Multi-omics analyses reveal stone cell distribution pattern in pear fruit.

Stone cells are the brachysclereid cells in pear (Pyrus) fruit, consisting almost entirely of lignified secondary cell walls. They are distributed mainly near the fruit core and spread radially in the whole fruit. However, the development of stone cells has not been comprehensively characterized, and little is known about the regulation of stone cell formation at the transcriptomic, proteomic, and metabolomic levels. In the present study, we performed phenomic analysis on the stone cells and their associated vascular bundles distributed near the fruit cores. Transcriptomic, proteomic, and metabolomic analyses revealed a significant positive regulation of biological processes which contribute to the lignification and lignin deposition in stone cells near the fruit core, including sucrose metabolism and phenylalanine, tyrosine, tryptophan, and phenylalanine biosynthesis. We found many metabolites generated from the phenylpropanoid pathway contributing to the cell wall formation of stone cells near the fruit core. Furthermore, we identified a key transcription factor, PbbZIP48, which was highly expressed near the fruit core and was shown to regulate lignin biosynthesis in stone cells. In conclusion, the present study provides insight into the mechanism of lignified stone cell formation near the pear fruit core at multiple levels.

Genome-wide characterisation of HD-Zip transcription factors and functional analysis of PbHB24 during stone cell formation in Chinese white pear (Pyrus bretschneideri).

BACKGROUND: The homodomain-leucine zipper (HD-Zip) is a conserved transcription factor family unique to plants that regulate multiple developmental processes including lignificaion. Stone cell content is a key determinant negatively affecting pear fruit quality, which causes a grainy texture of fruit flesh, because of the lignified cell walls. RESULTS: In this study, a comprehensive bioinformatics analysis of HD-Zip genes in Chinese white pear (Pyrus bretschneideri) (PbHBs) was performed. Genome-wide identification of the PbHB gene family revealed 67 genes encoding PbHB proteins, which could be divided into four subgroups (I, II, III, and IV). For some members, similar intron/exon structural patterns support close evolutionary relationships within the same subgroup. The functions of each subgroup of the PbHB family were predicted through comparative analysis with the HB genes in Arabidopsis and other plants. Cis-element analysis indicated that PbHB genes might be involved in plant hormone signalling and external environmental responses, such as light, stress, and temperature. Furthermore, RNA-sequencing data and quantitative real-time PCR (RT-qPCR) verification revealed the regulatory roles of PbHB genes in pear stone cell formation. Further, co-expression network analysis revealed that the eight PbHB genes could be classified into different clusters of co-expression with lignin-related genes. Besides, the biological function of PbHB24 in promoting stone cell formation has been demonstrated by overexpression in fruitlets. CONCLUSIONS: This study provided the comprehensive analysis of PbHBs and highlighted the importance of PbHB24 during stone cell development in pear fruits.

Automatic Branch-Leaf Segmentation and Leaf Phenotypic Parameter Estimation of Pear Trees Based on Three-Dimensional Point Clouds.

The leaf phenotypic traits of plants have a significant impact on the efficiency of canopy photosynthesis. However, traditional methods such as destructive sampling will hinder the continuous monitoring of plant growth, while manual measurements in the field are both time-consuming and laborious. Nondestructive and accurate measurements of leaf phenotypic parameters can be achieved through the use of 3D canopy models and object segmentation techniques. This paper proposed an automatic branch-leaf segmentation pipeline based on lidar point cloud and conducted the automatic measurement of leaf inclination angle, length, width, and area, using pear canopy as an example. Firstly, a three-dimensional model using a lidar point cloud was established using SCENE software. Next, 305 pear tree branches were manually divided into branch points and leaf points, and 45 branch samples were selected as test data. Leaf points were further marked as 572 leaf instances on these test data. The PointNet++ model was used, with 260 point clouds as training input to carry out semantic segmentation of branches and leaves. Using the leaf point clouds in the test dataset as input, a single leaf instance was extracted by means of a mean shift clustering algorithm. Finally, based on the single leaf point cloud, the leaf inclination angle was calculated by plane fitting, while the leaf length, width, and area were calculated by midrib fitting and triangulation. The semantic segmentation model was tested on 45 branches, with a mean Precisionsem, mean Recallsem, mean F1-score, and mean Intersection over Union (IoU) of branches and leaves of 0.93, 0.94, 0.93, and 0.88, respectively. For single leaf extraction, the Precisionins, Recallins, and mean coverage (mCoV) were 0.89, 0.92, and 0.87, respectively. Using the proposed method, the estimated leaf inclination, length, width, and area of pear leaves showed a high correlation with manual measurements, with correlation coefficients of 0.94 (root mean squared error: 4.44°), 0.94 (root mean squared error: 0.43 cm), 0.91 (root mean squared error: 0.39 cm), and 0.93 (root mean squared error: 5.21 cm2), respectively. These results demonstrate that the method can automatically and accurately measure the phenotypic parameters of pear leaves. This has great significance for monitoring pear tree growth, simulating canopy photosynthesis, and optimizing orchard management.

