stripe formation on pear fruit

Fruit with stripes, which are generally longitudinal, can occur naturally, but the bioprocesses underlying this phenomenon are unclear. Previously, we observed an atypical anthocyanin distribution that caused red-striped fruit on the spontaneous pear bud sport “Red Zaosu” (Pyrus bretschneideri Rehd.). In this study, comparative transcriptome analysis of the sport and wild-type “Zaosu” revealed that this atypical anthocyanin accumulation was tightly correlated with abnormal overexpression of the gene-encoding gibberellin (GA) 2-beta-dioxygenase 8, PbGA2ox8. Consistently, decreased methylation was also observed in the promoter region of PbGA2ox8 from “Red Zaosu” compared with “Zaosu”. Moreover, the GA levels in “Red Zaosu” seedlings were lower than those in “Zaosu” seedlings, and the application of exogenous GA4 reduced abnormal anthocyanin accumulation in “Red Zaosu”. Transient overexpression of PbGA2ox8 reduced the GA4 level and caused anthocyanin accumulation in pear fruit skin. Moreover, the presence of red stripes indicated anthocyanin accumulation in the hypanthial epidermal layer near vascular branches (VBs) in “Red Zaosu”. Transient overexpression of PbGA2ox8 resulting from vacuum infiltration induced anthocyanin accumulation preferentially in calcium-enriched areas near the vascular bundles in pear leaves. We propose a fruit-striping mechanism, in which the abnormal overexpression of PbGA2ox8 in “Red Zaosu” induces the formation of a longitudinal array of anthocyanin stripes near vascular bundles in fruit.

Molecular, serological and biological characterization of a novel Apple stem pitting virus strain from a local pear variety grown in China

Apple stem pitting virus (ASPV) is an important causal agent of pear diseases. Nowadays, the infection status and molecular characteristics of the virus in old pear trees have never been investigated. In this study, we provide the first complete genome sequence of an ASPV isolate LYC from an over 300-year-old tree of a local Pyrus bretschneideri cultivar ‘Chili’ specifically grown at Laiyang area in China. ASPV-LYC possesses a chimeric genome consisting of 9 273 nucleotides excluding a poly(A) tail at its 3’ end and harboring a recombination region in its open reading frame (ORF1) with Aurora-1 and KL9 identified as the major and minor parents. Western blot analysis with antisera against recombinant coat proteins (CPs) of three ASPV isolates from pear indicates that ASPV-LYC is serologically related to these ASPV isolates, but with differential activities. Further biological tests on indicator plants of Pyronia veitchii show that ASPV-LYC can induce serious leaf and stem symptoms as other ASPV isolates. The results provide an important information for understanding molecular evolution of ASPV and suggest a need to prevent dissemination of the isolate among pear trees.

Transcriptomic analysis of early fruit development in Chinese white pear (Pyrus bretschneideri Rehd.) and functional identification of PbCCR1 in lignin biosynthesis

BackgroundThe content of stone cells and lignin is one of the key factors affecting the quality of pear fruit. In a previous study, we determined the developmental regularity of stone cells and lignin in ‘Dangshan Su’ pear fruit 15-145 days after pollination (DAP). However, the development of fruit stone cells and lignin before 15 DAP has not been heavily researched.ResultsIn this study, we found that primordial stone cells began to appear at 7 DAP and that the fruit had formed a large number of stone cells at 15 DAP. Subsequently, transcriptome sequencing was performed on fruits at 0, 7, and 15 DAP and identified 3834 (0 vs. 7 DAP), 4049 (7 vs. 15 DAP) and 5763 (0 vs. 15 DAP) DEGs. During the 7-15 DAP period, a large number of key enzyme genes essential for lignin biosynthesis are gradually up-regulated, and their expression pattern is consistent with the accumulation of lignin in this period. Further analysis found that the biosynthesis of S-type lignin in ‘Dangshan Su’ pear does not depend on the catalytic activity of PbSAD but is primarily generated by the catalytic activity of caffeoyl-CoA through CCoAOMT, CCR, F5H, and CAD. We cloned PbCCR1, 2 and analysed their functions in Chinese white pear lignin biosynthesis. PbCCR1 and 2 have a degree of functional redundancy; both demonstrate the ability to participate in lignin biosynthesis. However, PbCCR1 may be the major gene for lignin biosynthesis, while PbCCR2 has little effect on lignin biosynthesis.ConclusionsOur results revealed that ‘Dangshan Su’ pear began to form a large number of stone cells and produce lignin after 7 DAP and mainly accumulated materials from 0 to 7 DAP. PbCCR1 is mainly involved in the biosynthesis of lignin in ‘Dangshan Su’ pear and plays a positive role in lignin biosynthesis.

