Pear fruit typically contains abundant highly lignified cells, known as stone cells, which have a negative impact on the fruit's edibility and processing quality. Despite extensive physiological and molecular research, there remains a limited understanding of the precise spatiotemporal aspects of lignification in flesh cells during pear development, particularly regarding the initiation of lignification and expansion of stone cell clusters. Here, an emerging bioorthogonal chemistry-based imaging technique was employed to in vivo visualize cell lignification dynamics in developing pear fruit. Specific identification of active sites undergoing lignification revealed that initial lignification of flesh cells occurred at 10 days after full bloom (DAFB), resulting in the formation of primordial stone cells (PSCs). These PSCs exhibited a random distribution and showed significantly larger diameter and area compared to normal parenchyma cells. Subsequently, PSCs developed pit canals and initiated lignification process in their neighboring cells at 15 DAFB. A cascading effect in the formation of stone cell aggregations was visualized by tracing of the lignification trajectory. This expansion process exhibited a domino effect, whereby lignification progressively spread from one cell to the next, creating a cascading pattern of stone cell formation. Finally, a cellular developmental model was proposed for stone cell formation. This study presented a procedure for applying the cutting-edge technology, click chemistry imaging, to get insights into practical scientific questions. The findings elucidated the spatiotemporal dynamics of active lignification sites in pear fruit at the cellular level, thereby enhancing our understanding of the initiation and aggregation processes in stone cell formation.

Putrescine plays a role in superficial scald development during the cold storage of pear fruit. However, the molecular mechanism behind this phenomenon has not been un-fully clarified until recently. In this study, a conjoint analysis of metabolites and gene expression profiles in the putrescine-metabolic pathway of P. bretschneideri Rehd. fruit followed by experimental validation revealed that PbrADC1, forming a homodimer in the chloroplast, was involved in putrescine biosynthesis and thus fruit chilling resistance. Additionally, the substrate-binding residue Cys⁵⁴⁶ in PbrADC1, whose activity was modified by H₂O₂, played a crucial role in arginine decarboxylation into agmatine. Through a combined analysis of the distribution of cis-acting elements in the PbrADC1 promoter as well as the expression profiles of related transcription factors (TFs), several TFs were identified as upstream regulators of PbrADC1 gene. Further investigation revealed that the nuclear PbrWRKY62 could directly bind to the W-box elements in the PbrADC1 promoter, activate its expression, enhance putrescine accumulation, and thus increase fruit chilling tolerance. In conclusion, our results suggest that the PbrWRKY62-PbrADC1 module is involved in the development of superficial scald in P. bretschneideri Rehd. fruit via regulating putrescine biosynthesis. Consequently, these findings could serve as valuable genetic resources for breeding scald-resistant pear fruit.

Due to self-incompatibility (SI) prevents self-fertilization, natural or artificial cross-pollination has been conducted in many orchards to stabilize fruit yield. However, it is still puzzled which routes of self S-RNase arresting pollen tube growth. Herein, 17 COBRA genes were isolated from pear genome. Of these genes, the pollen-specifically expressed PbCOB.A.1 and PbCOB.A.2 positively mediates pollen tube growth. The promoters of PbCOB.A.1 and/or PbCOB.A.2 were bound and activated by PbABF.E.2 (an ABRE-binding factor) and PbC2H2.K16.2 (a C2H2-type zinc finger protein). Notably, the expressions of PbCOB.A.1, PbCOB.A.2, and PbC2H2.K16.2 were repressed by self S-RNase, suggesting that self S-RNase reduces the expression of PbCOB.A.1 and PbCOB.A.2 by decreasing the expression of their upstream factors, such as PbC2H2.K16.2, to arrest pollen tube growth. PbCOB.A.1 or PbCOB.A.2 accelerates the growth of pollen tubes treated by self S-RNase, but can hardly affect level of reactive oxygen species and deploymerization of actin cytoskeleton in pollen tubes and cannot physically interact with any reported proteins involved in SI. These results indicate that PbCOB.A.1 and PbCOB.A.2 may not relieve S-RNase toxicity in incompatible pollen tube. The information provides a new route to elucidate the arresting pollen tube growth during SI reaction.

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

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.

