Ascorbic acid releases dormancy and promotes germination by an integrated regulation of abscisic acid and gibberellin in Pyrus betulifolia seeds.

Seed dormancy is an important life history state in which intact viable seeds delay or prevent germination under suitable conditions. Ascorbic acid (AsA) acts as a small molecule antioxidant, and breaking seed dormancy and promoting subsequent growth are among its numerous functions. In this study, a germination test using Pyrus betulifolia seeds treated with exogenous AsA or AsA synthesis inhibitor lycorine (Lyc) and water absorption was conducted. The results indicated that AsA released dormancy and increased germination and 20 mmol L-1 AsA promoted cell division, whereas Lyc reduced germination. Seed germination showed typical three phases of water absorption; and seeds at five key time points were sampled for transcriptome analysis. It revealed that multiple pathways were involved in breaking dormancy and promoting germination through transcriptome data, and 12 differentially expressed genes (DEGs) related to the metabolism and signal transduction of abscisic acid (ABA) and gibberellins (GA) were verified by subsequent RT-qPCR. For metabolites, exogenous AsA increased endogenous AsA and GA3 but reduced ABA and the ABA/GA3 ratio. In addition, three genes regulating ABA synthesis were downregulated by AsA, while five genes mediating ABA degradation were upregulated. Taken together, AsA regulates the pathways associated with ABA and GA synthesis, catalysis, and signal transduction, with subsequent reduction in ABA and increase in GA and further the balance of ABA/GA, ultimately releasing dormancy and promoting germination.

Introduction of a diverse genetic background of Pyrus into Malus through intergeneric hybridization.

Wide hybridizations across species and genera have been employed to enhance agriculturally important traits in crops. Within the tribe Maleae of the Rosaceae family, different genera and species exhibit several traits useful for increasing diversity and gene pool through hybridization. This study aimed to develop and characterize intergeneric hybrid individuals between Malus and Pyrus. Through seed germination, shoot multiplication, and rooting in vitro, acclimatized seedlings showing vegetative growth on their own roots were obtained from crosses of Malus × domestica pollinated by Pyrus communis, P. bretschneideri, and the Pyrus interspecific hybrid (P. communis × P. pyrifolia). Comparative analysis of leaf morphology, flow cytometry, and molecular genotyping confirmed the hybrid status of the individuals. Genome-wide genotyping revealed that all the hybrid individuals inherited genomic fragments symmetrically from the Malus and Pyrus parents. To the best of our knowledge, this is the first report on the development of intergeneric hybrid seedlings between Malus × domestica and P. bretschneideri. Furthermore, the Pyrus interspecific hybrid individual served as a bridge plant for introducing the genetic background of P. pyrifolia into Malus × domestica. The results of this study provided a crucial foundation for breeding through intergeneric hybridization between Malus and Pyrus, facilitating the incorporation of valuable traits from diverse gene pools.

A novel fluorescent probe with a large Stokes shift for colorimetric and selective detection of cysteine in water, milk, cucumber, pear and tomato.

Cysteine is an important amino acid that is related to human health and food safety. How to effectively detect Cys in food has received widespread attention. Compared with other methods, fluorescent probes have the advantages of simple operation, high sensitivity, and good selectivity. Therefore, a selective fluorescence probe 2 for Cys in food was designed and synthesized. Probe 2 employed the acrylate group as a thiol-recognition site for Cys, which endowed probe 2 with better selectivity for Cys over Hcy and GSH. The recognition pathway underwent Michael addition, intramolecular cyclization, and concomitant release of the piperideine-based fluorophore, along with a chromogenic change from yellow to orange. This pathway was supported by 1H NMR analysis and DFT calculations. In addition, probe 2 displays a linear response to Cys concentrations (0-30 µM), low detection limit (0.89 µM), and large Stokes shift (125 nm). Overall, probe 2 showed great application potential for the quantitative determination of Cys in water, milk, cucumber, pear and tomato.

Sucrose, cell wall, and polyamine metabolisms involve in preserving postharvest quality of 'Zaosu' pear fruit by L-glutamate treatment.

