Phosphorylated transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis in pear exposed to high light.

Plants are increasingly vulnerable to environmental stresses because of global warming and climate change. Stress-induced reactive oxygen species (ROS) accumulation results in plant cell damage, even cell death. Anthocyanins are important antioxidants that scavenge ROS to maintain ROS homeostasis. However, the mechanism underlying ROS-induced anthocyanin accumulation is unclear. In this study, we determined that the HD-Zip I family member transcription factor PuHB40 mediates ROS-dependent anthocyanin biosynthesis under high-light stress in pear (Pyrus ussuriensis). Specifically, PuHB40 induces the PuMYB123-like-PubHLH3 transcription factor complex for anthocyanin biosynthesis. The PuHB40-mediated transcriptional activation depends on its phosphorylation level, which is regulated by protein phosphatase PP2A. Elevated ROS content maintains high PuHB40 phosphorylation levels while also enhancing the PuHB40-induced PuMYB123-like transcription by decreasing the PuPP2AA2 expression, ultimately leading to increased anthocyanin biosynthesis. Our study reveals a pathway regulating the ROS-induced anthocyanin biosynthesis in pears, further clarifying the mechanism underlying the abiotic stress-induced anthocyanin biosynthesis, which may have implications for improving plant stress tolerance.

Acetylation of inorganic pyrophosphatase by S-RNase signaling induces pollen tube tip swelling by repressing pectin methylesterase.

Self-incompatibility (SI) is a widespread genetically determined system in flowering plants that prevents self-fertilization to promote gene flow and limit inbreeding. S-RNase-based SI is characterized by the arrest of pollen tube growth through the pistil. Arrested pollen tubes show disrupted polarized growth and swollen tips, but the underlying molecular mechanism is largely unknown. Here, we demonstrate that the swelling at the tips of incompatible pollen tubes in pear (Pyrus bretschneideri [Pbr]) is mediated by the SI-induced acetylation of the soluble inorganic pyrophosphatase (PPA) PbrPPA5. Acetylation at Lys-42 of PbrPPA5 by the acetyltransferase GCN5-related N-acetyltransferase 1 (GNAT1) drives accumulation of PbrPPA5 in the nucleus, where it binds to the transcription factor PbrbZIP77, forming a transcriptional repression complex that inhibits the expression of the pectin methylesterase (PME) gene PbrPME44. The function of PbrPPA5 as a transcriptional repressor does not require its PPA activity. Downregulating PbrPME44 resulted in increased levels of methyl-esterified pectins in growing pollen tubes, leading to swelling at their tips. These observations suggest a mechanism for PbrPPA5-driven swelling at the tips of pollen tubes during the SI response. The targets of PbrPPA5 include genes encoding cell wall-modifying enzymes, which are essential for building a continuous sustainable mechanical structure for pollen tube growth.

The ethylene-responsive transcription factor PpERF9 represses PpRAP2.4 and PpMYB114 via histone deacetylation to inhibit anthocyanin biosynthesis in pear.

Ethylene induces anthocyanin biosynthesis in most fruits, including apple (Malus domestica) and plum (Prunus spp.). By contrast, ethylene inhibits anthocyanin biosynthesis in pear (Pyrus spp.), but the underlying molecular mechanism remains unclear. In this study, we identified and characterized an ethylene-induced ETHYLENE RESPONSE FACTOR (ERF) transcription factor, PpETHYLENE RESPONSE FACTOR9 (PpERF9), which functions as a transcriptional repressor. Our analyses indicated PpERF9 can directly inhibit expression of the MYB transcription factor gene PpMYB114 by binding to its promoter. Additionally, PpERF9 inhibits the expression of the transcription factor gene PpRELATED TO APETALA2.4 (PpRAP2.4), which activates PpMYB114 expression, by binding to its promoter, thus forming a PpERF9-PpRAP2.4-PpMYB114 regulatory circuit. Furthermore, PpERF9 interacts with the co-repressor PpTOPLESS1 (PpTPL1) via EAR motifs to form a complex that removes the acetyl group on histone H3 and maintains low levels of acetylated H3 in the PpMYB114 and PpRAP2.4 promoter regions. The resulting suppressed expression of these 2 genes leads to decreased anthocyanin biosynthesis in pear. Collectively, these results indicate that ethylene inhibits anthocyanin biosynthesis by a mechanism that involves PpERF9-PpTPL1 complex-mediated histone deacetylation of PpMYB114 and PpRAP2.4. The data presented herein will be useful for clarifying the relationship between chromatin status and hormone signaling, with implications for plant biology research.

