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Recent evidence indicated that interspecific hybridization was the major mode of evolution in Pyrus. The genetic relationships and origins of the Asian pear are still unclear because of frequent hybrid events, fast radial evolution, and lack of informative data. Here, we developed fluorescent sequence-specific amplification polymorphism (SSAP) markers with lots of informative sites and high polymorphism to analyze the population structure among 93 pear accessions, including nearly all species native to Asia. Results of a population structure analysis indicated that nearly all Asian pear species experienced hybridization, and originated from five primitive genepools. Four genepools corresponded to four primary Asian species: P. betulaefolia, P. pashia, P. pyrifolia, and P. ussuriensis. However, cultivars of P. ussuriensis were not monophyletic and introgression occurred from P. pyrifolia. The specific genepool detected in putative hybrids between occidental and oriental pears might be from occidental pears. The remaining species, including P. calleryana, P. xerophila, P. sinkiangensis, P. phaeocarpa, P. hondoensis, and P. hopeiensis in Asia, were inferred to be of hybrid origins and their possible genepools were identified. This study will be of great help for understanding the origin and evolution of Asian pears.

Pear ( Pyrus L.) is a significant commercial fruit globally, with diverse species exhibiting variations in their flowering periods due to environmental factors. CONSTANS-like ( COL ) genes, known from previous studies in Arabidopsis , are key regulators of flowering time by sensing photoperiod. However, the evolutionary history and functions of COL genes in different pear species remain unclear. In this study, we identified a total of 79 COL genes in different pear species, including 12 COL genes in Pyrus bretschneideri ‘DangshanSuli’, 9 in Pyrus ussuriensis  ×  hybrid ‘Zhongai 1’, 11 in Pyrus communis ‘Bartlett’, 13 in Pyrus betulifolia , 18 in Pyrus pyrifolia ‘Cuiguan’, 16 in Pyrus pyrifolia ‘Nijisseiki’. Analysis of gene structure, phylogenetic tree, and multiple sequences provided valuable insights into the fundamental understanding of COL genes in pear. The impact of selection pressure on the PbrCOLs in Chinese white pear was assessed using Ka / Ks , revealing that the evolution rate of PbrCOLs was influenced by purification selection factors. The study also revealed different tissue-specific expression patterns of PbrCOLs under varying light quality. Real-time quantitative PCR revealed that under natural light conditions, the expression patterns of PbrCOL2 , PbrCOL3 , and PbrCOL4 are similar to previous studies on CONSTANS gene in Arabidopsis , with increased expression levels during the day and decreased levels at night. However, PbrCOL1 , PbrCOL6 , and PbrCOL9 exhibit different expression patterns, with decreased expression levels both during the day and at night. After red light treatment, high expression of PbrCOL3 and PbrCOL4 was observed at night, while the expression patterns of the other four genes did not show significant changes. Following blue light treatment, the expression peaks of PbrCOL1 and PbrCOL6 occurred during the night, showing opposite expression patterns compared to the study in Arabidopsis . The overexpression of PbrCOL3 significantly increase the chlorophyll content in pear seedlings, and its expression significantly affected the expression of other key flowering-related genes. Also, overexpression of PbrCOL3 resulted in a late-flowering phenotype in Arabidopsis. These findings indicate diverse responsive mechanisms and functions of PbrCOL genes on flowering time in pear. In conclusion, this study established a foundation for a deeper understanding of the specific roles of PbrCOLs in regulating the reproductive development of pear, particularly in the context of the photoperiodic flowering process.