'Zaosu' pear fruit is prone to yellowing of the surface and softening of the flesh after harvest. This work was performed to assess the influences of L-glutamate treatment on the quality of 'Zaosu' pears and elucidate the underlying mechanisms involved. Results demonstrated that L-glutamate immersion reduced ethylene release, respiratory intensity, weight loss, brightness (L*), redness (a*), yellowness (b*), and total coloration difference (ΔE); enhanced ascorbic acid, soluble solids, and soluble sugar contents; maintained chlorophyll content and flesh firmness of pears. L-glutamate also restrained the activities of neutral invertase and acid invertase, while enhancing sucrose phosphate synthetase and sucrose synthase activities to facilitate sucrose accumulation. The transcriptions of PbSGR1, PbSGR2, PbCHL, PbPPH, PbRCCR, and PbNYC were suppressed by L-glutamate, resulting in a deceleration of chlorophyll degradation. L-glutamate concurrently suppressed the transcription levels and enzymatic activities of polygalacturonases, pectin methylesterases, cellulase, and ß-glucosidase. It restrained polygalacturonic acid trans-eliminase and pectin methyl-trans-eliminase activities as well as inhibited the transcription levels of PbPL and Pbß-gal. Moreover, the gene transcriptions and enzymatic activities of arginine decarboxylase, ornithine decarboxylase, S-adenosine methionine decarboxylase, glutamate decarboxylase, γ-aminobutyric acid transaminase, glutamine synthetase along with the PbSPDS transcription was promoted by L-glutamate. L-glutamate also resulted in the down-regulation of PbPAO, PbDAO, PbSSADH, PbGDH, and PbGOGAT transcription levels, while enhancing γ-aminobutyric acid, glutamate, and pyruvate acid contents in pears. These findings suggest that L-glutamate immersion can effectively maintain the storage quality of 'Zaosu' pears via modulating key enzyme activities and gene transcriptions involved in sucrose, chlorophyll, cell wall, and polyamine metabolism.

PbRbohH/J mediates ROS generation to regulate the growth of pollen tube in pear.

Respiratory burst oxidase homolog (Rboh) family genes play crucial functions in development and growth. However, comprehensive and systematic investigation of Rboh family members in Rosaceae and their specific functions during pear pollen development are still limited. In the study, 63 Rboh genes were identified from eight Rosaceae genomes (Malus domestica, Pyrus bretschneideri, Pyrus communis, Prunus persica, Rubus occidentalis, Fragaria vesca, Prunus mume and Prunus avium) and divided into seven main subfamilies (I-VII) according to phylogenetic and structural features. Different modes of gene duplication led to the expansion of Rboh family, with purifying selection playing a vital role in the evolution of Rboh genes. In addition, RNA sequencing and qRT-PCR results indicated that PbRbohH and PbRbohJ were specifically high-expressed in pear pollen. Subsequently, subcellular localization revealed that PbRbohH/J distributed at the plasma membrane. Furthermore, by pharmacological analysis and antisense oligodeoxynucleotide assay, PbRbohH/J were demonstrated to mediate the formation of reactive oxygen species (ROS) to manage pollen tube growth. In conclusion, our results provide useful insights into the functions, expression patterns, evolutionary history of the Rboh genes in pear and other Rosaceae species.

Enhancing productivity, modifying biochemical parameters, and regulating the phenylpropanoid pathway in 'Le-Conte' pears through optimal protocatechuic acid treatments.