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.

Transcription factors PuPRE6/PuMYB12 and histone deacetylase PuHDAC9-like regulate sucrose levels in pear.

The improvement of fruit quality, in particular sugar content, has been a major goal of plant breeding programmes for many years. Here, 2 varieties of the Ussurian pear (Pyrus ussuriensis), Nanguo, and its high-sucrose accumulation bud sport, Nanhong, were used to study the molecular mechanisms regulating sucrose transport in fruits. Comparative transcriptome analysis showed that in Nanhong fruit, an MYB transcription factor, PuMYB12, and a sucrose transporter protein, PuSUT4-like, were expressed at higher levels, while a paclobutrazol resistance transcription factor, PuPRE6, and a histone deacetylase (HDAC), PuHDAC9-like, were expressed at lower levels in Nanguo fruit. PuSUT4-like silencing and overexpression experiments in Nanguo pear showed that PuSUT4-like is essential for sucrose transportation. PuPRE6 and PuMYB12 act as antagonistic complexes to regulate PuSUT4-like transcription and sucrose accumulation. The histone deacetylation levels of the PuMYB12 and PuSUT4-like promoters were higher in Nanguo fruit than in Nanhong fruit, and Y1H assays showed that HDAC PuHDAC9-like bound directly to the promoters of PuMYB12 and PuSUT4-like. Our results uncovered transcription regulation and epigenetic mechanisms underlying sucrose accumulation in pears.

Transcription factors BZR2/MYC2 modulate brassinosteroid and jasmonic acid crosstalk during pear dormancy.

Bud dormancy is an important physiological process during winter. Its release requires a certain period of chilling. In pear (Pyrus pyrifolia), the abscisic acid (ABA)-induced expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes represses bud break, whereas exogenous gibberellin (GA) promotes dormancy release. However, with the exception of ABA and GA, the regulatory effects of phytohormones on dormancy remain largely uncharacterized. In this study, we confirmed brassinosteroids (BRs) and jasmonic acid (JA) contribute to pear bud dormancy release. If chilling accumulation is insufficient, both 24-epibrassinolide (EBR) and methyl jasmonic acid (MeJA) can promote pear bud break, implying that they positively regulate dormancy release. BRASSINAZOLE RESISTANT 2 (BZR2), which is a BR-responsive transcription factor, inhibited PpyDAM3 expression and accelerated pear bud break. The transient overexpression of PpyBZR2 increased endogenous GA, JA, and JA-Ile levels. In addition, the direct interaction between PpyBZR2 and MYELOCYTOMATOSIS 2 (PpyMYC2) enhanced the PpyMYC2-mediated activation of Gibberellin 20-oxidase genes PpyGA20OX1L1 and PpyGA20OX2L2 transcription, thereby increasing GA3 contents and accelerating pear bud dormancy release. Interestingly, treatment with 5 µm MeJA increased the bud break rate, while also enhancing PpyMYC2-activated PpyGA20OX expression and increasing GA3,4 contents. The 100 µm MeJA treatment decreased the PpyMYC2-mediated activation of the PpyGA20OX1L1 and PpyGA20OX2L2 promoters and suppressed the inhibitory effect of PpyBZR2 on PpyDAM3 transcription, ultimately inhibiting pear bud break. In summary, our data provide insights into the crosstalk between the BR and JA signaling pathways that regulate the BZR2/MYC2-mediated pathway in the pear dormancy release process.

Transcription factor PbNAC71 regulates xylem and vessel development to control plant height.

