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.

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.

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.

PearMODB: a multiomics database for pear (Pyrus) genomics, genetics and breeding study.

Pear (Pyrus ssp.) belongs to Rosaceae and is an important fruit tree widely cultivated around the world. Currently, challenges to cope with the burgeoning sets of multiomics data are rapidly increasing. Here, we constructed the Pear Multiomics Database (PearMODB) by integrating genome, transcriptome, epigenome and population variation data, and aimed to provide a portal for accessing and analyzing pear multiomics data. A variety of online tools were built including gene search, BLAST, JBrowse, expression heatmap, synteny analysis and primer design. The information of DNA methylation sites and single-nucleotide polymorphisms can be retrieved through the custom JBrowse, providing an opportunity to explore the genetic polymorphisms linked to phenotype variation. Moreover, different gene families involving transcription factors, transcription regulators and disease resistance (nucleotide-binding site leucine-rich repeat) were identified and compiled for quick search. In particular, biosynthetic gene clusters (BGCs) were identified in pear genomes, and specialized webpages were set up to show detailed information of BGCs, laying a foundation for studying metabolic diversity among different pear varieties. Overall, PearMODB provides an important platform for pear genomics, genetics and breeding studies. Database URL http://pearomics.njau.edu.cn.

Comprehensive Genomic Analysis of SnRK in Rosaceae and Expression Analysis of RoSnRK2 in Response to Abiotic Stress in Rubus occidentalis.

The sucrose nonfermenting 1-related protein kinase (SnRK) plays an important role in responding to abiotic stresses by phosphorylating the target protein to regulate various signaling pathways. However, little is known about the characteristics, evolutionary history, and expression patterns of the SnRK family in black raspberry (Rubus occidentalis L.) or other Rosaceae family species. In this study, a total of 209 SnRK genes were identified in 7 Rosaceae species and divided into 3 subfamilies (SnRK1, SnRK2, and SnRK3) based on phylogenetic analysis and specific motifs. Whole-genome duplication (WGD) and dispersed duplication (DSD) were considered to be major contributions to the SnRK family expansion. Purifying selection was the primary driving force in the SnRK family evolution. The spatial expression indicated that the RoSnRK genes may play important roles in different tissues. In addition, the expression models of 5 RoSnRK2 genes in response to abiotic stresses were detected by qRT-PCR. The proteins encoded by RoSnRK2 genes localize to the cytoplasm and nucleus in order to perform their respective functions. Taken together, this study provided an analysis of the SnRK gene family expansion and evolution, and contributed to the current knowledge of the function of 5 RoSnRK2 genes, which in turn expanded understanding of the molecular mechanisms of black raspberry responses to abiotic stress.

The genomic basis of the plant island syndrome in Darwin's giant daisies.

The repeated, rapid and often pronounced patterns of evolutionary divergence observed in insular plants, or the 'plant island syndrome', include changes in leaf phenotypes, growth, as well as the acquisition of a perennial lifestyle. Here, we sequence and describe the genome of the critically endangered, Galápagos-endemic species Scalesia atractyloides Arnot., obtaining a chromosome-resolved, 3.2-Gbp assembly containing 43,093 candidate gene models. Using a combination of fossil transposable elements, k-mer spectra analyses and orthologue assignment, we identify the two ancestral genomes, and date their divergence and the polyploidization event, concluding that the ancestor of all extant Scalesia species was an allotetraploid. There are a comparable number of genes and transposable elements across the two subgenomes, and while their synteny has been mostly conserved, we find multiple inversions that may have facilitated adaptation. We identify clear signatures of selection across genes associated with vascular development, growth, adaptation to salinity and flowering time, thus finding compelling evidence for a genomic basis of the island syndrome in one of Darwin's giant daisies.

The Origin and Evolution of RNase T2 Family and Gametophytic Self-incompatibility System in Plants.

Ribonuclease (RNase) T2 genes are found widely in both eukaryotes and prokaryotes, and genes from this family have been revealed to have various functions in plants. In particular, S-RNase is known to be the female determinant in the S-RNase-based gametophytic self-incompatibility (GSI) system. However, the origin and evolution of the RNase T2 gene family and GSI system are not well understood. In this study, 785 RNase T2 genes were identified in 81 sequenced plant genomes representing broad-scale diversity and divided into three subgroups (Class I, II, and III) based on phylogenetic and synteny network analysis. Class I was found to be of ancient origin and to emerge in green algae, Class II was shown to originate with the appearance of angiosperms, while Class III was discovered to be eudicot-specific. Each of the three major classes could be further classified into several subclasses of which some subclasses were found to be lineage-specific. Furthermore, duplication, deletion, or inactivation of the S/S-like-locus was revealed to be linked to repeated loss and gain of self-incompatibility in different species from distantly related plant families with GSI. Finally, the origin and evolutionary history of S-locus in Rosaceae species was unraveled with independent loss and gain of S-RNase occurred in different subfamilies of Rosaceae. Our findings provide insights into the origin and evolution of the RNase T2 family and the GSI system in plants.

