Integrated metabolomic and transcriptomic analysis revealed the role of PbrCYP94B in wax accumulation in pear fruit after bagging treatment.

Preharvest fruit bagging is a safe and environmentally friendly production measure. Cuticular wax, as the first protective layer on the fruit surface, has important functions. However, the effects of preharvest bagging on cuticular wax synthesis in pears and the related molecular mechanisms are still unclear. Here, the impact of fruit bagging with different materials on cuticular wax synthesis in pear fruit, and the underlying molecular mechanism, were revealed from metabolomic, transcriptomic, morphological, and molecular biological perspectives. Our results revealed that, compared with that in the not bagged (NB) treatment group (0.59 mg/cm2), the total wax concentration was 1.32- and 1.37-fold greater in the single-layered white paper bag (WPB, 1.37 mg/cm2) and double-layered yellow-white paper bag, (YWPB, 1.40 mg/cm2) treatment groups, while it was slightly lower in the double-layered yellow-black paper bag (YBPB, 0.45 mg/cm2) group, which was consistent with the scanning electron microscopy (SEM) results. Integrated metabolomic and transcriptomic analysis revealed 29 genes associated with cuticular wax synthesis. Overexpression of PbrCYP94B, which is a key gene in the wax synthesis pathway in pear fruit, increased the total wax and alkane contents. This study provides valuable insights for the creation of new pear germplasms with high wax contents.

Synergistic Interaction Between PbbZIP88 and PbSRK2E Enhances Drought Resistance in Pear Through Regulation of PbATL18 Expression and Stomatal Closure.

Drought poses significant challenges to agricultural production, ecological stability and global food security. While wild pear trees exhibit strong drought resistance, cultivated varieties show weaker drought tolerance. This study aims to elucidate the molecular mechanisms underlying pear trees' response to drought stress. We identified a drought resistance-related transcription factor, PbbZIP88, which binds to and activates the expression of the drought-responsive gene PbATL18. Overexpression of PbbZIP88 in Arabidopsis and pear seedlings resulted in enhanced drought resistance and significantly improved physiological parameters under drought stress. We discovered that PbbZIP88 interacts with the key protein PbSRK2E in the ABA signalling pathway. This interaction enhances PbbZIP88's ability to activate PbATL18 expression, leading to higher levels of PbATL18. Furthermore, the PbbZIP88 and PbSRK2E interaction accelerates the regulation of stomatal closure under ABA treatment conditions, reducing water loss more effectively. Experimental evidence showed that silencing PbbZIP88 and PbSRK2E genes significantly decreased drought resistance in pear seedlings. In conclusion, this study reveals the synergistic role of PbbZIP88 and PbSRK2E in enhancing drought resistance in pear trees, particularly in the upregulation of PbATL18 expression, and the accelerated promotion of stomatal closure. These findings provide new candidate genes for breeding drought-resistant varieties and offer a theoretical foundation and technical support for achieving sustainable agriculture.

Molecular characterization of PSEUDO RESPONSE REGULATOR family in Rosaceae and function of PbPRR59a and PbPRR59b in flowering regulation.

BACKGROUND: PSEUDO RESPONSE REGULATOR (PRR) genes are essential components of circadian clock, playing vital roles in multiple processes including plant growth, flowering and stress response. Nonetheless, little is known about the evolution and function of PRR family in Rosaceae species. RESULTS: In this study, a total of 43 PRR genes in seven Rosaceae species were identified through comprehensive analysis. The evolutionary relationships were analyzed with phylogenetic tree, duplication events and synteny. PRR genes were classified into three groups (PRR1, PRR5/9, PRR3/7). The expansion of PRR family was mainly derived from dispersed and whole-genome duplication events. Purifying selection was the major force for PRR family evolution. Synteny analysis indicated the existence of multiple orthologous PRR gene pairs between pear and other Rosaceae species. Moreover, the conserved motifs of eight PbPRR proteins supported the phylogenetic relationship. PRR genes showed diverse expression pattern in various tissues of pear (Pyrus bretschneideri). Transcript analysis under 12-h light/ dark cycle and constant light conditions revealed that PRR genes exhibited distinct rhythmic oscillations in pear. PbPRR59a and PbPRR59b highly homologous to AtPRR5 and AtPRR9 were cloned for further functional verification. PbPRR59a and PbPRR59b proteins were localized in the nucleus. The ectopic overexpression of PbPRR59a and PbPRR59b significantly delayed flowering in Arabidopsis transgenic plants by repress the expression of AtGI, AtCO and AtFT under long-day conditions. CONCLUSIONS: These results provide information for exploring the evolution of PRR genes in plants, and contribute to the subsequent functional studies of PRR genes in pear and other Rosaceae species.