BACKGROUND: This study aimed to investigate the impact of protocatechuic acid (PRC) treatments on the productivity and fruit quality of 'Le-Conte' pears, with a specific focus on productivity, stone cells content, and antioxidant activity. The research spanned over three consecutive cultivating seasons, with the first season serving as a preliminary study to determine the optimal PRC concentrations and the most effective number of spray applications. During the initial season, response surface methodology (RSM) was employed to optimize PRC concentration and application frequency. PRC was evaluated at concentrations ranging from 50 to 400 ppm, with treatment frequencies of either once or twice. Considering the optimal conditions obtained from RSM results, PRC treatments at 200 ppm and 300 ppm were applied twice, and their respective effects were studied in comparison to the control in the following seasons. RESULTS: RSM results indicated that PRC at 200 and 300 ppm, applied twice, once during full bloom and again three weeks later, yielded the most significant effects. Subsequent studies revealed that PRC treatments had a substantial impact on various aspects of fruit production and quality. Applying 300 ppm PRC once during full bloom and again three weeks later resulted in higher fruit set percentages, lower fruit abscission, and enhanced fruit yield compared to untreated trees. Additionally, the 200 ppm PRC treatment maintained physicochemical characteristics such as fruit color, increased total soluble solids (TSS), and total sugar, and maintained higher ascorbic acid content and antioxidant capacity in the fruits while reducing stone cells content and lignin. Notably, enzyme activities related to phenylpropanoid metabolism and stone cells, including phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-Coumarate-CoA Ligase (4CL), cinnamyl alcohol dehydrogenase (CAD), and cinnamoyl-CoA reductase (CCR), as well as peroxidase, polyphenol oxidase, and laccase, were significantly regulated by PRC treatments. CONCLUSION: Overall, this study suggests that PRC treatments are suitable for enhancing pear yield and quality, with PRC at 200 ppm being the more recommended option over 300 ppm. This approach serves as an effective strategy for achieving a balance between enhancing the productivity and fruit quality of 'Le-Conte' pears.

PbrbZIP15 promotes sugar accumulation in pear via activating the transcription of the glucose isomerase gene PbrXylA1.

The composition and abundance of soluble sugars in mature pear (Pyrus) fruit are important for its acceptance by consumers. However, our understanding of the genes responsible for soluble sugar accumulation remains limited. In this study, a S1-group member of bZIP gene family, PbrbZIP15, was characterized from pear genome through the combined analyses of metabolite and transcriptome data followed by experimental validation. PbrbZIP15, located in nucleus, was found to function in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli. After analyzing the expression profiles of sugar-metabolism-related genes and the distribution of cis-acting elements in their promoters, the glucose isomerase 1 gene (PbrXylA1), whose corresponding protein catalyzed the isomerization of glucose and fructose in vitro, was identified as a downstream target gene of PbrbZIP15. PbrbZIP15 could directly bind to the G-box element in PbrXylA1 promoter and activate its transcription, as evidenced by chromatin immunoprecipitation-quantitative PCR, yeast one-hybrid, electrophoretic mobility shift assay, and dual-luciferase assay. PbrXylA1, featuring a leucine-rich signal peptide in its N-terminal, was localized to the endoplasmic reticulum. It was validated to play a significant role in fructose, sucrose, and total soluble sugar accumulation in pear fruit and calli, which was associated with the upregulated fructose/glucose ratio. Further studies revealed a positive correlation between the sucrose content and the expression levels of several sucrose-biosynthesis-related genes (PbrFRK3/8, PbrSPS1/3/4/8, and PbrSPP1) in PbrbZIP15-/PbrXylA1-transgenic fruit/calli. In conclusion, our results suggest that PbrbZIP15-induced soluble sugar accumulation during pear development is at least partly attributed to the activation of PbrXylA1 transcription.

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.

Transcription factor PbMYB80 regulates lignification of stone cells and undergoes RING finger protein PbRHY1-mediated degradation in pear fruit.

The Chinese white pear (Pyrus bretschneideri) fruit carries a high proportion of stone cells, adversely affecting fruit quality. Lignin is a main component of stone cells in pear fruit. In this study, we discovered that a pear MYB transcription factor, PbMYB80, binds to the promoters of key lignin biosynthesis genes and inhibits their expression. Stable overexpression of PbMYB80 in Arabidopsis showed that lignin deposition and secondary wall thickening were inhibited, and the expression of the lignin biosynthesis genes in transgenic Arabidopsis was decreased. Transient overexpression of PbMYB80 in pear fruit inhibited lignin metabolism and stone cell development, and the expression of some genes in the lignin metabolism pathway was reduced. In contrast, silencing PbMYB80 with VIGS increased the lignin and stone cell content in pear fruit, and increased expression of genes in the lignin metabolism pathway. By screening a pear fruit cDNA library in yeast, we found that PbMYB80 binds to a RING finger (PbRHY1) protein. We also showed that PbRHY1 exhibits E3 ubiquitin ligase activity and degrades ubiquitinated PbMYB80 in vivo and in vitro. This investigation contributes to a better understanding of the regulation of lignin biosynthesis in pear fruit, and provides a theoretical foundation for increasing pear fruit quality at the molecular level.