Genome-wide identification and comparative analysis of the PYL gene family in eight Rosaceae species and expression analysis of seeds germination in pear.

Abscisic acid (ABA) is a phytohormone that plays important roles in the regulation of plant growth, seed germination, and stress responses. The pyrabactin resistance 1-like (PYR/PYL) protein, an ABA receptor, was involved in the initial step in ABA signal transduction. However, the evolutionary history and characteristics of PYL genes expression remain unclear in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. In this study, 67 PYL genes were identified in eight Rosaceae species, and have been classified into three subgroups based on specific motifs and phylogenetic analysis. Intriguingly, we observed that whole-genome duplication (WGD) and dispersed duplication (DSD) have a major contribution to PYL family expansion. Purifying selection was the major force in PYL genes evolution. Expression analysis finds that PYL genes may function in multiple pear tissues. qRT-PCR validation of 11 PbrPYL genes indicates their roles in seed germination and abiotic stress responses. Our study provides a basis for further elucidation of the function of PYL genes and analysis of their expansion, evolution and expression patterns, which helps to understand the molecular mechanism of pear response to seed germination and seedling abiotic stress.

Transcriptome and Resequencing Analyses Provide Insight into Differences in Organic Acid Accumulation in Two Pear Varieties.

Fruit acidity is one of the main determinants of fruit flavor and a target trait in fruit breeding. However, the genomic mechanisms governing acidity variation among different pear varieties remain poorly understood. In this study, two pear varieties with contrasting organic acid levels, 'Dangshansuli' (low-acidity) and 'Amute' (high-acidity), were selected, and a combination of transcriptome and population genomics analyses were applied to characterize their patterns of gene expression and genetic variation. Based on RNA-seq data analysis, differentially expressed genes (DEGs) involved in organic acid metabolism and accumulation were identified. Weighted correlation network analysis (WGCNA) revealed that nine candidate TCA (tricarboxylic acid)-related DEGs and three acid transporter-related DEGs were located in three key modules. The regulatory networks of the above candidate genes were also predicted. By integrating pear resequencing data, two domestication-related genes were found to be upregulated in 'Amute', and this trend was further validated for other pear varieties with high levels of organic acid, suggesting distinct selective sweeps during pear dissemination and domestication. Collectively, this study provides insight into organic acid differences related to expression divergence and domestication in two pear varieties, pinpointing several candidate genes for the genetic manipulation of acidity in pears.

Characterization of the pectin methylesterase inhibitor gene family in Rosaceae and role of PbrPMEI23/39/41 in methylesterified pectin distribution in pear pollen tube.

MAIN CONCLUSION: Pectin methylesterase inhibitor gene family in the seven Rosaceae species (including three pear cultivars) is characterized and three pectin methylesterase inhibitor genes are identified to regulate pollen tube growth in pear. Pectin methylesterase inhibitor (PMEI) participates in a variety of biological processes in plants. However, the information and function of PMEI genes in Rosaceae are largely unknown. In this study, a total of 423 PMEI genes are identified in the genomes of seven Rosaceae species. The PMEI genes in pear are categorized into five subfamilies based on structural analysis and evolutionary analysis. WGD and TD are the main duplication events in the PMEI gene family of pear. Quantitative real-time PCR analysis indicates that PbrPMEI23, PbrPMEI39, and PbrPMEI41 are increasingly expressed during pear pollen tube growth. Under the treatment of recombinant proteins PbrPMEI23, PbrPMEI39 or PbrPMEI41, the content of methylesterified pectin at the region 5-20 µm from the pollen tube tip significantly increases, and the growth of pear pollen tubes is promoted. These results indicate that PMEI regulates the growth of pollen tubes by changing the distribution of methylesterified pectin in the apex.

Genome-wide identification of lysin motif containing protein family genes in eight rosaceae species, and expression analysis in response to pathogenic fungus Botryosphaeria dothidea in Chinese white pear.