PbGBF3 enhances salt response in pear by upregulating PbAPL2 and PbSDH1 and reducing ABA-mediated salt sensitivity.

Pear is a widely cultivated fruit crop, but its distribution and sustainable production are significantly limited by salt stress. This study used RNA-Seq time-course analysis, WGCNA, and functional enrichment analysis to uncover the molecular mechanisms underlying salt stress tolerance in Pyrus ussuriensis. We identified an ABA-related regulatory module, PbGBF3-PbAPL2-PbSDH1, as crucial in this response. PbGBF3, a bZIP transcription factor, enhances salt tolerance by upregulating PbAPL2 and PbSDH1. Overexpression of PbGBF3 improved salt tolerance in Pyrus communis calli and Arabidopsis, while silencing it reduced tolerance in Pyrus betulifolia. Functional assays showed that PbGBF3 binds to the promoters of PbAPL2 and PbSDH1, increasing their expression. PbAPL2 and PbSDH1, key enzymes in starch synthesis and the sorbitol pathway, respectively, enhance salt tolerance by increasing AGPase activity, soluble sugar content, and SDH activity, improving ROS scavenging and ion balance. Our findings suggest that the PbGBF3-PbAPL2 and PbGBF3-PbSDH1 modules positively regulate salt tolerance by enhancing ABA signaling and reducing ABA-mediated growth inhibition. These insights provide a foundation for developing salt-tolerant pear cultivars.

Haplotype-resolved T2T genome assemblies and pangenome graph of pear reveal diverse patterns of allele-specific expression and the 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, 'Yuluxiang' (YLX) and 'Hongxiangsu' (HXS), which share the same maternal parent but differ in their paternal parents. We then used these assemblies to explore the genome-scale landscape of allele-specific expression (ASE) and create a pangenome graph for pear. ASE was observed for close to 6000 genes in both hybrid cultivars. A subset of ASE genes related to aspects of fruit quality such as sugars, organic acids, and cuticular wax were identified, suggesting their important contributions to heterosis. Specifically, Ma1, a gene regulating fruit acidity, is absent in the paternal haplotypes of HXS and YLX. A pangenome graph was built based on our assemblies and seven published pear genomes. Resequencing data for 139 cultivated pear genotypes (including 97 genotypes sequenced here) were subsequently aligned to the pangenome graph, revealing numerous structural variant hotspots and selective sweeps during pear diversification. As predicted, the Ma1 allele was found to be absent in varieties with low organic acid content, and this association was functionally validated by Ma1 overexpression in pear fruit and calli. Overall, these results reveal the contributions of ASE to fruit-quality heterosis and provide a robust pangenome reference for high-resolution allele discovery and association mapping.

The combined effect of handgrip strength and obesity phenotype on the risk of stroke in Chinese middle-aged and elderly: A cohort study.

OBJECTIVE: The aim of this study was to investigate the combined effect of handgrip strength (HGS) and obesity phenotype on the risk of stroke in Chinese middle-aged and elderly people. METHODS: The data was used from the China Health and Retirement Longitudinal Study (CHARLS). Middle-aged and older adults who participated in surveys between 2011 and 2018 were included in the study. They were divided into 4 different types of obesity phenotypes based on obesity and metabolic status: metabolically healthy non-overweight/obesity (MHNO), metabolically healthy overweight/obesity (MHO), metabolically abnormal non-overweight/obesity (MANO), and metabolically abnormal overweight/obesity (MAO). The HGS level was divided into low and high groups according to the median values. Cox proportional risk regression model was used to analyze the joint effect of HGS and obesity phenotype on the risk of stroke among participants. RESULTS: A total of 7904 participants aged 58.89±9.08 years were included in this study. After adjusting for potential confounders, high HGS&MHO (HR=1.86, 95 % CI=1.12-3.09), high HGS&MANO (HR=2.01, 95 %CI=1.42-2.86), high HGS&MAO (HR=2.01, 95 % CI=1.37-2.93), low HGS&MHNO (HR=1.57, 95 % CI=1.00-2.46), low HGS&MHO (HR=2.09, 95 % CI=1.29-3.38), low HGS&MANO (HR=2.02, 95 % CI=1.35-3.03), and low HGS&MAO (HR=2.48, 95 % CI=1.72-3.58) group had significantly higher risks of stroke than the high HGS&MHNO group. CONCLUSION: The coexistence of metabolically unhealthy and low HGS can synergistically increase the risk of stroke in Chinese middle-aged and elderly people.