Genome-wide identification of the Pyrus R2R3-MYB gene family and PhMYB62 regulation analysis in Pyrus hopeiensis flowers at low temperature.

The R2R3-MYB gene family play an important role in plant growth, development and stress responses. In this study, a total of 122 PcoR2R3-MYB genes were identified and grouped into 26 clades in pear. And these PcoMYBs were unevenly distributed among 17 chromosomes. The sequence characteristics, conversed motifs, exon/intron structures, classification, duplication events and cis-acting elements were also investigated. The gene duplication events showed that segmental duplication may play key roles in expansion of the PcoMYB gene family. Pyrus hopeiensis, which is a valuable wild resource, has strong cold resistance. An integrative analyses of miRNA and mRNA showed that PhMYB62 was involved in regulating low-temperature stress in P. hopeiensis flower organs. Subcellular localization analysis showed that PhMYB62 protein was specifically localized to the nucleus. The result of DAP-seq showed that PhMYB62 responded to low-temperature stress in P. hopeiensis by regulating TFs, which were associated with plant stress resistance, and POD, GAUT12, AUX28 and CHS genes. Subsequently, yeast one-hybrid verified that PhMYB62 could bind and activate the promoter of POD gene. The current study would provide a comprehensive information for further functional research on the stress-responsive R2R3-MYB gene candidates in pear, and may help to identify the genes associated with cold resistance for the cultivation of cold-resistant pear varieties.

Genome wide and comprehensive analysis of the cytochrome P450 (CYPs) gene family in Pyrus bretschneideri: Expression patterns during Sporidiobolus pararoseus Y16 enhanced with ascorbic acid (VC) treatment.

Cytochrome P450s (CYPs) constitute the largest group of enzymes in plants and are involved in a variety of processes related to growth and protection. However, the CYP gene superfamily in pear (Pyrus bretschneideri) and their characteristics is unclear. Through a comprehensive genome-wide analysis, this article identified a total of 74 CYP genes in the P. bretschneideri genome, which were categorized into fourteen families. Motif analysis reveals that most of the ten motifs predicted were with the p450 conserved domain. The majority of the CYP genes have exon arrangements. Furthermore, promoter analysis unveiled a multitude of cis-acting elements associated with diverse responsiveness including hormones, light responsive, anoxic specific inducibility and anaerobic induction. Analysis of the transcriptome data reveal that about 80% of the pear CYPs genes were upregulated and they were positively correlated with the antioxidant's parameters such as total flavonoids and total phenol content as well as ABTS and DPPH radicals. RT-qPCR analysis confirmed that the CYP genes could be regulated in pear. Collectively, our results reveal comprehensive insights into the CYP superfamily in pear and make a valuable contribution to the ongoing process of functional validation.

The Odorant-Binding Protein 1 Mediates the Foraging Behavior of Grapholita molesta Larvae.

Since eggs are laid directly on fruit skin, it is typically believed that food odor has little impact on the foraging of Grapholita molesta larvae. It is crucial to note that larvae that hatch on twigs and leaves could need some sort of identification system when foraging. Here, 22 GmolOBP genes were identified from the G. molesta larval transcriptome via the comparison of conserved domain and homology in the protein level. GmolOBP1 had strong affinities for important pear-fruit volatiles, which caused larvae strong behavioral responses. However, after GmolOBP1 silencing, the larvae lost their attraction to methyl salicylate, α-farnesene, butyl acetate, ethyl butanoate, and ethyl hexanoate, and the effects of larvae seeking various pears were significantly reduced. Consequently, GmolOBP1 was required for the reception of pear volatiles and was involved in mediating how G. molesta larvae foraged. Our research revealed the GmolOBP1 foraging signal recognition mechanism as well as potential molecular targets for field pest management.

bZIP transcription factor PubZIP914 enhances production of fatty acid-derived volatiles in pear.

