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.

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.

Cellulose accumulation mediated by PbrCSLD5, a cellulose synthase-like protein, results in cessation of pollen tube growth in Pyrus bretschneideri.

Cellulose, a key component of the cell wall, plays an important role in maintaining the growth of pollen tubes. However, the molecular mechanism of cellulose participating in the cessation of pear pollen tube growth remains unclear. Here, we reported that at 15 h post-cultured (HPC), the slow-growth pear pollen tubes showed thickened cell walls and cellulose accumulation in the inner wall. Transcriptome data and quantitative real-time PCR analysis showed that PbrCSLD5, a cellulose synthesis-like gene, was highly expressed in the 15 HPC pear pollen tubes. Knockdown of PbrCSLD5 caused a decrease in cellulose content in pear pollen tubes. Moreover, PbrCSLD5 overexpression in Arabidopsis resulted in the accumulation of cellulose and disruption of normal pollen tube growth. Transcription factor PbrMADS52 was found to bind to the promoter of PbrCSLD5 and enhanced its expression. Our results suggested that the PbrMADS52-PbrCSLD5 signaling pathway led to increased cellulose content in the pear pollen tube cell wall, thereby inhibiting pollen tube growth. These results provided new insights into the regulation of pollen tube growth.

PbrMYB169 positively regulates lignification of stone cells in pear fruit.

Stone cells negatively affect fruit quality because of their firm and lignified cell walls, so are targets for reduction in pear breeding programmes. However, there is only limited knowledge of the molecular mechanisms underlying the formation of stone cells. Here, we show that PbrMYB169, an R2R3 MYB transcription factor, of Pyrus bretschneideri positively regulates lignification of stone cells in pear fruit. PbrMYB169 was shown to be co-expressed with lignin biosynthesis genes during pear fruit development, and this co-expression pattern was coincident with stone cell formation in the fruit of Pyrus bretschneideri 'Dangshansuli'. The PbrMYB169 expression level was also positively correlated with stone cell content in 36 pear cultivars tested. PbrMYB169 protein significantly activated the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 via binding to AC elements [ACC(T/A)ACC] in these promoters. Furthermore, overexpression of PbrMYB169 in transgenic Arabidopsis plants enhanced the expression of lignin genes, and increased lignin deposition and cell wall thickness of vessel elements, but did not change the ratio of syringyl and guaiacyl lignin monomers. In conclusion, PbrMYB169 appears to be a transcriptional activator of lignin biosynthesis and regulates secondary wall formation in fruit stone cells. This study advances the understanding of the regulation of lignin biosynthesis and provides valuable molecular genetic information for reducing stone cell content in pear fruit.

Identification of miRNAs involved in pear fruit development and quality.

BACKGROUND: MicroRNAs (miRNAs) are a class of small, endogenous RNAs that take part in regulating genes through mediating gene expressions at the post-transcriptional level in plants. Previous studies have reported miRNA identification in various plants ranging from model plants to perennial fruit trees. However, the role of miRNAs in pear (Pyrus bretschneideri) fruit development is not clear. Here, we investigated the miRNA profiles of pear fruits from different time stages during development with Illumina HiSeq 2000 platform and bioinformatics analysis. Quantitative real-time PCR was used to validate the expression levels of miRNAs. RESULTS: Both conserved and species-specific miRNAs in pear have been identified in this study. Total reads, ranging from 19,030,925 to 25,576,773, were obtained from six small RNA libraries constructed for different stages of fruit development after flowering. Comparative profiling showed that an average of 90 miRNAs was expressed with significant differences between various developmental stages. KEGG pathway analysis on 2,216 target genes of 188 known miRNAs and 1,127 target genes of 184 novel miRNAs showed that miRNAs are widely involved in the regulation of fruit development. Among these, a total of eleven miRNAs putatively participate in the pathway of lignin biosynthesis, nine miRNAs were identified to take part in sugar and acid metabolism, and MiR160 was identified to regulate auxin response factor. CONCLUSION: Comparative analysis of miRNAomes during pear fruit development is presented, and miRNAs were proved to be widely involved in the regulation of fruit development and formation of fruit quality, for example through lignin synthesis, sugar and acid metabolism, and hormone signaling. Combined with computational analysis and experimental confirmation, the research contributes valuable information for further functional research of microRNA in fruit development for pear and other species.

Comparative genomic analysis of the RabGAP gene family in seven Rosaceae species, and functional identification of PbrRabGAP10 in controlling pollen tube growth by mediating cellulose deposition in pear.

Rab GTPase-activating proteins (RabGAPs), serving as crucial signaling switches, play essential roles in several physiological processes related to plant growth and development. However, despite their importance, information regarding the RabGAP gene family and their biological functions remains unknown in the Rosaceae. In this study, we identified a total of 127 RabGAP genes in seven Rosaceae species, which were divided into five subfamilies. Our findings indicate that whole genome duplication (WGD) events or dispersed duplication events largely contributed to the expansion of RabGAP family members within Rosaceae species. Through tissue-specific expression analyses, we revealed that the PbrRabGAP genes exhibited distinct expression patterns in different pear tissues. Furthermore, by examining the expression pattern during pollen development and employing an antisense oligonucleotide approach, we demonstrated that PbrRabGAP10, located in the cytoplasm, mediates the imbalance of cellulose distribution, thus regulating pollen tube elongation. In conclusion, the present study offers an overview of the RabGAP family in Rosaceae genomes and serves as the basis for further functional studies.

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.

Comprehensive genomic analysis of the Rho GTPases regulators in seven Rosaceae species revealed that PbrGDI1 controls pollen tube growth in Pyrus via mediating cellulose deposition.