Evolutionary origin and gradual accumulation with plant evolution of the LACS family.

BACKGROUND: LACS (long-chain acyl-CoA synthetase) genes are widespread in organisms and have multiple functions in plants, especially in lipid metabolism. However, the origin and evolutionary dynamics of the LACS gene family remain largely unknown. RESULTS: Here, we identified 1785 LACS genes in the genomes of 166 diverse plant species and identified the clades (I, II, III, IV, V, VI) of six clades for the LACS gene family of green plants through phylogenetic analysis. Based on the evolutionary history of plant lineages, we found differences in the origins of different clades, with Clade IV originating from chlorophytes and representing the origin of LACS genes in green plants. The structural characteristics of different clades indicate that clade IV is relatively independent, while the relationships between clades (I, II, III) and clades (V, VI) are closer. Dispersed duplication (DSD) and transposed duplication (TRD) are the main forces driving the evolution of plant LACS genes. Network clustering analysis further grouped all LACS genes into six main clusters, with genes within each cluster showing significant co-linearity. Ka/Ks results suggest that LACS family genes underwent purifying selection during evolution. We analyzed the phylogenetic relationships and characteristics of six clades of the LACS gene family to explain the origin, evolutionary history, and phylogenetic relationships of different clades and proposed a hypothetical evolutionary model for the LACS family of genes in plants. CONCLUSIONS: Our research provides genome-wide insights into the evolutionary history of the LACS gene family in green plants. These insights lay an important foundation for comprehensive functional characterization in future research.

Interaction among homeodomain transcription factors mediates ethylene biosynthesis during pear fruit ripening.

Fruit ripening is manipulated by the plant phytohormone ethylene in climacteric fruits. While the transcription factors (TFs) involved in ethylene biosynthesis and fruit ripening have been extensively studied in tomato, their identification in pear remains limited. In this study, we identified and characterized a HOMEODOMAIN TF, PbHB.G7.2, through transcriptome analysis. PbHB.G7.2 could directly bind to the promoter of the ethylene biosynthetic gene, 1-aminocyclopropane-1-carboxylic acid synthase (PbACS1b), thereby enhancing its activity and resulting in increased ethylene production during pear fruit ripening. Yeast-two-hybrid screening revealed that PbHB.G7.2 interacted with PbHB.G1 and PbHB.G2.1. Notably, these interactions disrupted the transcriptional activation of PbHB.G7.2. Interestingly, PbHB.G1 and PbHB.G2.1 also bind to the PbACS1b promoter, albeit different regions from those bound by PbHB.G7.2. Moreover, the regions of PbHB.G1 and PbHB.G2.1 involved in their interaction with PbHB.G7.2 differ from the regions responsible for binding to the PbACS1b promoter. Nonetheless, these interactions also disrupt the transcriptional activation of PbHB.G1 and PbHB.G2.1. These findings offer a new mechanism of ethylene biosynthesis during climacteric fruit ripening.

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.

Transcription factors Pbr3RAV2 and PbrTTG1 regulate pear resistance to Botryosphaeria dothidea via the autophagy pathway.

Pear ring rot, caused by Botryosphaeria dothidea, is the most serious disease of pear (Pyrus spp.) trees. However, the molecular mechanisms underlying pear resistance to B. dothidea remain elusive. In this study, we demonstrated that the pear AuTophagy-related Gene 1a (PbrATG1a) plays a key role in autophagic activity and resistance to B. dothidea. Stable overexpression of PbrATG1a enhanced resistance to B. dothidea in pear calli. Autophagy activity was greater in PbrATG1a-overexpressing calli than in wild-type calli. We used yeast 1-hybrid screening to identify a transcription factor, related to ABI3 and VP1 (Pbr3RAV2), that binds the promoter of PbrATG1a and enhances pear resistance to B. dothidea by regulating autophagic activity. Specifically, the overexpression of Pbr3RAV2 enhanced resistance to B. dothidea in pear calli, while transient silencing of Pbr3RAV2 resulted in compromised resistance to B. dothidea in Pyrus betulifolia. In addition, we identified Transparent Testa Glabra 1 (PbrTTG1), which interacts with Pbr3RAV2. Pathogen infection enhanced the interaction between Pbr3RAV2 and PbrTTG1. The Pbr3RAV2-PbrTTG1 complex increased the binding capacity of Pbr3RAV2 and transcription of PbrATG1a. In addition to providing insights into the molecular mechanisms underlying pear disease resistance, these findings suggest potential genetic targets for enhancing disease resistance in pear.