Transcriptome analysis provides new ideas for studying the regulation of glucose-induced lignin biosynthesis in pear calli.

BACKGROUND: Glucose can be involved in metabolic activities as a structural substance or signaling molecule and plays an important regulatory role in fruit development. Glucose metabolism is closely related to the phenylpropanoid pathway, but the specific role of glucose in regulating lignin biosynthesis in pear fruit is still unclear. The transcriptome of pear calli generated from fruit and treated with glucose was analyzed to investigate the role of glucose in lignin biosynthesis. RESULTS: The treatment of exogenous glucose significantly enhanced the accumulation of lignin in pear calli. A total of 6566 differentially expressed genes were obtained by transcriptome sequencing. Glycolysis was found to be the pathway with significant changes. Many differentially expressed genes were enriched in secondary metabolic pathways, especially the phenylpropanoid pathway. Expression of structural genes (PbPAL, PbHCT, PbCOMT, PbPRX) in lignin biosynthesis was up-regulated after glucose treatment. In addition, glucose might regulate lignin biosynthesis through interactions with ABA, GA, and SA signaling. Several candidate MYB transcription factors involved in glucose-induced lignin biosynthesis have also been revealed. The qRT-PCR analyses showed that the expression pattern of PbPFP at early developmental stage in 'Dangshansuli' fruits was consistent with the trend of lignin content. Transient expression of PbPFP resulted in a significant increase of lignin content in 'Dangshansuli' fruits at 35 days after full bloom (DAB) and tobacco leaves, indicating that PbPFP (Pbr015118.1) might be associated with the enhancement of lignin biosynthesis in response to glucose treatment. CONCLUSIONS: PbPFP plays a positive role in regulating lignin biosynthesis in response to glucose treatment. This study may reveal the regulatory pathway related to lignin accumulation in pear calli induced by glucose.

Transcriptome provides potential insights into how calcium affects the formation of stone cell in Pyrus.

BACKGROUND: The content of stone cells in pears has a great influence on taste. Stone cells are formed by the accumulation of lignin. The treatment of exogenous calcium can affect the lignin synthesis, but this Ca-mediated mechanism is still unclear. In this study, the author performed a comparative transcriptomic analysis of callus of pears (Pyrus x bretschneideri) treated with calcium nitrate Ca (NO3)2 to investigate the role of calcium in lignin synthesis. RESULTS: There were 2889 differentially expressed genes (DEGs) detected between the Control and Ca (NO3)2 treatment in total. Among these 2889 DEGs, not only a large number of genes related to Ca single were found, but also many genes were enriched in secondary metabolic pathway, especially in lignin synthesis. Most of them were up-regulated during the development of callus after Ca (NO3)2 treatment. In order to further explore how calcium nitrate treatment affects lignin synthesis, the author screened genes associated with transduction of calcium signal in DEGs, and finally found CAM, CML, CDPK, CBL and CIPK. Then the author identified the PbCML3 in pears and conducted relevant experiments finding the overexpression of PbCML3 would increase the content of pear stone cells, providing potential insights into how Ca treatment enhances the stone cell in pears. CONCLUSIONS: Our deep analysis reveals the effects of exogenous calcium on calcium signal and lignin biosynthesis pathway. The function of PbCML3 on stone cells formation was verified in pear.

Genome-wide analyses and expression patterns under abiotic stress of NAC transcription factors in white pear (Pyrus bretschneideri).