Investigation and analysis of relationship between mineral elements alteration and cork spot physiological disorder of Chinese pear ‘Chili’ (Pyrus bretschneideri Rehd.)

Cork spot is one of the most damaging physiological disorders in pear. However, the mechanism of cork spot is unclear. Ca2+ deficiency is considered as the major agent to cork spot. In this study, we investigated cork spot characteristics in Chinese ‘Chili’ (Pyrus bretschneideri Rehd.) and analyzed the relationship between mineral elements alteration and cork spot occurrence. We found early initiation of cork spot disorder in ‘Chili’ at 80 days after full bloom (DAFB). Cork spot in disordered fruit mostly distributed around the fruit eye with necrotic and brown tissues at the spotted site in flesh. At harvest time, cork spot disordered fruit had a smaller size, higher firmness, lower titratable acidity, higher lignin and cellulose content than healthy fruit, while starch concentration was the same between disordered and healthy fruit. Differences of mineral elements content were also found in both leaves and fruits, and Ca2+ distribution was visualized to be concentrated inside cell wall in disordered fruit cells. Principal components analysis (PCA) of mineral elements data showed that fruit samples could be separated according to their healthy or disordered condition at 80 and 160 DAFB. However, no significant correlation was found between cork spot disorder and mineral elements data. Ca2+ concentration was not suggested as the cork spot biochemical marker in our study. At last, we hypothesized the relationship between cork spot occurrence and Ca2+ distribution in ‘Chili’ and summarized the factors probably contributing to Ca2+ deficiency disorder, which would be useful in designing orchard strategies against cork spot.

Integrative Analysis of the Core Fruit Lignification Toolbox in Pear Reveals Targets for Fruit Quality Bioengineering.

Stone cell content is an important factor affecting pear fruit flavor. Lignin, a major component of pear stone cells, hinders the quality and value of commercial fruit. The completion of the Chinese white pear (Pyrus bretschneideri) genome sequence provides an opportunity to perform integrative analysis of the genes encoding the eleven protein families (i.e., PAL, C4H, 4CL, HCT, C3H, CSE, CCoAOMT, CCR, F5H, COMT, and CAD) in the phenylpropanoid pathway. Here, a systematic study based on expression patterns and phylogenetic analyses was performed to identify the members of each gene family potentially involved in the lignification in the Chinese white pear. The phylogenetic analysis suggested that 35 P. bretschneideri genes belong to bona fide lignification clade members. Compared to other plants, some multigene families are expanded by tandem gene duplication, such as HCT, C3H, COMT, and CCR. RNA sequencing was used to study the expression patterns of the genes in different tissues, including leaf, petal, bud, sepal, ovary, stem, and fruit. Eighteen genes presented a high expression in fruit, indicating that these genes may be involved in the biosynthesis of lignin in pear fruit. Similarly to what has been observed for Populus trichocarpa, a bimolecular fluorescence complementation (BiFC) experiment indicated that P. bretschneideri C3H and C4H might also interact with each other to regulate monolignol biosynthesis in P. bretschneideri, ultimately affecting the stone cell content in pear fruits. The identification of the major genes involved in lignin biosynthesis in pear fruits provides the basis for the development of strategies to improve fruit quality.

Identification and functional characterization of SOC1-like genes in Pyrus bretschneideri

Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear.

