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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., (© 2024 John Wiley & Sons Ltd.)

Lignification of the cell wall in pear (Pyrus) fruit results in the formation of stone cells, which affects the texture and quality of the fruit. However, it is still unclear that how different transcription factors (TFs) work together to coordinate the synthesis and deposition of lignin. Here, we examined the transcriptome of pear varieties with different stone cell contents and found a key TF (PbAGL7) that can promote the increase of stone cell contents and secondary cell wall thicknesses. In addition, PbAGL7 can facilitate the expression level of lignin biosynthesis-related genes and accelerate the lignin biosynthesis in pear fruit and Arabidopsis. However, PbAGL7 did not directly bind to the promoters of PbC3H1 and PbHCT17 which are crucial genes involved in lignin biosynthesis. On the other hand, yeast two-hybrid (Y2H) library showed that PbNAC47 and PbMYB73 interacted with PbAGL7 in the nucleus. PbNAC47 and PbMYB73 also increased the stone cell and lignin contents, and upregulated the expressions of PbC3H1 and PbHCT17 by binding to the SNBE and AC elements, respectively. Moreover, PbNAC47 also interacted with PbMYB73 to form PbAGL7-PbNAC47-PbMYB73 complex. This complex significantly activated the expression levels of PbC3H1 and PbHCT17 and promoted lignin biosynthesis to form stone cells in pear fruit. Overall, our study provides new insights into the molecular mechanism of TFs that coordinately regulate the stone cell formation in pear fruit and extend our knowledge to understand cell wall lignification in plants., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Background: Flowering is a critical agronomic trait in fruit tree cultivation, essential for sexual reproduction and fruit yield. Circadian clock system, governing processes such as flowering, growth, and hormone signaling, plays a key role in plant adaptability. While some clock-related genes influencing pear flowering have been studied, the role of the PbELF4 (EARLY FLOWERING 4) family remains largely unexplored., Results: In this study, we identified five ELF4 homologous genes within the pear (Pyrus bretschneideri) genome. Phylogenetic analysis delineated two distinct groups within the PbELF4 genes, with PbELF4a and PbELF4b clustering with AtELF4. Expression profiling across various pear tissues revealed diverse expression patterns. Diurnal rhythms of PbELF4 genes were discernible in pear leaves, suggesting potential regulatory roles. Ectopic overexpression of PbELF4a and PbELF4b in Arabidopsis significantly delayed flowering and suppressed the expression of flowering-related genes. Additionally, PbELF4b overexpression induced premature leaf senescence, evidenced by reduced chlorophyll content and increased expression of senescence-associated genes. Nuclear localization of PbELF4a and PbELF4b proteins was observed, and interaction assays revealed that PbELF4a interacted with PbELF3α., Conclusions: These findings underscore the conserved function of PbELF4a and PbELF4b as negative regulators of flowering time, with PbELF4b also demonstrating a positive role in leaf senescence., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Autophagy is vital for plant defense against pathogens, with ATG6 being a key gene in this process. At present, little has been reported on the potential function and molecular mechanisms of ATG6 mediated pathogen resistance in pear. This study investigates the function of the pear homolog of ATG6 (PbrATG6) in resistance to Botryosphaeria dothidea. PbrATG6 is expressed differentially in pear tissues and its expression increases upon infection. Overexpression of PbrATG6 enhances resistance in Arabidopsis and pear calli, while silencing it increases susceptibility. PbrTLP15, a pathogenesis-related protein belonging to the PR5 family, was found that interacts with PbrATG6 by a yeast two-hybrid screening. Yeast two-hybrid, luciferase complementation imaging, bimolecular fluorescence complementation assays and pull-down assays showed that PbrATG6 interacts with PbrTLP15. The transient silencing transgenic assays of PbrATG6 and PbrTLP15 revealed that PbrATG6 could cooperate with PbrTLP15 to regulate pear B. dothidea resistance. In addition, transcriptional analyses of autophagy key genes in pTRV-PbrTLP15 and transmission electron microscopy (TEM) assays also implied that PbrTLP15 does affect autophagy. Hence, PbrATG6 and PbrTLP15 may synergistically enhance pear B. dothidea disease resistance. It provides a new strategy for the study of autophagy in pear disease resistance and enriches the research on pear disease resistance mechanism., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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., (© 2024. The Author(s).)