BACKGROUND: Although the genome of Chinese white pear ('Dangshansuli') has been released, little is known about the functions, evolutionary history and expression patterns of NAC families in this species to date. RESULTS: In this study, we identified a total of 183 NAC transcription factors (TFs) in the pear genome, among which 146 pear NAC (PbNAC) members were mapped onto 16 chromosomes, and 37 PbNAC genes were located on scaffold contigs. No PbNAC genes were mapped to chromosome 2. Based on gene structure, protein motif analysis, and topology of the phylogenetic tree, the pear PbNAC family was classified into 33 groups. By comparing and analyzing the unique NAC subgroups in Rosaceae, we identified 19 NAC subgroups specific to pear. We also found that whole-genome duplication (WGD)/segmental duplication played critical roles in the expansion of the NAC family in pear, such as the 83 PbNAC duplicated gene pairs dated back to the two WGD events. Further, we found that purifying selection was the primary force driving the evolution of PbNAC family genes. Next, we used transcriptomic data to study responses to drought and cold stresses in pear, and we found that genes in groups C2f, C72b, and C100a were related to drought and cold stress response. CONCLUSIONS: Through the phylogenetic, evolutionary, and expression analyses of the NAC gene family in Chinese white pear, we indentified 11 PbNAC TFs associated with abiotic stress in pear.

Characterization and Quantification of Polyphenols and Triterpenoids in Thinned Young Fruits of Ten Pear Varieties by UPLC-Q TRAP-MS/MS.

Large quantities of thinned young pears, a natural source of bioactive compounds, are abandoned as agricultural by-products in many orchards. Hence, ten thinned young pear varieties were systematically investigated in terms of their chemical composition and antioxidant potential. Through ultra-performance liquid chromatography coupled with electrospray ionization triple quadrupole mass spectrometry (UPLC-Q TRAP-MS/MS), 102 polyphenols and 16 triterpenoids were identified and individually quantified within a short time using multiple reaction monitoring (MRM). Subsequently, the antioxidant capacities of these pears were determined with DPPH assays, and the correlation between total antioxidant activity and each component was analyzed. The results indicated that the bioactive compound content and antioxidant capacity in thinned pears were considerably high. Regarding chemical composition, chlorogenic acid, quinic acid and arbutin were the primary polyphenols and ursolic acid was the predominant triterpenoid, whereas 27 polyphenolic compounds, especially chlorogenic acid and most of the flavan-3-ols, were the main antioxidants in young pears. These findings should provide a scientific basis for the further use of pear fruit by-products.

The mining and evolutionary investigation of AP2/ERF genes in pear (Pyrus).

BACKGROUND: In plants, ERF genes participate in a variety of regulatory pathways, such as plant growth and biotic and/or abiotic stress responses. Although the genome of Chinese white pear ('Dangshansuli') has been released, knowledge regarding the ERF family in pear, such as gene functions, evolutionary history and expression patterns, remains limited. RESULTS: In our study, a total of 155 members of ERF families were identified in pear (Pyrus bretschneideri). The Ka and Ks values suggested that whole-genome duplication (WGD) and dispersed duplication have effectively contributed to the expansion of the pear ERF family. Gene structure and phylogeny analysis divided the PbrERF family into 12 groups, and their gene functions were predicted by comparative analysis. qRT-PCR was carried out to verify the relative expression levels of 7 genes in group III using wild and cultivated pear fruits at three key developmental stages. Wild samples had higher expression of these genes than cultivated samples, especially at the enlarged fruit stage. The transcriptome data of pear seedlings subjected to dehydration treatment further revealed that 4 of the 7 genes responded to drought conditions. CONCLUSION: The AP2/ERF gene family is greatly expanded in pear. Comparative analysis revealed the probability of ERF genes performing functional roles in multiple pathways. Expression analysis at different stages of pear fruit development in wild and cultivated samples indicated that genes in group III might be involved in abiotic and/or biotic stresses. Further transcriptome data on seedlings subjected to drought treatment verified the potential role of ERF genes in stress response. These results will provide a valuable reference for understanding the function and evolution of the ERF family in higher plants.

The unique evolutionary pattern of the Hydroxyproline-rich glycoproteins superfamily in Chinese white pear (Pyrus bretschneideri).