Dwarfism is an important agronomic trait in fruit breeding programs. However, the germplasm resources required to generate dwarf pear (Pyrus spp.) varieties are limited. Moreover, the mechanisms underlying dwarfism remain unclear. In this study, "Yunnan" quince (Cydonia oblonga Mill.) had a dwarfing effect on "Zaosu" pear. Additionally, the dwarfism-related NAC transcription factor gene PbNAC71 was isolated from pear trees comprising "Zaosu" (scion) grafted onto "Yunnan" quince (rootstock). Transgenic Nicotiana benthamiana and pear OHF-333 (Pyrus communis) plants overexpressing PbNAC71 exhibited dwarfism, with a substantially smaller xylem and vessel area relative to the wild-type controls. Yeast one-hybrid, dual-luciferase, chromatin immunoprecipitation-qPCR, and electrophoretic mobility shift assays indicated that PbNAC71 downregulates PbWalls are thin 1 expression by binding to NAC-binding elements in its promoter. Yeast two-hybrid assays showed that PbNAC71 interacts with the E3 ubiquitin ligase PbRING finger protein 217 (PbRNF217). Furthermore, PbRNF217 promotes the ubiquitin-mediated degradation of PbNAC71 by the 26S proteasome, thereby regulating plant height as well as xylem and vessel development. Our findings reveal a mechanism underlying pear dwarfism and expand our understanding of the molecular basis of dwarfism in woody plants.

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.

Self S-RNase inhibits ABF-LRX signaling to arrest pollen tube growth to achieve self-incompatibility in pear.

Gametophytic self-incompatibility (GSI) has been widely studied in flowering plants, but studies of the mechanisms underlying pollen tube growth arrest by self S-RNase in GSI species are limited. In the present study, two leucine-rich repeat extensin genes in pear (Pyrus bretschneideri), PbLRXA2.1 and PbLRXA2.2, were identified based on transcriptome and quantitative real-time PCR analyses. The expression levels of these two LRX genes were significantly higher in the pollen grains and pollen tubes of the self-compatible cultivar 'Jinzhui' (harboring a spontaneous bud mutation) than in those of the self-incompatible cultivar 'Yali'. Both PbLRXA2.1 and PbLRXA2.2 stimulated pollen tube growth and attenuated the inhibitory effects of self S-RNase on pollen tube growth by stabilizing the actin cytoskeleton and enhancing cell wall integrity. These results indicate that abnormal expression of PbLRXA2.1 and PbLRXA2.2 is involved in the loss of self-incompatibility in 'Jinzhui'. The PbLRXA2.1 and PbLRXA2.2 promoters were directly bound by the ABRE-binding factor PbABF.D.2. Knockdown of PbABF.D.2 decreased PbLRXA2.1 and PbLRXA2.2 expression and inhibited pollen tube growth. Notably, the expression of PbLRXA2.1, PbLRXA2.2, and PbABF.D.2 was repressed by self S-RNase, suggesting that self S-RNase can arrest pollen tube growth by restricting the PbABF.D.2-PbLRXA2.1/PbLRXA2.2 signal cascade. These results provide novel insight into pollen tube growth arrest by self S-RNase.

Multi-omics analyses reveal stone cell distribution pattern in pear fruit.

Stone cells are the brachysclereid cells in pear (Pyrus) fruit, consisting almost entirely of lignified secondary cell walls. They are distributed mainly near the fruit core and spread radially in the whole fruit. However, the development of stone cells has not been comprehensively characterized, and little is known about the regulation of stone cell formation at the transcriptomic, proteomic, and metabolomic levels. In the present study, we performed phenomic analysis on the stone cells and their associated vascular bundles distributed near the fruit cores. Transcriptomic, proteomic, and metabolomic analyses revealed a significant positive regulation of biological processes which contribute to the lignification and lignin deposition in stone cells near the fruit core, including sucrose metabolism and phenylalanine, tyrosine, tryptophan, and phenylalanine biosynthesis. We found many metabolites generated from the phenylpropanoid pathway contributing to the cell wall formation of stone cells near the fruit core. Furthermore, we identified a key transcription factor, PbbZIP48, which was highly expressed near the fruit core and was shown to regulate lignin biosynthesis in stone cells. In conclusion, the present study provides insight into the mechanism of lignified stone cell formation near the pear fruit core at multiple levels.