Drought is one of the crucial abiotic stresses which affects growth, development, and performance of pear trees. This research was performed to investigate responses of five pear species including Pyrus communis L., Pyrus boissieriana Bushe., Pyrus glabra Boiss., Pyrus syriaca Boiss., and Pyrus salicifolia Pall. to different levels of drought stress. The potted trees were irrigated with water volume of 100%, 60%, or 30% of field capacity (FC) during 90 days. Based on the visual observation, the plant growth was restricted by severe drought in all species. Malondialdehyde (MDA) and glycine betaine (GB) contents, as well as the ascorbate peroxidase (APX), guaiacol peroxidase (GPX) and catalase (CAT) activities were indicated an uprising behavior under drought stress. Principal component analysis (PCA) analysis revealed P. glabra as tolerant and P. communis as sensitive to drought stress. The expression rates of stress-responsive transcription factors (TFs); WRKY29 and DREB6 and their responding genes, including LEA29 and Dehydrin1 , were analyzed in the two differentially drought-responding pear species i.e., P. glabra and P. communis . The expression of the studied TFs was induced when both species were exposed to higher drought levels. The species P . glabra exhibited better osmoregulation, antioxidant response, and higher up-regulation of WRKY29 , DREB6 , LEA29 and Dehydrin1 . In conclusion, among the studied pear species, P. glabra could best tolerate drought stress by boosting protective mechanisms.

The fruit of the 'Dangshansuli' pear has a greenish yellow skin, whereas its mutant, the 'Xiusu' pear, has a russet skin, which represents a genetic variation. It has been demonstrated that the formation of russet fruit in the 'Xiusu' pear is related to lignin accumulation in skin exocarp cells. In this study, we localized hydrogen peroxide (H sub(2)O sub(2)) to the cell wall using transmission electron microscopy (TEM) and quantified the concentrations of H sub(2)O sub(2) and polyamines. In addition, the expression levels of genes involved in polyamine biosynthesis were measured in the exocarps of samples of young fruits of 'Dangshansuli', 'Xiusu', 'Xiusu' treated with methylglyoxal bis(guanylhydrazone), and 'Xiusu' treated with ethephon. The results obtained could explain the mechanism by which H sub(2)O sub(2) participates in polyamine metabolism in the lignification of exocarp cells in the russet fruit mutant. The TEM results showed that free H sub(2)O sub(2) is present near the cell wall, where lignin is primarily synthesized, and the H sub(2)O sub(2) concentration was highly positively correlated with the lignin concentration. Although H sub(2)O sub(2) related to lignification showed no significant correlation with the putrescine or spermine concentration, it was highly positively correlated with the spermidine (Spd) concentration. Additionally, the Spd concentration was significantly positively correlated with altered expression of the polyamine oxidase gene (PbPAO). Taken together, these results have demonstrated that H sub(2)O sub(2) involved in lignification originates from the oxidation of Spd by the enzyme PAO, with high expression of the PbPAO gene, which suggests that H sub(2)O sub(2) from polyamine metabolism affects lignification in the exocarp of the russet mutant pear.

Key messagePp4ERF24 and Pp12ERF96 fine tune blue light-induced anthocyanin biosynthesis via interacting with PpMYB114 and promoting the interaction between PpMYB114 and PpbHLH3, which enhances the expression of PpMYB114-induced PpUFGT.The red coloration of pear fruit is attributed to anthocyanin accumulation, which is transcriptionally regulated by the MYB-bHLH-WD40 complex. A number of ethylene response factors (ERF) have been identified to regulate anthocyanin biosynthesis in different plants. In pear, several ERF transcription factor genes were identified to be potentially involved in the light-induced anthocyanin biosynthesis according to transcriptome data. But the molecular mechanism of these ERFs underlying the regulation of anthocyanin accumulation is unknown. In this study, exposure of ‘Red Zaosu’ pear, a mutant of ‘Zaosu’ pear, to blue light significantly induced the anthocyanin accumulation by increasing the expression levels of anthocyanin biosynthetic genes. Gene expression analysis confirmed that the expression of Pp4ERF24 and Pp12ERF96 genes were up-regulated in the process of blue light-induced anthocyanin biosynthesis. Yeast two-hybrid and bimolecular fluorescence complementation assay revealed that Pp4ERF24 and Pp12ERF96 interacted with PpMYB114, but not with PpMYB10. Bimolecular fluorescence complementation assay demonstrated that the interaction between these two ERFs and PpMYB114 enhanced the interaction between PpMYB114 and PpbHLH3. Further analysis by dual luciferase assay verified that these two ERFs increased the up-regulation of PpMYB114-mediated PpUFGT expression. Furthermore, co-transformation of Pp12ERF96 with PpMYB114 and PpbHLH3 in tobacco leaves led to enhanced anthocyanin accumulation. Transient overexpression of Pp4ERF24 or Pp12ERF96 alone in ‘Red Zaosu’ pear fruit also induced anthocyanin biosynthesis in pear peel. Our findings provide insights into a mechanism involving the synergistic interaction of ERFs with PpMYB114 to regulate light-dependent coloration and anthocyanin biosynthesis in pear fruits.