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., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

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., (© 2024. The Author(s).)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Stone cell, a type of lignified cell, is a unique trait in pear and one of the key factors affects pear fruit quality and economic value. The transmissibility of cell lignification process has been proven to exist, however the effects of callose on the permeability of plasmodesmata (PD) and how to influence cell lignification processes are still unknown. In this study, the genome-wide analysis of PD callose binding proteins (PDCB) gene family in pear genome was performed, and 25 PbPDCB genes were identified and divided into four branches. Similar intron/exon structural patterns were observed in the same branch, strongly supporting their close evolutionary relationship. The expression of PbPDCB16 was negatively correlated with lignin accumulation through qRT-PCR analysis. With transient expression in pear fruit and stable expression in pear calli, the increased callose content accompanied by decreased lignin content was further observed. Besides, compared with wild type Arabidopsis, the transgenic plants grew slowly, and cell walls in the stem were thinner, while fewer PDs were observed on the cell walls, and the interspore filaments were also blocked in transgenic Arabidopsis through the transmission electron microscope (TEM). In summary, overexpression of PbPDCB16 could promote accumulation of callose at PD to affect the PD-mediated intercellular connectivity, and inhibit the intercellular communication. This study will provide new insight in reducing the lignin content through callose deposition, and also provide the theoretical basis for further exploration of lignin metabolism and cell wall lignification to form stone cells in pear fruit., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Flowering is a prerequisite for pear fruit production. Therefore, the development of flower buds and the control of flowering time are important for pear trees. However, the molecular mechanism of pear flowering is unclear. SOC1, a member of MADS-box family, is known as a flowering signal integrator in Arabidopsis. We identified eight SOC1-like genes in Pyrus bretschneideri and analyzed their basic information and expression patterns. Some pear SOC1-like genes were regulated by photoperiod in leaves. Moreover, the expression patterns were diverse during the development of pear flower buds. Two members of the pear SOC1-like genes, PbSOC1d and PbSOC1g, could lead to early flowering phenotype when overexpressed in Arabidopsis. PbSOC1d and PbSOC1g were identified as activators of the floral meristem identity genes AtAP1 and AtLFY and promote flowering time. These results suggest that PbSOC1d and PbSOC1g are promoters of flowering time and may be involved in flower bud development in pear., Competing Interests: Declaration of Competing Interest The authors declare that they have no conflict of interest., (Copyright © 2019. Published by Elsevier Inc.)

Main Conclusion: PbrKAT1, which is inhibited by external Na + in Xenopus laevis oocytes, is characterized as encoding a typical inward rectifying channel that is mainly expressed in guard cells. Potassium (K + ) is the most abundant cation in plant cells necessary for plant growth and development. The uptake and transport of K + are mainly completed through transporters and channels, and the Shaker family genes are the most studied K + channels in plants. However, there is far less information about this family in Rosaceae species. We performed a genome-wide analysis and identified Shaker K + channel gene family members in Rosaceae. We cloned and characterized a Shaker K + channel KAT1 from pear (Pyrus × bretschneideri). In total, 36 Shaker K + channel genes were identified from Rosaceae species and were classified into five subgroups based on structural characteristics and a phylogenetic analysis. Whole-genome and dispersed duplications were the primary forces underlying Shaker K + channel gene family expansion in Rosaceae, and purifying selection played a key role in the evolution of Shaker K + channel genes. β-Glucuronidase and qRT-PCR assays revealed that PbrKAT1 was mainly expressed in leaves, especially in guard cells. PbrKAT1 displayed a typical inward-rectifying current when expressed in Xenopus laevis oocytes. The activity of PbrKAT1 was inhibited by external sodium ions, possibly playing an important role in the regulation of salt tolerance in pear. These results provide valuable information on evolution, expression and functions of the Shaker K + channel gene family in plants.

WRKY comprises a large family of transcription factors in plants, but most WRKY members are still poorly understood. In this study, we report the identification and functional characterization of PbrWRKY53 isolated from Pyrus betulaefolia. PbrWRKY53 was greatly up-regulated by drought and abscisic acid, but slightly induced by salt and cold. Subcellar localization analyses showed that PbrWRKY53 was located in the nucleus. Ectopic expression of PbrWRKY53 in tobacco and Pyrus ussuriensis conferred enhanced tolerance to drought stress. The transgenic plants exhibited better water status, less reactive oxygen species generation and higher levels of antioxidant enzyme activities and metabolites than the wild type. In addition, overexpression of PbrWRKY53 in transgenic tobacco resulted in enhanced expression level of PbrNCED1, and led to the increase in larger amount of vitamin C accumulation in comparison to WT. Knock-down of PbrWRKY53 in P. ussuriensis down-regulated PbrNCED1 abundance, accompanied by compromised drought tolerance. Yeast one-hybrid assay, EMSA and transient expression analysis demonstrated that PbrWRKY53 could bind to the W-box element in the promoter region of PbrNCED1. Taken together, these results demonstrated that PbrWRKY53 plays a positive role in drought tolerance, which might be, at least in part, promoting production of vitamin C via regulating PbrNCED1 expression., (© 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)