The mitogen-activated protein kinase (MAPK) cascade is crucial to plant growth, development, and stress responses. MAPK kinases (MAPKK) play a vital role in linking upstream MAPKK kinases (MAPKKK) with the downstream MAPK. Black spot is one of the most serious fungal diseases of pear which is an important part of the fruit industry in China. The MAPKK genes have been identified in many plants, however, none has been reported in pear (Pyrus bretschneideri). In order to explore whether MAPK gene of pear is related to black spot disease, we designed this experiment. The present study investigated eight putative PbrMAPKK genes obtained from the Chinese white pear genome. The phylogenetic analysis revealed that PbrMAPKK genes were divided into A, B, C, and D groups. These PbrMAPKK genes are randomly distributed on 7 out of 17 chromosomes and mainly originated from the whole-genome duplication (WGD) event. The expression analysis of PbrMAPKK genes in seven pear tissues and the leaves of susceptible and resistant varieties after Alternaria alternata infection by quantitative real-time PCR (qRT-PCR) identified seven candidate genes associated with resistance. Furthermore, virus-induced gene silencing (VIGS) indicated that PbrMAPKK6 gene enhanced resistance to pear black spot disease in pear.

N⁶-methylated adenine (m⁶A) is an emerging epigenetic marker in eukaryotic organisms that plays an important role in biological functions and in enriching genetic information. m⁶A exerts these functions via the dynamic interplay among m⁶A writers, erasers, and readers. However, little is known about the underlying mechanisms of m⁶A in plant growth and stress responses. Here, we identified 276 masked m⁶A regulators from nine Rosaceae species (Pyrus bretschneideri, Pyrus betulifolia, Pyrus communis, Malus domestica, Fragaria vesca, Prunus avium, Prunus mume, Prunus persica, and Rubus occidentalis). We classified and named these genes in more detail based on phylogenetic and synteny analysis. The expansion of m⁶A regulators in Maloideae was dated back to the recent whole-genome duplication (WGD) in Rosaceae. Based on the expression pattern analysis and gene structure analysis of m⁶A regulators, m⁶A was shown to be a significant factor in regulating plant development and resistance. In addition, PbrMTA1-silenced pear plants displayed significantly reduced drought tolerance and chlorophyll content, as well as increased electrolyte leakage and concentrations of malondialdehyde and H₂O₂.

The Chinese pear (Pyrus spp.) exhibits typical gametophytic self-incompatibility (GSI), which inhibits self-crossing and promotes out-crossing, similar to other fruit species in the Rosaceae family. Thus, S-compatible cultivars are required in pear orchards to ensure successful pollination and stable yields. In this study, 84 native Chinese pear accessions were genotyped by allele-specific PCR using one pair allele consensus primers and 29 pairs of S-allele-specific primers that were designed in this study. After cloning and sequencing the PCR products, the S-genotypes of all 84 pear accessions, including wild and cultivated accessions, were determined. The reported 34 S-alleles and a novel S-allele were isolated from these pear accessions. These S-alleles were expressed specifically in the style. Sequence analysis identified that six pear cultivars originated in China shared the same S-RNases with P. communis (Pc). These findings supported the hypothesis that oriental and occidental Pyrus spp. may share the same pool of alleles at the S-locus. A novel S-RNase was isolated in 'Putiandouli', 'Daguoshanli', 'Yunhongli 1′, and 'Dianli' and deposited as S₆₇-RNase under accession number MT773568. Furthermore, the deduced amino acid sequences exhibited high similarity (99.56%) to S₃₂-RNase in Malus. The high similarity between S-RNase in Pyrus and Malus indicated that the existence of S-RNase could have predated speciation between Pyrus and Malus. Furthermore, S-allele information was rearranged in Asian and European pears to provide information for selecting the best pollinator for widely cultivated pear cultivars in China. This information is useful for pear production, cross-breeding, and understanding the mechanism of the self-incompatibility reaction.

With-No-Lysine kinases (WNK) have been reported to be associated with plant growth regulation in Arabidopsis, soybean, and rice, but little is known of their roles in fruits. In this study, a total of 114 WNK genes were identified from 8 fruit trees species, and these WNK genes belonged to 2 classes (Ⅰ and Ⅱ) that respectively contain 4 (A, B, C, D) and 2 groups (E, F). The WNK genes had variable exon-intron structures and were randomly distributed among most chromosomes of each genus. The expression levels of six of the 18 WNK genes in peach were almost undetectable in fruits, suggesting that they may not be associated with fruit development and ripening. Of the other 12 WNK genes, PpWNK.B1 exhibited stronger stability of expression levels than the best reference genes reported previously in fruits from two peach cultivars. These results suggested that PpWNK.B1 could be a reliable reference gene for gene expression studies of peach fruit. Moreover, qRT-PCR assays yielded evidence that when using single reference gene as internal control, mistakes were more possible, indicating that the use of three reference genes is necessary for gene expression studies. Based on the gene expression profiles validated by qRT-PCR, PpWNK.A1 is predicted to likely be involved in fruit ripening, while PpWNK.A2 and PpWNK.E3.1 are likely associated with early fruit development. The present study represented the WNK genes in fruits, and it will be a valuable resource in continuing investigation of gene regulation network during fruit development and ripening.