The PbbHLH62/PbVHA-B1 module confers salt tolerance through modulating intracellular Na+/K+ homeostasis and reactive oxygen species removal in pear.

The vacuolar H+-ATPase (V-ATPase) is a multi-subunit membrane protein complex, which plays pivotal roles in building up an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this study, a B subunit of V-ATPase gene, PbVHA-B1 was discovered and isolated from stress-induced P. betulaefolia combining with RT-PCR method. The RT-qPCR analysis revealed that the expression level of PbVHA-B1 was upregulated by salt, drought, cold, and exogenous ABA treatment. Subcellular localization analyses showed that PbVHA-B1 was located in the cytoplasm and nucleus. Moreover, overexpression of PbVHA-B1 gene noticeably increased the ATPase activity and the tolerance to salt in transgenic Arabidopsis plants. In contrast, knockdown of PbVHA-B1 gene in P.betulaefolia by virus-induced gene silencing had reduced resistance to salt stress. In addition, using yeast one-hybride (Y1H) and yeast two-hybride (Y2H) screens, PbbHLH62, a bHLH transcription factor, was identified as a partner of the PbVHA-B1 promoter and protein. Then, we also found that PbbHLH62 positively regulate the expression of PbVHA-B1 and the ATPase activity after salt stress treatment. These findings provide evidence that PbbHLH62 played a critical role in the salt response. Collectively, our results demonstrate that a PbbHLH62/PbVHA-B1 module plays a positive role in salt tolerance by maintain intracellular ion and ROS homeostasis in pear.

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.

Multi-omics provide insights into the regulation of DNA methylation in pear fruit metabolism.

BACKGROUND: Extensive research has been conducted on fruit development in crops, but the metabolic regulatory networks underlying perennial fruit trees remain poorly understood. To address this knowledge gap, we conduct a comprehensive analysis of the metabolome, proteome, transcriptome, DNA methylome, and small RNAome profiles of pear fruit flesh at 11 developing stages, spanning from fruitlet to ripening. Here, we systematically investigate the metabolic landscape and regulatory network involved. RESULTS: We generate an association database consisting of 439 metabolites and 14,399 genes to elucidate the gene regulatory network of pear flesh metabolism. Interestingly, we detect increased DNA methylation in the promoters of most genes within the database during pear flesh development. Application of a DNA methylation inhibitor to the developing fruit represses chlorophyll degradation in the pericarp and promotes xanthophyll, ß-carotene, and abscisic acid (ABA) accumulation in the flesh. We find the gradual increase in ABA production during pear flesh development is correlated with the expression of several carotenoid pathway genes and multiple transcription factors. Of these transcription factors, the zinc finger protein PbZFP1 is identified as a positive mediator of ABA biosynthesis in pear flesh. Most ABA pathway genes and transcription factors are modified by DNA methylation in the promoters, although some are induced by the DNA methylation inhibitor. These results suggest that DNA methylation inhibits ABA accumulation, which may delay fruit ripening. CONCLUSION: Our findings provide insights into epigenetic regulation of metabolic regulatory networks during pear flesh development, particularly with regard to DNA methylation.

PuNDH9, a subunit of ETC Complex I regulates plant defense by interacting with PuPR1.

NAD+ and NADH play critical roles in energy metabolism, cell death, and gene expression. The NADH-ubiquinone oxidoreductase complex (Complex I) has been long known as a key enzyme in NAD+ and NADH metabolism. In the present study, we found and analyzed a new subunit of Complex I (NDH9), which was isolated from Pyrus ussuriensis combined with RT-PCR. Following infection with A. alternata, RT-qPCR analysis demonstrated an increase in the expression of PuNDH9. Genetic manipulation of PuNDH9 levels suggested that PuNDH9 plays key roles in NADH/NAD+ homeostasis, defense enzyme activities, ROS generation, cell death, gene expression, energy metabolism, and mitochondrial functions during the pear- A. alternata interaction. Furthermore, Y2H, GST-pull down, and a split-luciferase complementation imaging assays revealed that PuNDH9 interacts with PuPR1. We discover that PuNDH9 and PuPR1 synergistically activate defense enzyme activities, ROS accumulation, cell death, and plant defenses. Collectively, our findings reveal that PuNDH9 is likely important for plant defenses.