BACKGROUND: The hydroxyproline-rich glycoprotein (HRGP) superfamily, comprising three families (arabinogalactan-proteins, AGPs; extensins, EXTs; proline-rich proteins, PRPs), is a class of proline-rich proteins that exhibit high diversity and are involved in many aspects of plant biology. RESULTS: In this study, 838 HRGPs were identified from Chinese white pear (Pyrus bretschneideri) by searching for biased amino acid composition and conserved motifs. 405 HRGPs were derived from whole genome duplication (WGD) events which is suggested to be the major force of driving HRGPs expansion and the recent WGD event shared by apple and pear generated most duplicated HRGPs in pear. This duplication event drived the structural variation of the HRGPs encoding hydroxyproline (Hyp)-rich motifs. The rate of HRGPs evolution mainly impacted the Hyp-rich motifs even in chimeric HRGPs. During the evolution of 53 PRPs that are also typified by 7-deoxyloganetin glucosyltransferase-like genes, the duplication from PRP to non-PRP was indirectly modified by positive selection. These results suggested that the rate of HRGP evolution mainly influenced the Hyp-rich motifs even in chimeric HRGPs. The expression divergence of HRGPs was higher than that of other commonly duplicated genes. In pear pistil, 601 HRGPs exhibited expression, while in pear pollen, 285 HRGPs were expressed. The qPCR results revealed that Pbr036330.1 and Pbr010506.1 showed different expression profile in self-incompatibility of pear pistil. CONCLUSIONS: The researches indicated that WGD events was the main duplication type during the evolution of HRGPs, and the highly variable Hyp-motifs might be accountable for the expansion, evolution and expression divergence of HRGPs and that this divergence may be responsible for the gain of new functions in plants.

The genome of the pear (Pyrus bretschneideri Rehd.).

The draft genome of the pear (Pyrus bretschneideri) using a combination of BAC-by-BAC and next-generation sequencing is reported. A 512.0-Mb sequence corresponding to 97.1% of the estimated genome size of this highly heterozygous species is assembled with 194× coverage. High-density genetic maps comprising 2005 SNP markers anchored 75.5% of the sequence to all 17 chromosomes. The pear genome encodes 42,812 protein-coding genes, and of these, ~28.5% encode multiple isoforms. Repetitive sequences of 271.9 Mb in length, accounting for 53.1% of the pear genome, are identified. Simulation of eudicots to the ancestor of Rosaceae has reconstructed nine ancestral chromosomes. Pear and apple diverged from each other ~5.4-21.5 million years ago, and a recent whole-genome duplication (WGD) event must have occurred 30-45 MYA prior to their divergence, but following divergence from strawberry. When compared with the apple genome sequence, size differences between the apple and pear genomes are confirmed mainly due to the presence of repetitive sequences predominantly contributed by transposable elements (TEs), while genic regions are similar in both species. Genes critical for self-incompatibility, lignified stone cells (a unique feature of pear fruit), sorbitol metabolism, and volatile compounds of fruit have also been identified. Multiple candidate SFB genes appear as tandem repeats in the S-locus region of pear; while lignin synthesis-related gene family expansion and highly expressed gene families of HCT, C3'H, and CCOMT contribute to high accumulation of both G-lignin and S-lignin. Moreover, alpha-linolenic acid metabolism is a key pathway for aroma in pear fruit.

Long-chain base phosphates modulate pollen tube growth via channel-mediated influx of calcium.

Long-chain base phosphates (LCBPs) have been correlated with amounts of crucial biological processes ranging from cell proliferation to apoptosis in animals. However, their functions in plants remain largely unknown. Here, we report that LCBPs, sphingosine-1-phosphate (S1P) and phytosphingosine-1-phosphate (Phyto-S1P), modulate pollen tube growth in a concentration-dependent bi-phasic manner. The pollen tube growth in the stylar transmitting tissue was promoted by SPHK1 overexpression (SPHK1-OE) but dampened by SPHK1 knockdown (SPHK1-KD) compared with wild-type of Arabidopsis; however, there was no detectable effect on in vitro pollen tube growth caused by misexpression of SPHK1. Interestingly, exogenous S1P or Phyto-S1P applications could increase the pollen tube growth rate in SPHK1-OE, SPHK1-KD and wild-type of Arabidopsis. Calcium ion (Ca(2+) )-imaging analysis showed that S1P triggered a remarkable increase in cytosolic Ca(2+) concentration in pollen. Extracellular S1P induced hyperpolarization-activated Ca(2+) currents in the pollen plasma membrane, and the Ca(2+) current activation was mediated by heterotrimeric G proteins. Moreover, the S1P-induced increase of cytosolic free Ca(2+) inhibited the influx of potassium ions in pollen tubes. Our findings suggest that LCBPs functions in a signaling cascade that facilitates Ca(2+) influx and modulates pollen tube growth.