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.

Natural variations in the PbCPK28 promoter regulate sugar content through interaction with PbTST4 and PbVHA-A1 in pear.

Soluble sugars play an important role in plant growth, development and fruit quality. Pear fruits have demonstrated a considerable improvement in sugar quality during their long history of selection. However, little is known about the underlying molecular mechanisms accompanying the changes in fruit sugar content as a result of selection by horticulturists. Here, we identified a calcium-dependent protein kinase (PbCPK28), which is located on LG15 and is present within a selective sweep region, thus linked to the quantitative trait loci for soluble solids. Association analysis indicates that a single nucleotide polymorphism-13 variation (SNP13T/C ) in the PbCPK28 regulatory region led to fructose content diversity in pear. Elevated expression of PbCPK28 resulted in significantly increased fructose levels in pear fruits. Furthermore, PbCPK28 interacts with and phosphorylates PbTST4, a proton antiporter, thereby coupling the sugar import into the vacuole with proton export. We demonstrated that residues S277 and S314 of PbTST4 are crucial for its function. Additionally, PbCPK28 interacts with and phosphorylates the vacuolar hydrogen proton pump PbVHA-A1, which could provide proton motive forces for PbTST4. We also found that the T11 and Y120 phosphorylation sites in PbVHA-A1 are essential for its function. Evolution analysis and yeast-two-hybrid results support that the CPK-TST/CPK-VHA-A regulatory network is highly conserved in plants, especially the corresponding phosphorylation sites. Together, our work identifies an agriculturally important natural variation and an important regulatory network, allowing genetic improvement of fruit sugar contents in pears through modulation of PbCPK28 expression and phosphorylation of PbTST4 and PbVHA-A1.

Transcriptome analysis reveals that PbMYB61 and PbMYB308 are involved in the regulation of lignin biosynthesis in pear fruit stone cells.

Pear fruit stone cells have thick walls and are formed by the secondary deposition of lignin in the primary cell wall of thin-walled cells. Their content and size seriously affect fruit characteristics related to edibility. To reveal the regulatory mechanism underlying stone cell formation during pear fruit development and to identify hub genes, we examined the stone cell and lignin contents of 30 'Shannongsu' pear flesh samples and analyzed the transcriptomes of 15 pear flesh samples collected at five developmental stages. On the basis of the RNA-seq data, 35 874 differentially expressed genes were detected. Additionally, two stone cell-related modules were identified according to a WGCNA. A total of 42 lignin-related structural genes were subsequently obtained. Furthermore, nine hub structural genes were identified in the lignin regulatory network. We also identified PbMYB61 and PbMYB308 as candidate transcriptional regulators of stone cell formation after analyzing co-expression networks and phylogenetic relationships. Finally, we experimentally validated and characterized the candidate transcription factors and revealed that PbMYB61 regulates stone cell lignin formation by binding to the AC element in the PbLAC1 promoter to upregulate expression. However, PbMYB308 negatively regulates stone cell lignin synthesis by binding to PbMYB61 to form a dimer that cannot activate PbLAC1 expression. In this study, we explored the lignin synthesis-related functions of MYB family members. The results presented herein are useful for elucidating the complex mechanisms underlying lignin biosynthesis during pear fruit stone cell development.

Identification and expression analysis of ATP-binding cassette (ABC) transporters revealed its role in regulating stress response in pear (Pyrus bretchneideri).