The transcription factor PbbHLH164 is destabilized by PbRAD23C/D.1 and mediates ethylene biosynthesis during pear fruit ripening.

The phytohormone ethylene plays an important role in climacteric fruit ripening. However, the knowledge on molecular regulation of ethylene biosynthesis remains limited in pear fruit. Herein, a new basic helix-loop-helix transcription factor, PbbHLH164, was identified based on the transcriptome analysis of different developing and ripening fruits of two pear cultivars 'Sucui No. 1' and 'Cuiguan'. PbbHLH164 was more highly expressed in ripening fruit than in developing fruit and positively correlated with ethylene production in both cultivars. PbbHLH164 could directly bind to the promoter of 1-aminocyclopropane-1-carboxylate synthase, PbACS1b, to enhance the expression, leading to the increase of ethylene production and the acceleration of fruit ripening. Interestingly, PbbHLH164 physically interacted with an ubiquitin-like/ubiquitin-associated protein PbRAD23C/D.1, and the interaction of PbbHLH164 with PbRAD23C/D.1 attenuated the function of PbbHLH164 in enhancing the activity of the PbACS1b promoter. Notably, PbRAD23C/D.1 was involved in the degradation of PbbHLH164, and this degradation was inhibited by an ubiquitin proteasome inhibitor MG132. Different from PbbHLH164, PbRAD23C/D.1 was more highly expressed in developing fruit than in ripening fruit of both cultivars. These results suggest that the increase of ethylene production during pear fruit ripening results from the up-regulated expression of PbbHLH164 and the down-regulated expression of PbRAD23C/D.1. This information provided new insights into the molecular regulation of ethylene biosynthesis during fruit ripening.

Physiological and autophagy evaluation of different pear varieties (Pyrus spp.) in response to Botryosphaeria dothidea infection.

Ring rot disease is one of the most common diseases in pear orchards. To better understand the physiology, biochemistry and autophagic changes of different pear varieties after Botryosphaeria dothidea (B.dothidea) infection, we evaluated eight different pear varieties for B. dothidea resistance. The susceptible varieties had larger spot diameters, lower chlorophyll contents and higher malondialdehyde contents than the resistant varieties. In disease-resistant varieties, reactive oxygen species (ROS) levels were relatively lower, while the ROS metabolism (antioxidant enzyme activities and the ascorbic acid-glutathione cycle) was also maintained at higher levels, and it induced a significant upregulation of related gene expression. In addition, autophagy, as an important evaluation index, was found to have more autophagic activity in disease-resistant varieties than in susceptible varieties, suggesting that pathogen infestation drives plants to increase autophagy to defend against pathogens. In summary, the results of this study reveal that different resistant pear varieties enhance plant resistance to the disease through a series of physio-biochemical changes and autophagic activity after inoculation with B. dothidea. This study provides clear physiological and biochemical traits for pear disease resistance selection, potential genetic resources and material basis for pear disease control and disease resistance, breeding and points out the direction for research on the mechanism of pear resistance to B. dothidea.

Self S-RNase reduces the expression of two pollen-specific COBRA genes to inhibit pollen tube growth in pear.

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.

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.

PbrChiA: a key chitinase of pear in response to Botryosphaeria dothidea infection by interacting with PbrLYK1b2 and down-regulating ROS accumulation.

Pear ring rot, caused by the pathogenic fungi Botryosphaeria dothidea, seriously affects pear production. While the infection-induced reactive oxygen species (ROS) burst of infected plants limits the proliferation of B. dothidea during the early infection stage, high ROS levels can also contribute to their growth during the later necrotrophic infection stage. Therefore, it is important to understand how plants balance ROS levels and resistance to pathogenic B. dothidea during the later stage. In this study, we identified PbrChiA, a glycosyl hydrolases 18 (GH18) chitinase-encoding gene with high infection-induced expression, through a comparative transcriptome analysis. Artificial substitution, stable overexpression, and virus induced gene silencing (VIGS) experiments demonstrated that PbrChiA can positively regulate pear resistance as a secreted chitinase to break down B. dothidea mycelium in vitro and that overexpression of PbrChiA suppressed infection-induced ROS accumulation. Further analysis revealed that PbrChiA can bind to the ectodomain of PbrLYK1b2, and this interaction suppressed PbrLYK1b2-mediated chitin-induced ROS accumulation. Collectively, we propose that the combination of higher antifungal activity from abundant PbrChiA and lower ROS levels during later necrotrophic infection stage confer resistance of pear against B. dothidea.