The primary regulators of Rho GTPases are GTPase-activating protein (GAP), guanine nucleotide exchange factor (GEF), and GDP dissociation inhibitor (GDI), which function as signaling switches in several physiological processes involved in plant growth and development. This study compared how the Rho GTPase regulators functioned in seven Rosaceae species. Seven Rosaceae species, divided into three subgroups, had a total of 177 regulators of Rho GTPases. According to duplication analysis, the expansion of GEF, GAP, and GDI families was facilitated by whole genome duplication or a dispersed duplication event. The balance of cellulose deposition to control the growth of the pear pollen tube, as demonstrated by the expression profile and antisense oligonucleotide approach. Moreover, protein-protein interactions indicated that PbrGDI1 and PbrROP1 could directly interact, suggesting that PbrGDI1 regulated the growth of the pear pollen tube through PbrROP1 signaling downstream. These results lay the foundations for future functional characterization of the GAP, GEF, and GDI gene families in Pyrus bretschneideri.

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.

PbCSE1 promotes lignification during stone cell development in pear (Pyrus bretschneideri) fruit.

Pear [Pyrus bretschneideri cv. Dangshan Su] fruit quality is not always satisfactory owing to the presence of stone cells, and lignin is the main component of stone cells in pear fruits. Caffeoyl shikimate esterase (CSE) is a key enzyme in the lignin biosynthesis. Although CSE-like genes have been isolated from a variety of plant species, their orthologs are not characterized in pear. In this study, the CSE gene family (PbCSE) from P. bretschneideri was identified. According to the physiological data and quantitative RT-PCR (qRT-PCR), PbCSE1 was associated with lignin deposition and stone cell formation. The overexpression of PbCSE1 increased the lignin content in pear fruits. Relative to wild-type (WT) Arabidopsis, the overexpression of PbCSE1 delayed growth, increased the lignin deposition and lignin content in stems. Simultaneously, the expression of lignin biosynthetic genes were also increased in pear fruits and Arabidopsis. These results demonstrated that PbCSE1 plays an important role in cell lignification and will provide a potential molecular strategy to improve the quality of pear fruits.

Genome-wide association studies provide insights into the genetic determination of fruit traits of pear.

Pear is a major fruit tree crop distributed worldwide, yet its breeding is a very time-consuming process. To facilitate molecular breeding and gene identification, here we have performed genome-wide association studies (GWAS) on eleven fruit traits. We identify 37 loci associated with eight fruit quality traits and five loci associated with three fruit phenological traits. Scans for selective sweeps indicate that traits including fruit stone cell content, organic acid and sugar contents might have been under continuous selection during breeding improvement. One candidate gene, PbrSTONE, identified in GWAS, has been functionally verified to be involved in the regulation of stone cell formation, one of the most important fruit quality traits in pear. Our study provides insights into the complex fruit related biology and identifies genes controlling important traits in pear through GWAS, which extends the genetic resources and basis for facilitating molecular breeding in perennial trees.

Low-density microparticles with petaloid surface structure for pulmonary drug delivery.

The morphology of spray-dried particles composed of psicose and hydroxypropyl methylcellulose was modified by adding ammonium bicarbonate (ABC) to the solution. The surface structure of the particles was altered by immediate transformation of ABC to gaseous components during the spray drying. As a result, low-density microparticles with a petaloid surface structure, which was controllable by changing the evaporation rate of ABC, was obtained. This technique should be useful for modifying characteristics of solid particles for pulmonary drug delivery.

Insulin adsorption into porous charged membranes: effect of the electrostatic interaction.

Insulin adsorption into a series of porous charged membranes was investigated by batch adsorption experiments, and the experimental results were analyzed by the homogeneous diffusion model. The membranes used in this study were prepared by pore-surface modification of porous poly(acrylonitrile) (PAN) membranes by grafting with weak acidic and basic functional groups. The amount of insulin adsorbed into the membrane was determined from the material balance of insulin. The insulin partition coefficient K between the membrane and solution was estimated from the equilibrium adsorption amount, and the effective diffusion coefficient D was estimated by matching the model with the experimental data as a fitting parameter. The dependence of K and D on the charge properties of the insulin and membrane is observed and discussed. The partition coefficient K increased when the insulin and the membrane carried opposite charges, on the other hand, the effective diffusion coefficient D was reduced. These results indicate that the electrostatic interaction between the insulin and the membranes played an important role in the insulin adsorption.

Insulin transport across porous charged membranes: Effect of the electrostatic interaction.

Insulin transport phenomena across a series of porous charged membranes were studied at two pH conditions (pH 3.3 and pH 7.4) in this article. The membranes were prepared by pore-surface modification of porous poly(acrylonitrile) (PAN) membranes by grafting with weak acidic and basic functional groups. The insulin partition coefficient K between the membrane and solution was estimated from the equilibrium adsorption amount in the batch adsorption experiment. The insulin effective diffusion coefficient D inside the membrane was determined as a fitting parameter by matching the diffusion model with the experimental data of the diffusion measurement. Both K and D correlated well with the charge properties of the insulin and membrane: when the insulin and membrane carried opposite net charge, the partition coefficient showed relatively larger values, while the effective diffusion coefficient was reduced. The insulin permeability coefficient P obtained from the experimental results agreed with that estimated from the partition coefficient and effective diffusion coefficient. These results suggested that the combined effects of the solubility and diffusivity on the permeability coefficient complicated the relationship between the permeability and the charge properties of the insulin and membrane. Additionally, our calculation supported that insulin permeability was reduced by the boundary layer between the membrane and solution.