BACKGROUND: Lysin motif-containing proteins (LYP), which act as pattern-recognition receptors, play central roles in growth, node formation, and responses to biotic stresses. The sequence of Chinese white pear genome (cv. 'Dangshansuli') along with the seven other species of Rosaceae has already been reported. Although, in these fruit crops, there is still a lack of clarity regarding the LYP family genes and their evolutionary history. RESULTS: In the existing study, eight Rosaceae species i.e., Pyrus communis, Prunus persica, Fragaria vesca, Pyrus bretschneideri, Prunus avium, Prunus mume, Rubus occidentalis, and Malus × domestica were evaluated. Here, we determined a total of 124 LYP genes from the underlined Rosaceae species. While eighteen of the genes were from Chinese white pear, named as PbrLYPs. According to the LYPs structural characteristics and their phylogenetic analysis, those genes were classified into eight groups (group LYK1, LYK2, LYK3, LYK4/5, LYM1/3, LYM2, NFP, and WAKL). Dispersed duplication and whole-genome duplication (WGD) were found to be the most contributing factors of LYP family expansion in the Rosaceae species. More than half of the duplicated PbrLYP gene pairs were dated back to the ancient WGD (~ 140 million years ago (MYA)), and PbrLYP genes have experienced long-term purifying selection. The transcriptomic results indicated that the PbrLYP genes expression was tissue-specific. Most PbrLYP genes showed differential expression in leaves under fungal pathogen infection with two of them located in the plasmalemma. CONCLUSION: A comprehensive analysis identified 124 LYP genes in eight Rosaceae species. Our findings have provided insights into the functions and characteristics of the Rosaceae LYP genes and a guide for the identification of other candidate LYPs for further genetic improvements for pathogen-resistance in higher plants.

Genome-wide identification and comparative analysis of the ADH gene family in Chinese white pear (Pyrus bretschneideri) and other Rosaceae species.

Alcohol dehydrogenase (ADH) is essential to the formation of aromatic compounds in fruits. However, the evolutionary history and characteristics of ADH gene expression remain largely unclear in Rosaceae fruit species. In this study, 464 ADH genes were identified in eight Rosaceae fruit species, 68 of the genes were from pear and which were classified into four subgroups. Frequent single gene duplication events were found to have contributed to the formation of ADH gene clusters and the expansion of the ADH gene family in these eight Rosaceae species. Purifying selection was the major force in ADH gene evolution. The younger genes derived from tandem and proximal duplications had evolved faster than those derived from other types of duplication. RNA-Seq and qRT-PCR analysis revealed that the expression levels of three ADH genes were closely correlated with the content of aromatic compounds detected during fruit development.

Characterization of the pectin methyl-esterase gene family and its function in controlling pollen tube growth in pear (Pyrus bretschneideri).

Pectin methyl-esterases (PMEs) play crucial roles in plant growth. In this study, we identified 81 PbrPMEs in pear. Whole-genome duplication and purifying selection drove the evolution of PbrPME gene family. The expression of 47 PbrPMEs was detected in pear pollen tube, which were assigned to 13 clusters by an expression tendency analysis. One of the 13 clusters presented opposite expression trends towards the changes of methyl-esterified pectins at the apical cell wall. PbrPMEs were localized in the cytoplasm and plasma membrane. Repression of PbrPME11, PbrPME44, and PbrPME59 resulted in the inhibition of pear pollen tube growth and abnormal deposition of methyl-esterified pectins at pollen tube tip. Pharmacological analysis confirmed that reduced PbrPME activities repressed the pollen tube growth. Overall, we have explored the evolutionary characteristics of PbrPME gene family and found the key PbrPME genes that control the growth of pollen tube, which deepened the understanding of pear fertility regulation.

Comparative analysis of the P-type ATPase gene family in seven Rosaceae species and an expression analysis in pear (Pyrus bretschneideri Rehd.).

P-type ATPases are integral membrane transporters that play important roles in transmembrane transport in plants. However, a comprehensive analysis of the P-type ATPase gene family has not been conducted in Chinese white pear (Pyrus bretschneideri) or other Rosaceae species. Here, we identified 419 P-type ATPase genes from seven Rosaceae species (Pyrus bretschneideri, Malus domestica, Prunus persica, Fragaria vesca, Prunus mume, Pyrus communis and Pyrus betulifolia). Structural and phylogenetic analyses revealed that P-type ATPase genes can be divided into five subfamilies. Different subfamilies have different conserved motifs and cis-acting elements, which may lead to functional divergence within one gene family. Dispersed duplication and whole-genome duplication may play critical roles in the expansion of the P-type ATPase family. Purifying selection was the primary force driving the evolution of P-type ATPase family genes. Based on the dynamic transcriptome analysis and transient transformation of Chinese white pear fruit, Pbr029767.1 in the P3A subfamily were found to be associated with malate accumulation during pear fruit development. Using a co-expression network, we identified several transcription factors that may have regulatory relationships with the P-type ATPase gene family. Overall, this study lays a solid foundation for understanding the evolution and functions of P-type ATPase genes in Chinese white pear and six other Rosaceae species.