BACKGROUND: ATP-binding cassette (ABC) transporter proteins constitute a plant gene superfamily crucial for growth, development, and responses to environmental stresses. Despite their identification in various plants like maize, rice, and Arabidopsis, little is known about the information on ABC transporters in pear. To investigate the functions of ABC transporters in pear development and abiotic stress response, we conducted an extensive analysis of ABC gene family in the pear genome. RESULTS: In this study, 177 ABC transporter genes were successfully identified in the pear genome, classified into seven subfamilies: 8 ABCAs, 40 ABCBs, 24 ABCCs, 8 ABCDs, 9 ABCEs, 8 ABCFs, and 80 ABCGs. Ten motifs were common among all ABC transporter proteins, while distinct motif structures were observed for each subfamily. Distribution analysis revealed 85 PbrABC transporter genes across 17 chromosomes, driven primarily by WGD and dispersed duplication. Cis-regulatory element analysis of PbrABC promoters indicated associations with phytohormones and stress responses. Tissue-specific expression profiles demonstrated varied expression levels across tissues, suggesting diverse functions in development. Furthermore, several PbrABC genes responded to abiotic stresses, with 82 genes sensitive to salt stress, including 40 upregulated and 23 downregulated genes. Additionally, 91 genes were responsive to drought stress, with 22 upregulated and 36 downregulated genes. These findings highlight the pivotal role of PbrABC genes in abiotic stress responses. CONCLUSION: This study provides evolutionary insights into PbrABC transporter genes, establishing a foundation for future research on their functions in pear. The identified motifs, distribution patterns, and stress-responsive expressions contribute to understanding the regulatory mechanisms of ABC transporters in pear. The observed tissue-specific expression profiles suggest diverse roles in developmental processes. Notably, the significant responses to salt and drought stress emphasize the importance of PbrABC genes in mediating adaptive responses. Overall, our study advances the understanding of PbrABC transporter genes in pear, opening avenues for further investigations in plant molecular biology and stress physiology.

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.

Genome-wide identification of GA2ox genes family and analysis of PbrGA2ox1-mediated enhanced chlorophyll accumulation by promoting chloroplast development in pear.

BACKGROUND: Chlorophyll (Chl) is an agronomic trait associated with photosynthesis and yield. Gibberellin 2-oxidases (GA2oxs) have previously been shown to be involved in Chl accumulation. However, whether and how the PbrGA2ox proteins (PbrGA2oxs) mediate Chl accumulation in pear (Pyrus spp.) is scarce. RESULTS: Here, we aimed to elucidate the role of the pear GA2ox gene family in Chl accumulation and the related underlying mechanisms. We isolated 13 PbrGA2ox genes (PbrGA2oxs) from the pear database and identified PbrGA2ox1 as a potential regulator of Chl accumulation. We found that transiently overexpressing PbrGA2ox1 in chlorotic pear leaves led to Chl accumulation, and PbrGA2ox1 silencing in normal pear leaves led to Chl degradation, as evident by the regreening and chlorosis phenomenon, respectively. Meanwhile, PbrGA2ox1-overexpressing (OE) tobacco plants discernably exhibited Chl built-up, as evidenced by significantly higher Pn and Fv/Fm. In addition, RNA sequencing (RNA-seq), physiological and biochemical investigations revealed an increase in abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) concentrations and signaling pathways; a marked elevation in reducing and soluble sugar contents; and a marginal decline in the starch and sucrose levels in OE plants. Interestingly, PbrGA2ox1 overexpression did not prominently affect Chl synthesis. However, it indeed facilitated chloroplast development by increasing chloroplast number per cell and compacting the thylakoid granum stacks. These findings might jointly contribute to Chl accumulation in OE plants. CONCLUSION: Overall, our results suggested that GA2oxs accelerate Chl accumulation by stimulating chloroplast development and proved the potential of PbrGA2ox1 as a candidate gene for genetically breeding biofortified pear plants with a higher yield.

Determination of anthracnose (Colletotrichum fructicola) resistance mechanism using transcriptome analysis of resistant and susceptible pear (Pyrus pyrifolia).

BACKGROUND: Anthracnose, mainly caused by Colletotrichum fructicola, leads to severe losses in pear production. However, there is limited information available regarding the molecular response to anthracnose in pears. RESULTS: In this study, the anthracnose-resistant variety 'Seli' and susceptible pear cultivar 'Cuiguan' were subjected to transcriptome analysis following C. fructicola inoculation at 6 and 24 h using RNA sequencing. A total of 3186 differentially expressed genes were detected in 'Seli' and 'Cuiguan' using Illumina sequencing technology. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the transcriptional response of pears to C. fructicola infection included responses to reactive oxygen species, phytohormone signaling, phenylpropanoid biosynthesis, and secondary metabolite biosynthetic processes. Moreover, the mitogen-activated protein kinase (MAPK) signaling pathway and phenylpropanoid biosynthesis were involved in the defense of 'Seli'. Furthermore, the gene coexpression network data showed that genes related to plant-pathogen interactions were associated with C. fructicola resistance in 'Seli' at the early stage. CONCLUSION: Our results showed that the activation of specific genes in MAPK, calcium signaling pathways and phenylpropanoid biosynthesis was highly related to C. fructicola resistance in 'Seli' and providing several potential candidate genes for breeding anthracnose-resistant pear varieties.