Mitochondrial dysfunction mediated by cytoplasmic acidification results in pollen tube growth cessation in Pyrus pyrifolia.

The length of pollen tubes grown in synthetic media is normally shorter than those grown in vivo. However, the mechanism(s) underlying the cessation of pollen tube growth under culture conditions remain(s) largely unknown. Here, we report a previously unknown correlation between vacuolar function and the cell's ability to sustain mitochondrial functions in pear pollen tubes. The pear pollen tubes in vitro grew slowly after 15 hours post-cultured (HPC) and nearly ceased growth at 18 HPC. There was increased malondialdehyde content and membrane ion leakage at 15 HPC compared with 12 HPC. Furthermore, cytoplasmic acidification mainly mediated by decreased vacuolar H(+)-ATPase [V-ATPase, Enzyme Commission (EC) 3.6.1.3] activity was observed in pollen tubes after 15 HPC, and this further resulted in mitochondrial dysfunction, including mitochondrial structure disruption, mitochondrial membrane potential collapse and decreases in both oxygen consumption and ATP production. Our findings suggest that vacuoles and mitochondria intimately linked in regulating pollen tube elongation.

Evolution of the aroma volatiles of pear fruits supplemented with fatty acid metabolic precursors.

To examine the biochemical metabolism of aroma volatiles derived from fatty acids, pear fruits were incubated in vitro with metabolic precursors of these compounds. Aroma volatiles, especially esters, were significantly increased, both qualitatively and quantitatively, in pear fruits fed on fatty acid metabolic precursors. Cultivars having different flavor characteristics had distinctly different aroma volatile metabolisms. More esters were formed in fruity-flavored "Nanguoli" fruits than in green-flavored "Dangshansuli" fruits fed on the same quantities of linoleic acid and linolenic acid. Hexanal and hexanol were more efficient metabolic intermediates for volatile synthesis than linoleic acid and linolenic acid. Hexyl esters were the predominant esters produced by pear fruits fed on hexanol, and their contents in "Dangshansuli" fruits were higher than in "Nanguoli" fruits. Hexyl esters and hexanoate esters were the primary esters produced in pear fruits fed on hexanal, however the content of hexyl ester in "Dangshansuli" was approximately three times that in "Nanguoli". The higher contents of hexyl esters in "Dangshansuli" may have resulted from a higher level of hexanol derived from hexanal. In conclusion, the synthesis of aroma volatiles was largely dependent on the metabolic precursors presented.

PbARF19-mediated auxin signaling regulates lignification in pear fruit stone cells.

The stone cells in pear fruits cause rough flesh and low juice, seriously affecting the taste. Lignin has been demonstrated as the main component of stone cells. Auxin, one of the most important plant hormone, regulates most physiological processes in plants including lignification. However, the concentration effect and regulators of auxin on pear fruits stone cell formation remains unclear. Here, endogenous indole-3-acetic acid (IAA) and stone cells were found to be co-localized in lignified cells by immunofluorescence localization analysis. The exogenous treatment of different concentrations of IAA demonstrated that the application of 200 µM IAA significantly reduced stone cell content, while concentrations greater than 500 µM significantly increased stone cell content. Besides, 31 auxin response factors (ARFs) were identified in pear genome. Putative ARFs were predicted as critical regulators involved in the lignification of pear flesh cells by phylogenetic relationship and expression analysis. Furthermore, the negative regulation of PbARF19 on stone cell formation in pear fruit was demonstrated by overexpression in pear fruitlets and Arabidopsis. These results illustrated that the PbARF19-mediated auxin signal plays a critical role in the lignification of pear stone cell by regulating lignin biosynthetic genes. This study provides theoretical and practical guidance for improving fruit quality in pear production.