Genome-wide comparative analysis of the BAHD superfamily in seven Rosaceae species and expression analysis in pear (Pyrus bretschneideri).

BACKGROUND: The BAHD acyltransferase superfamily exhibits various biological roles in plants, including regulating fruit quality, catalytic synthesizing of terpene, phenolics and esters, and improving stress resistance. However, the copy numbers, expression characteristics and associations with fruit aroma formation of the BAHD genes remain unclear. RESULTS: In total, 717 BAHD genes were obtained from the genomes of seven Rosaceae, (Pyrus bretschneideri, Malus domestica, Prunus avium, Prunus persica, Fragaria vesca, Pyrus communis and Rubus occidentalis). Based on the detailed phylogenetic analysis and classifications in model plants, we divided the BAHD family genes into seven groups, I-a, I-b, II-a, II-b, III-a, IV and V. An inter-species synteny analysis revealed the ancient origin of BAHD superfamily with 78 syntenic gene pairs were detected among the seven Rosaceae species. Different types of gene duplication events jointly drive the expansion of BAHD superfamily, and purifying selection dominates the evolution of BAHD genes supported by the small Ka/Ks ratios. Based on the correlation analysis between the ester content and expression levels of BAHD genes at different developmental stages, four candidate genes were selected for verification as assessed by qRT-PCR. The result implied that Pbr020016.1, Pbr019034.1, Pbr014028.1 and Pbr029551.1 are important candidate genes involved in aroma formation during pear fruit development. CONCLUSION: We have thoroughly identified the BAHD superfamily genes and performed a comprehensive comparative analysis of their phylogenetic relationships, expansion patterns, and expression characteristics in seven Rosaceae species, and we also obtained four candidate genes involved in aroma synthesis in pear fruit. These results provide a theoretical basis for future studies of the specific biological functions of BAHD superfamily members and the improvement of pear fruit quality.

Unbiased subgenome evolution following a recent whole-genome duplication in pear (Pyrus bretschneideri Rehd.).

Genome fractionation (also known as diploidization) frequently occurs following paleopolyploidization events. Biased fractionation between subgenomes has been found in some paleo-allopolyploids, while this phenomenon is absent in paleo-autopolyploids. Pear (Pyrus bretschneideri Rehd.) experienced a recent whole-genome duplication (WGD, ~30 million years ago); however, the evolutionary fate of the two subgenomes derived from this WGD event is not clear. In this study, we identified the two paleo-subgenomes in pear using peach (Prunus persica) as an outgroup and investigated differences in the gene loss rate, evolutionary rate, gene expression level, and DNA methylation level between these two subgenomes. Fractionation bias was not found between the two pear subgenomes, which evolved at similar evolutionary rates. The DNA methylation level of the two subgenomes showed little bias, and we found no expression dominance between the subgenomes. However, we found that singleton genes and homeologous genes within each subgenome showed divergent evolutionary patterns of selective constraints, expression and epigenetic modification. These results provide insights into subgenome evolution following paleopolyploidization in pear.

Gene duplication and evolution in recurring polyploidization-diploidization cycles in plants.

BACKGROUND: The sharp increase of plant genome and transcriptome data provide valuable resources to investigate evolutionary consequences of gene duplication in a range of taxa, and unravel common principles underlying duplicate gene retention. RESULTS: We survey 141 sequenced plant genomes to elucidate consequences of gene and genome duplication, processes central to the evolution of biodiversity. We develop a pipeline named DupGen_finder to identify different modes of gene duplication in plants. Genes derived from whole-genome, tandem, proximal, transposed, or dispersed duplication differ in abundance, selection pressure, expression divergence, and gene conversion rate among genomes. The number of WGD-derived duplicate genes decreases exponentially with increasing age of duplication events-transposed duplication- and dispersed duplication-derived genes declined in parallel. In contrast, the frequency of tandem and proximal duplications showed no significant decrease over time, providing a continuous supply of variants available for adaptation to continuously changing environments. Moreover, tandem and proximal duplicates experienced stronger selective pressure than genes formed by other modes and evolved toward biased functional roles involved in plant self-defense. The rate of gene conversion among WGD-derived gene pairs declined over time, peaking shortly after polyploidization. To provide a platform for accessing duplicated gene pairs in different plants, we constructed the Plant Duplicate Gene Database. CONCLUSIONS: We identify a comprehensive landscape of different modes of gene duplication across the plant kingdom by comparing 141 genomes, which provides a solid foundation for further investigation of the dynamic evolution of duplicate genes.