'Nanguo' pear emitted a rich aroma when entirely ripe. The six-carbon (C6) volatiles, including the aldehydes, 2-hexenal, and hexanal, as well as their corresponding alcohols and esters which are derived from lipoxygenase pathway are the important volatile components in 'Nanguo' pears. However, the transcriptional regulation mechanism of aroma synthesis of 'Nanguo' pears remains largely unknown. bZIP transcription factors (TFs) mediate different developmental processes in plants. In this study, we identified and characterized a bZIP TF that is highly expressed and induced in 'Nanguo' pear fruits at the mature stage. The content of fatty acid-derived volatiles increased significantly in transgenic pears and tomatoes of PubZIP914 overexpression. Meanwhile, PubZIP914 could regulate PuLOX3.1 by binding directly to PuLOX3.1 promoter. The results of this study provide evidence demonstrating how bZIP transcription factors regulate fatty acid-derived volatiles biosynthesis during pear fruit ripening.

Mass Spectrometric Identification of Metabolites after Magnetic-Pulse Treatment of Infected Pyrus communis L. Microplants.

The major goal of this study is to create a venue for further work on the effect of pulsed magnetic fields on plant metabolism. It deals with metabolite synthesis in the aforementioned conditions in microplants of Pyrus communis L. So far, there have been glimpses into the governing factors of plant biochemistry in vivo, and low-frequency pulsed magnestatic fields have been shown to induce additional electric currents in plant tissues, thus perturbing the value of cell membrane potential and causing the biosynthesis of new metabolites. In this study, sixty-seven metabolites synthesized in microplants within 3-72 h after treatment were identified and annotated. In total, thirty-one metabolites were produced. Magnetic-pulse treatment caused an 8.75-fold increase in the concentration of chlorogenic acid (RT = 8.33 ± 0.0197 min) in tissues and the perturbation of phenolic composition. Aucubin, which has antiviral and antistress biological activity, was identified as well. This study sheds light on the effect of magnetic fields on the biochemistry of low-molecular-weight metabolites of pear plants in vitro, thus providing in-depth metabolite analysis under optimized synthetic conditions. This study utilized high-resolution gas chromatography-mass spectrometry, metabolomics methods, stochastic dynamics mass spectrometry, quantum chemistry, and chemometrics, respectively. Stochastic dynamics uses the relationships between measurands and molecular structures of silylated carbohydrates, showing virtually identical mass spectra and comparable chemometrics parameters.

PbBPC4 involved in a xylem-deficient dwarf phenotype in pear by directly regulating the expression of PbXND1.

Dwarfing is an important agronomic trait in fruit breeding. At present, dwarf cultivars or dwarfing rootstocks are used for high-density planting. Although some dwarf rootstocks have been used in the cultivation of pear (Pyrus bretschneideri Rehd), the breeding of dwarf pear rootstocks or cultivars is still sorely lacking. A previous study reported that PbXND1 results in a xylem-dwarf phenotype in pear trees. However, the regulatory mechanism upstream of PbXND1 is unclear. In this study, we identified PbBPC4 as an upstream regulatory factor of PbXND1 in yeast one-hybrid assays. In ß-glucuronidase staining and dual-luciferase assays, PbBPC4 enhanced the activity of the PbXND1 promoter. Tobacco plants overexpressing PbBPC4 showed decreased plant height because of a reduced xylem size. Similar changes in the xylem was observed in transgenic pear roots; those overexpressing PbBPC4 showed reduced xylem size, and those with silencing PbBPC4 expression showed increased xylem size, greater density of xylem vessels, and a larger proportion of the xylem out of the total cross-section area. Expression analyses showed that PbBPC4 increases the transcription of PbXND1, leading to reduced transcript levels of genes involved in the positive regulation of xylem development, ultimately resulting in a xylem-deficient dwarf phenotype. Taken together, our results reveal the mechanism by which PbBPC4 participates in the regulation of xylem development via directly altering the expression of PbXND1, thus leading to the dwarf phenotype in pear. These findings have reference value for the breeding of dwarf pear trees.

Beyond apples and pears: sex-specific genetics of body fat percentage.