Pyrus betulaefolia ERF3 interacts with HsfC1a to coordinately regulate aquaporin PIP1;4 and NCED4 for drought tolerance.

Environmental disasters like drought reduce agricultural output and plant growth. Redox management significantly affects plant stress responses. An earlier study found that PbPIP1;4 transports H2O2 and promotes H2O2 downstream cascade signaling to restore redox equilibrium. However, this regulatory mechanism requires additional investigation. In this search, the AP2 domain-containing transcription factor was isolated by screening Y1H from the wild pear (Pyrus betulaefolia) cDNA library, named PbERF3. The overexpression of PbERF3 in pear callus and Arabidopsis enhanced plant resistance to drought and re-established redox balance. The transcripts of the NCEDs gene were upregulated under drought stress. The drought stress-related abscisic acid (ABA) signaling pathway modulates PbERF3. PbERF3 silencing lowered drought tolerance. Furthermore, yeast 2-hybrid, luciferase, bimolecular fluorescence complementation, and co-immunoprecipitation assays verified that PbERF3 physically interacted with PbHsfC1a. The PbERF3-PbHsfC1a heterodimer coordinately bound to PbPIP1;4 and PbNCED4 promoter, therefore activating both the H2O2 and the ABA signaling pathway. This work revealed a novel PbERF3-PbHsfC1a-PbNCED4-PbPIP1;4 regulatory module, in which PbERF3 interacts with PbHsfC1a to trigger the expression of target genes. This module establishes an interaction between the H2O2 signaling component PbPIP1;4 and the ABA pathways component PbNCED4, enabling a response to drought.

Haplotype-resolved T2T genome assemblies and pangenome graph of pear reveal diverse patterns of allele-specific expression and genomic basis of fruit quality traits.

Hybrid crops often exhibit increased yield and greater resilience, yet the genomic mechanism(s) underlying hybrid vigor or heterosis remain unclear, hindering our ability to predict the expression of phenotypic traits in hybrid breeding. Here, we generated haplotype-resolved T2T genome assemblies of two pear hybrid varieties 'Yuluxiangli' (YLX) and 'Hongxiangsu' (HXS) that share the same maternal parent, but differ in their paternal parents. We then used these assemblies to explore genome-scale landscape of allele-specific expression and create a pangenome graph for pear. Allele specific expression (ASE) was observed for close to 6000 genes in both hybrid cultivars. A subset of ASEGs related to fruit quality including sugar, organic acid and cuticular wax were identified, suggesting their important contributions to heterosis. Specifically, Ma1, a gene regulating fruit acidity, was absent in the paternal haplotypes of HXS and YLX. Further, a pangenome graph was built based on our assemblies and eight published pear genomes. Resequencing data for 139 cultivated pear genotypes (including 97 genotypes sequenced here) were subsequently aligned to the pangenome graph, revealing numerous SV hotspots and selective sweeps during pear diversification. As predicted, the Ma1 allele was found to be absent in varieties with low organic acid content, an association that was functionally validated by Ma1 over-expression in pear fruit and calli. Overall, the results unraveled contributions of allele-specific expression to heterosis involving fruit quality and provided a robust pangenome reference for high resolution allele discovery and association mapping.

BreedingEIS: An Efficient Evaluation Information System for Crop Breeding.

Crop breeding programs generate large datasets. Thus, it is difficult to ensure the accuracy and integrity of all the collected data in the breeding process. To improve breeding efficiency, we established an open source and free breeding evaluation information system (BreedingEIS). The full system is composed of a web client and a mobile client. The web client is used to name the individual breeding offspring plants and analyze data. The mobile client is based on the technology of widely used smartphones and is suitable for Android and iOS systems. Its functions focus on field evaluation, including quick response code recognition, evaluation data entry, and real-time viewing. In addition, near-field communication technology and portable label machines are introduced to enable breeders to quickly locate each individual plant and accurately label any samples collected from it. Generally, BreedingEIS enables users to accurately and conveniently register phenotypic data and quickly lock target individual plants from large volumes of data. The system provides a low-cost and highly efficient solution for crop information evaluation and enables breeders to better collect, manage, and use breeding data for decision making, which is a valuable resource for crop breeding.