Integrated genetic linkage maps for Korean pears (Pyrus hybrid) using GBS-based SNPs and SSRs

Integrated linkage maps are a valuable tool for comparative mapping between and within genus and species. Novel single-nucleotide polymorphism (SNP) linkage maps integrated with simple sequence repeats (SSRs) based on pseudo-chromosomes of the Chinese pear reference genome of ‘Dangshansuli’ (Pyrus bretschneideri) were constructed. In total, 4801 qualified SNP markers were obtained using a customized pipeline. The consensus map for ‘Whangkeumbae’ and ‘Minibae’ contained 321 SNP and 30 SSR markers spanning 1511.1 cM with an average genetic distance of 4.3 cM. A total of 30 SSR markers made it possible to compare our consensus map to other pear and apple maps. SSR markers originating from pear and apple maps showed high co-linearity. SNPs coordinated with pseudo-chromosomes, provide information on physical length coverage for 17 corresponding linkage groups, and enable easier genome annotation for genomic regions detected by quantitative trait loci analysis. Genotyping-by-sequencing-based SNP maps integrated with SSRs in the interspecific mapping population illustrate the genomic structure of Korean pear resources and will be used as our reference maps for tribe Maleae.

Genetic relationship between the ‘Korla fragrant pear’ and local pear varieties in xinjiang based on floral organ characteristics

The characters of floral organ and pollen of local pear varieties in Xinjiang, such as the ‘Korla fragrant pear’ and 39 other varieties or types, Pyrus bretschneideri Rehd., Pyrus pyrifolia Nakai., and Pyrus communis L. were measured and described. The genetic relationships between the ‘Korla fragrant pear’ and other local pear varieties in Xinjiang were analyzed by clustering. The number of flowers per inflorescence of varieties or types was approximately 7.6, and the difference in the number of stamens was small. Except for ‘Aiwenqieke’, ‘Qipanli’ and ‘Daxiangshui’, which had no pollen, the pollen quantity of the varieties or types was 1000.0–5000.0 grains. The pollen grains were isopolar and radiation symmetrical single-grain. The pollen grains had three germination furrows, and the main type of pollen grain exine ornamentation was stripe ornamentation. According to Erdtman’s NPC classification system, the pear pollen was of the N3P4C5 type. According to the clustering results, there was high similarity between the ‘Korla fragrant pear’ and Pyrus sinkiangensis Yü. The ‘Korla fragrant pear’ clustered with most local varieties in Xinjiang, and a genetic relationship between P. bretschneideri and P. pyrifolia was observed. There were interspecific and variety or type differences in floral organ and pollen morphology between P. sinkiangensis and P. bretschneideri, P. pyrifolia, Pyrus ussuriensis Maxim., P. communis and Pyrus betulifolia Bunge. There were many similarities between the ‘Korla fragrant pear’ and local pear varieties in Xinjiang, which should be classified within the P. sinkiangensis system.

In Silico Genome-Wide Analysis of the Pear (Pyrus bretschneideri) KNOX Family and the Functional Characterization of PbKNOX1, an Arabidopsis BREVIPEDICELLUS Orthologue Gene, Involved in Cell Wall and Lignin Biosynthesis.

Stone cells are a characteristic trait of pear fruit, but the contents and sizes of stone cells negatively correlate with fruit texture and flavor. Secondary cell wall thickening and lignification have been established as key steps of stone cell development. KNOTTED-LIKE HOMEOBOX (KNOX) proteins play important roles in plant cell growth and development, including cell wall formation and lignification. Although the characteristics and biological functions of KNOX proteins have been investigated in other plants, this gene family has not been functionally characterized in pear. Eighteen PbKNOX genes were identified in the present study, and all of the identified family members contained the KNOX I and/or KNOX II domains. Based on the phylogenetic tree and chromosomal localization, the 18 PbKNOX genes were divided into five subfamilies [SHOOT MERISTEMLESS (STM)-like, BREVIPEDICELLUS (BP)-like, KNOTTED ARABIDOPSIS THALIANA 2/6 (KNAT2/6)-like, KNAT7-like, and KNAT3-5-like] and were distributed among 10 chromosomes. In addition, we identified 9, 11, and 11 KNOX genes in the genomes of grape, mei, and strawberry, respectively, and the greatest number of collinear KNOX gene pairs formed between pears and peaches. Analyses of the spatiotemporal expression patterns showed that the tissue specificity of PbKNOX gene expression was not very significant and that the level of the PbKNOX1 transcript showed an opposite trend to the levels of stone cells and lignin accumulation. Furthermore, PbKNOX1 has high sequence identity and similarity with Arabidopsis BP. Compared with wild-type Arabidopsis, plants overexpressing PbKNOX1 not only showed an approximately 19% decrease in the secondary cell wall thickness of vessel cells but also exhibited an approximately 13% reduction in the lignin content of inflorescence stems. Moreover, the expression of several genes involved in lignin biosynthesis was downregulated in transgenic lines. Based on our results, PbKNOX1/BP participates in cell wall-thickening and lignin biosynthesis and represses the transcription of key structural genes involved in lignin synthesis, providing genetic evidence for the roles of KNOX in cell wall thickening and lignin biosynthesis in pear.