Introduction: Biological sex influences both overall adiposity and fat distribution. Further, testosterone and sex hormone binding globulin (SHBG) influence adiposity and metabolic function, with differential effects of testosterone in men and women. Here, we aimed to perform sex-stratified genome-wide association studies (GWAS) of body fat percentage (BFPAdj) (adjusting for testosterone and sex hormone binding globulin (SHBG)) to increase statistical power. Methods: GWAS were performed in white British individuals from the UK Biobank (157,937 males and 154,337 females). To avoid collider bias, loci associated with SHBG or testosterone were excluded. We investigated association of BFPAdj loci with high density cholesterol (HDL), triglyceride (TG), type 2 diabetes (T2D), coronary artery disease (CAD), and MRI-derived abdominal subcutaneous adipose tissue (ASAT), visceral adipose tissue (VAT) and gluteofemoral adipose tissue (GFAT) using publicly available data from large GWAS. We also performed 2-sample Mendelian Randomization (MR) using identified BFPAdj variants as instruments to investigate causal effect of BFPAdj on HDL, TG, T2D and CAD in males and females separately. Results: We identified 195 and 174 loci explaining 3.35% and 2.60% of the variation in BFPAdj in males and females, respectively at genome-wide significance (GWS, p<5x10-8). Although the direction of effect at these loci was generally concordant in males and females, only 38 loci were common to both sexes at GWS. Seven loci in males and ten loci in females have not been associated with any adiposity/cardiometabolic traits previously. BFPAdj loci generally did not associate with cardiometabolic traits; several had paradoxically beneficial cardiometabolic effects with favourable fat distribution. MR analyses did not find convincing supportive evidence that increased BFPAdj has deleterious cardiometabolic effects in either sex with highly significant heterogeneity. Conclusions: There was limited genetic overlap between BFPAdj in males and females at GWS. BFPAdj loci generally did not have adverse cardiometabolic effects which may reflect the effects of favourable fat distribution and cardiometabolic risk modulation by testosterone and SHBG.

Genome-wide identification, comparative analysis and functional roles in flavonoid biosynthesis of cytochrome P450 superfamily in pear (Pyrus spp.).

BACKGROUND: The cytochrome P450 (CYP) superfamily is the largest enzyme metabolism family in plants identified to date, and it is involved in many biological processes, including secondary metabolite biosynthesis, hormone metabolism and stress resistance. However, the P450 gene superfamily has not been well studied in pear (Pyrus spp.). RESULTS: Here, the comprehensive identification and a comparative analysis of P450 superfamily members were conducted in cultivated and wild pear genomes. In total, 338, 299 and 419 P450 genes were identified in Chinese white pear, European pear and the wild pear, respectively. Based on the phylogenetic analyses, pear P450 genes were divided into ten clans, comprising 48 families. The motif and gene structure analyses further supported this classification. The expansion of the pear P450 gene family was attributed to whole-genome and single-gene duplication events. Several P450 gene clusters were detected, which have resulted from tandem and proximal duplications. Purifying selection was the major force imposed on the long-term evolution of P450 genes. Gene dosage balance, subfunctionalization and neofunctionalization jointly drove the retention and functional diversification of P450 gene pairs. Based on the association analysis between transcriptome expression profiles and flavonoid content during fruit development, three candidate genes were identified as being closely associated with the flavonoid biosynthesis, and the expression of one gene was further verified using qRT-PCR and its function was validated through transient transformation in pear fruit. CONCLUSIONS: The study results provide insights into the evolution and biological functions of P450 genes in pear.

Genome-Wide Identification and Evolution of the GRF Gene Family and Functional Characterization of PbGRF18 in Pear.

Proteins encoded by the G-box regulating factor (GRF, also called 14-3-3) gene family are involved in protein-protein interactions and mediate signaling transduction, which play important roles in plant growth, development, and stress responses. However, there were no detailed investigations of the GRF gene family in pear at present. In this study, we identified 25 GRF family members in the pear genome. Based on a phylogenetic analysis, the 25 GRF genes were clustered into two groups; the ε group and the non-ε group. Analyses of the exon-intron structures and motifs showed that the gene structures were conserved within each of the ε and non-ε groups. Gene duplication analysis indicated that most of the PbGRF gene expansion that occurred in both groups was due to WGD/segmental duplication. Phosphorylation sites analysis showed that the main phosphorylation sites of PbGRF proteins were serine residues. For gene expression, five PbGRF genes (PbGRF7, PbGRF11, PbGRF16, PbGRF21, and PbGRF23) were highly expressed in fruits, and PbGRF18 was highly expressed in all tissues. Further analysis revealed that eight PbGRF genes were significantly differentially expressed after treatment with different sugars; the expression of PbGRF7, PbGRF8, and PbGRF11 significantly increased, implying the involvement of these genes in sugar signaling. In addition, subcellular localization studies showed that the tested GRF proteins localize to the plasma membrane, and transgenic analysis showed that PbGRF18 can increase the sugar content in tomato leaves and fruit. The results of our research establish a foundation for functional determination of PbGRF proteins, and will help to promote a further understanding of the regulatory network in pear fruit development.