PbPDCB16-mediated callose deposition affects the plasmodesmata blockage and reduces lignification in pear fruit.

Stone cell, a type of lignified cell, is a unique trait in pear and one of the key factors affects pear fruit quality and economic value. The transmissibility of cell lignification process has been proven to exist, however the effects of callose on the permeability of plasmodesmata (PD) and how to influence cell lignification processes are still unknown. In this study, the genome-wide analysis of PD callose binding proteins (PDCB) gene family in pear genome was performed, and 25 PbPDCB genes were identified and divided into four branches. Similar intron/exon structural patterns were observed in the same branch, strongly supporting their close evolutionary relationship. The expression of PbPDCB16 was negatively correlated with lignin accumulation through qRT-PCR analysis. With transient expression in pear fruit and stable expression in pear calli, the increased callose content accompanied by decreased lignin content was further observed. Besides, compared with wild type Arabidopsis, the transgenic plants grew slowly, and cell walls in the stem were thinner, while fewer PDs were observed on the cell walls, and the interspore filaments were also blocked in transgenic Arabidopsis through the transmission electron microscope (TEM). In summary, overexpression of PbPDCB16 could promote accumulation of callose at PD to affect the PD-mediated intercellular connectivity, and inhibit the intercellular communication. This study will provide new insight in reducing the lignin content through callose deposition, and also provide the theoretical basis for further exploration of lignin metabolism and cell wall lignification to form stone cells in pear fruit.

A large-scale proteogenomic atlas of pear.

Pear is an important fruit tree that is widely distributed around the world. The first pear genome map was reported from our laboratory approximately 10 years ago. To further study global protein expression patterns in pear, we generated pear proteome data based on 24 major tissues. The tissue-resolved profiles provided evidence of the expression of 17 953 proteins. We identified 4294 new coding events and improved the pear genome annotation via the proteogenomic strategy based on 18 090 peptide spectra with peptide spectrum matches >1. Among the eight randomly selected new short coding open reading frames that were expressed in the style, four promoted and one inhibited the growth of pear pollen tubes. Based on gene coexpression module analysis, we explored the key genes associated with important agronomic traits, such as stone cell formation in fruits. The network regulating the synthesis of lignin, a major component of stone cells, was reconstructed, and receptor-like kinases were implicated as core factors in this regulatory network. Moreover, we constructed the online database PearEXP (http://www.peardb.org.cn) to enable access to the pear proteogenomic resources. This study provides a paradigm for in-depth proteogenomic studies of woody plants.

Cryptochrome-mediated blue-light signal contributes to lignin biosynthesis in stone cells in pear fruit.

Light environment is an indispensable factor that regulates multitudinous developmental processes during the whole life cycle of plants, including fruit development. Stone cells which negatively influence pear fruit quality because of their strongly lignified cell wall are also affected by light, however, how light qualities influence lignin biosynthesis in pear remains unclear. Here, the calli of European pear (Pyrus communis L.) treated with different lights were used to explore the changes in phenotype, lignin content, and H2O2 content, coupled with RNA-Seq and quantitative real-time PCR (qRT-PCR) to investigate the possible regulation pathway of light on lignin biosynthesis in stone cells. Results showed that blue light increased the expression of lignin structure genes and promoted lignin accumulation. Besides, four blue light receptors cryptochromes (CRYs) were identified in white pear, named PbCRY1a (Pbr024556.1), PbCRY1b (Pbr001636.3), PbCRY2a (Pbr023037.1), and PbCRY2b (Pbr002655.4). qRT-PCR analysis showed that PbCRY1a is highly expressed in cultivars with a high content of stone cells. Furthermore, the molecular function of PbCRY1a on stone cell formation in pear fruit was demonstrated by genetic transformation of pear calli and Agrobacterium-mediated transient overexpression in pear fruitlets. Co-expression network analyses with RNA-seq data showed that 8 MYB and 5 NAC genes were classified into different co-expression clusters with lignin biosynthesis genes under blue light conditions. These results indicate that CRY-mediated blue-light signal plays an important role in cell wall lignification and promotes the formation of stone cells in pear by regulating downstream genes.