Different response to 1-methylcyclopropene in two cultivars of Chinese pear fruit with contrasting softening characteristics

In this study, the change in softening and its related genes expression under influence of 500 nl L-1 1-methylcyclopropene (1-MCP) was assessed in the two Chinese pear fruit, ‘Jingbaili’ (Pyrus ussuriensis Maxim) and ‘Yali’ (Pyrus bretschneideri Rehd), which exhibit different softening characteristics. ‘Jingbaili’ pear fruit softened rapidly after harvest, and was strongly inhibited by 1-MCP. In contrast, there was no obvious change of firmness compared to the control after 1-MCP treatment in ‘Yali’ pear fruit. The respiration and ethylene production rates were reduced by 1-MCP at early storage in both two cultivars. ‘Jingbaili’ pear fruit exhibited dramatically increased expression levels of the softening-related genes, i.e., polygalacturonase1 (PG1), polygalacturonase2 (PG2), β-Galactosidase4 (GAL4), α-arabinofuranosidase1 (ARF1) and α-arabinofuranosidase2 (ARF2), and these genes’ expression levels were significantly decreased by 1-MCP treatment. In contrast, ‘Yali’ pear fruit showed lower expression levels of the above-mentioned genes, as well as a relatively smaller inhibition effect by 1-MCP treatment before day 27. These results suggest that ‘Jingbaili’ pear fruit are more sensitive to 1-MCP/ethylene than ‘Yali’ pear fruit during ripening.

In Silico Genome-Wide Analysis of Respiratory Burst Oxidase Homolog (RBOH) Family Genes in Five Fruit-Producing Trees, and Potential Functional Analysis on Lignification of Stone Cells in Chinese White Pear.

The accumulation of lignin in fruit has a significant negative impact on the quality of fruit-producing trees, and in particular the lignin formation stimulates the development of stone cells in pear fruit. Reactive oxygen species (ROS) are essential for lignin polymerization. However, knowledge of the RBOH family, a key enzyme in ROS metabolism, remains unknown in most fruit trees. In this study, a total of 40 RBOHs were identified from five fruit-producing trees (Pyrus bretschneideri, Prunus persica, Citrus sinensis, Vitis vinifera, and Prunus mume), and 10 of these sequences came from Pyrus bretschneideri. Multiple sequence alignments revealed that all 10 PbRBOHs contained the NADPH_Ox domain and the six alpha-helical transmembrane domains (TM-I to TM-VI). Chromosome localization and interspecies phylogenetic tree analysis showed that 10 PbRBOHs irregularly distributed on 8 chromosomes and 3 PbRBOHs (PbRBOHA, PbRBOHB, and PbRBOHD) are closely related to known lignification-related RBOHs. Furthermore, hormone response pattern analysis showed that the transcription of PbRBOHs is regulated by SA, ABA and MeJA. Reverse transcription-quantitative real-time polymerase chain reaction (qRT-PCR) and transcriptome sequencing analysis showed that PbRBOHA, PbRBOHB, and PbRBOHD accumulated high transcript abundance in pear fruit, and the transcriptional trends of PbRBOHA and PbRBOHD was consistent with the change of stone cell content during fruit development. In addition, subcellular localization revealed that PbRBOHA and PbRBOHD are distributed on the plasma membrane. Combining the changes of apoplastic superoxide (O2.−) content and spatio-temporal expression analysis, these results indicate that PbRBOHA and PbRBOHD, which are candidate genes, may play an important role in ROS metabolism during the lignification of pear stone cells. This study not only provided insight into the molecular characteristics of the RBOH family in fruit-producing trees, but also lays the foundation for studying the role of ROS in plant lignification.