Chromosome-level genome provides new insight into the overwintering process of Korla pear (Pyrus sinkiangensis Yu).

Korla pear has a unique taste and aroma and is a breeding parent of numerous pear varieties. It is susceptible to Valsa mali var. pyri, which invades bark wounded by freezing injury. Its genetic relationships have not been fully defined and could offer insight into the mechanism for freezing tolerance and disease resistance. We generated a high-quality, chromosome-level genome assembly for Korla pear via the Illumina and PacBio circular consensus sequencing (CCS) platforms and high-throughput chromosome conformation capture (Hi-C). The Korla pear genome is ~ 496.63 Mb, and 99.18% of it is assembled to 17 chromosomes. Collinearity and phylogenetic analyses indicated that Korla might be derived from Pyrus pyrifolia and that it diverged ~ 3.9-4.6 Mya. During domestication, seven late embryogenesis abundant (LEA), two dehydrin (DHN), and 54 disease resistance genes were lost from Korla pear compared with P. betulifolia. Moreover, 21 LEA and 31 disease resistance genes were common to the Korla pear and P. betulifolia genomes but were upregulated under overwintering only in P. betulifolia because key cis elements were missing in Korla pear. Gene deletion and downregulation during domestication reduced freezing tolerance and disease resistance in Korla pear. These results could facilitate the breeding of novel pear varieties with high biotic and abiotic stress resistance.

Transcriptome and metabolome profiling reveal the effects of hormones on current-year shoot growth in Chinese 'Cuiguan' pear grafted onto vigorous rootstock 'Duli' and dwarf rootstock 'Quince A'.

BACKGROUND: Dwarf rootstocks have important practical significance for high-density planting in pear orchards. The shoots of 'Cuiguan' grafted onto the dwarf rootstock were shorter than those grafted onto the vigorous rootstock. However, the mechanism of shorter shoot formation is not clear. RESULTS: In this study, the current-year shoot transcriptomes and phytohormone contents of 'CG‒QA' ('Cuiguan' was grafted onto 'Quince A', and 'Hardy' was used as interstock) and 'CG‒DL' ('Cuiguan' was grafted onto 'Duli', and 'Hardy' was used as interstock) were compared. The transcriptome results showed that a total of 452 differentially expressed genes (DEGs) were identified, including 248 downregulated genes and 204 upregulated genes; the plant hormone signal transduction and zeatin biosynthesis pathways were significantly enriched in the top 20 KEGG enrichment terms. Abscisic acid (ABA) was the most abundant hormone in 'CG‒QA' and 'CG‒DL'; auxin and cytokinin (CTK) were the most diverse hormones; additionally, the contents of ABA, auxin, and CTK in 'CG‒DL' were higher than those in 'CG‒QA', while the fresh shoot of 'CG‒QA' accumulated more gibberellin (GA) and salicylic acid (SA). Metabolome and transcriptome co-analysis identified three key hormone-related DEGs, of which two (Aldehyde dehydrogenase gene ALDH3F1 and YUCCA2) were upregulated and one (Cytokinin oxidase/dehydrogenase gene CKX3) was downregulated. CONCLUSIONS: Based on the results of transcriptomic and metabolomic analysis, we found that auxin and CTK mainly regulated the shoot differences of 'CG-QA' and 'CG-DL', and other hormones such as ABA, GA, and SA synergistically regulated this process. Three hormone-related genes ALDH3F1, YUCCA2, and CKX3 were the key genes contributing to the difference in shoot growth between 'CG-QA' and 'CG-DL' pear. This research provides new insight into the molecular mechanism underlying shoot shortening after grafted onto dwarf rootstocks.

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.