Transcription factor PbbZIP4 is targeted for proteasome-mediated degradation by the ubiquitin ligase PbATL18 to influence pear's resistance to Colletotrichum fructicola by regulating the expression of PbNPR3.

Pear anthracnose caused by Colletotrichum fructicola is one of the main fungal diseases in all pear-producing areas. The degradation of ubiquitinated proteins by the 26S proteasome is a regulatory mechanism of eukaryotes. E3 ubiquitin ligase is substrate specific and is one of the most diversified and abundant enzymes in the regulation mechanism of plant ubiquitination. Although numerous studies in other plants have shown that the degradation of ubiquitinated proteins by the 26S proteasome is closely related to plant immunity, there are limited studies on them in pear trees. Here, we found that an E3 ubiquitin ligase, PbATL18, interacts with and ubiquitinates the transcription factor PbbZIP4, and this process is enhanced by C. fructicola infection. PbATL18 overexpression in pear callus enhanced resistance to C. fructicola infection, whereas PbbZIP4 overexpression increased sensitivity to C. fructicola infection. Silencing PbATL18 and PbbZIP4 in Pyrus betulaefolia seedlings resulted in opposite effects, with PbbZIP4 silencing enhancing resistance to C. fructicola infection and PbATL18 silencing increasing sensitivity to C. fructicola infection. Using yeast one-hybrid screens, an electrophoretic mobility shift assay, and dual-luciferase assays, we demonstrated that the transcription factor PbbZIP4 upregulated the expression of PbNPR3 by directly binding to its promoter. PbNPR3 is one of the key genes in the salicylic acid (SA) signal transduction pathway that can inhibit SA signal transduction. Here, we proposed a PbATL18-PbbZIP4-PbNPR3-SA model for plant response to C. fructicola infection. PbbZIP4 was ubiquitinated by PbATL18 and degraded by the 26S proteasome, which decreased the expression of PbNPR3 and promoted SA signal transduction, thereby enhancing plant C. fructicola resistance. Our study provides new insights into the molecular mechanism of pear response to C. fructicola infection, which can serve as a theoretical basis for breeding superior disease-resistant pear varieties.

PbWRKY26 positively regulates malate accumulation in pear fruit by activating PbMDH3.

Malate is the main organic acid that affects fruit acidity and flavor in pear (Pyrus spp.). However, the regulatory mechanism of malic acid accumulation in pear remains unclear. We identified PbWRKY26 as a candidate gene using mRNA-seq, and quantification analysis verified the expression level. The expression of PbWRKY26 was positively correlated with the malic acid content in two P. pyrifolia cultivars ('Cuiguan', 'Hongsucui') and two P. ussuriensis cultivars ('Qiuxiang', 'Hanhong'), with respective correlation coefficients of 0.748*, 0.871**, 0.889**, and 0.910** (*, P < 0.05; **, P < 0.01). The expression of PbWRKY26 enhanced the malate content in overexpression transgenic pear fruit and callus. In contrast, silencing PbWRKY26 decreased the pear fruit malic acid content. Analysis of the neighbor-joining phylogenetic tree indicated that PbWRKY26 was a PH3 homolog. The WRKY26 (PH3) has been identified to regulate a proton pump gene, PH5, in a lot of plant species, but the LUC and Y1H assays showed that PbWRKY26 could not bind to PbPH5 promoter in our study. Interestingly, a malate dehydrogenase gene, PbMDH3, was identified to be regulated by PbWRKY26. This study might be valuable to understand the metabolic regulatory network associated with malate accumulation.