Summary 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.

Peel color is an economically important characteristic that influences the appearance quality of red pear, whose red color is due to anthocyanin accumulation. The process of coloration in the fruit peel is strongly influenced by light. However, how light quality influences color development remains unclear. In this study, we analyzed the effects of different light qualities on color development in the red pear ‘Red Zaosu’, a mutant of the hybrid cultivar ‘Zaosu’ of Pyrus pyrifolia and P. communis. The results showed that blue light increased anthocyanin accumulation after 72 h of light treatment, while red light had almost no effect. The expression of anthocyanin biosynthesis-related genes showed a similar trend to the anthocyanin accumulation. To clarify the mechanism of blue-light induced coloration, PpCRYs, PpCOP1 and PpHY5 genes were cloned. Gene expression analysis showed that their transcript abundance did not correlate with the expression of anthocyanin-related genes or anthocyanin content, but the yeast two-hybrid system revealed conserved physical interactions among these proteins. In addition, PpHY5 directly bound to the promoters of the anthocyanin biosynthesis genes PpCHS, PpDFR, PpANS and PpMYB10, and activated the transcription of PpCHS in a Nicotiana benthamiana-based dual-luciferase assay. In summary, our results preliminarily revealed that the conserved blue light signal transduction module CRY–COP1–HY5 contributed to the anthocyanin biosynthesis induced by blue light in red pear. However, our results did not provide evidence for why red light had no effect on anthocyanin accumulation, which needs further study.

Stone cells negatively affect fruit quality because of their firm and lignified cell walls, so are targets for reduction in pear breeding programmes. However, there is only limited knowledge of the molecular mechanisms underlying the formation of stone cells. Here, we show that PbrMYB169, an R2R3 MYB transcription factor, of Pyrus bretschneideri positively regulates lignification of stone cells in pear fruit. PbrMYB169 was shown to be co-expressed with lignin biosynthesis genes during pear fruit development, and this co-expression pattern was coincident with stone cell formation in the fruit of Pyrus bretschneideri ‘Dangshansuli’. The PbrMYB169 expression level was also positively correlated with stone cell content in 36 pear cultivars tested. PbrMYB169 protein significantly activated the promoter of lignin genes C3H1, CCR1, CCOMT2, CAD, 4CL1, 4CL2, HCT2, and LAC18 via binding to AC elements [ACC(T/A)ACC] in these promoters. Furthermore, overexpression of PbrMYB169 in transgenic Arabidopsis plants enhanced the expression of lignin genes, and increased lignin deposition and cell wall thickness of vessel elements, but did not change the ratio of syringyl and guaiacyl lignin monomers. In conclusion, PbrMYB169 appears to be a transcriptional activator of lignin biosynthesis and regulates secondary wall formation in fruit stone cells. This study advances the understanding of the regulation of lignin biosynthesis and provides valuable molecular genetic information for reducing stone cell content in pear fruit.

Summary Lignified stone cells substantially reduce fruit quality. Therefore, it is desirable to inhibit stone cell development using genetic technologies. However, the molecular mechanisms regulating lignification are poorly understood in fruit stone cells. In this study, we have shown that microRNA (miR) miR397a regulates fruit cell lignification by inhibiting laccase (LAC) genes that encode key lignin biosynthesis enzymes. Transient overexpression of PbrmiR397a, which is the miR397a of Chinese pear (Pyrus bretschneideri), and simultaneous silencing of three LAC genes reduced the lignin content and stone cell number in pear fruit. A single nucleotide polymorphism (SNP) identified in the promoter of the PbrmiR397a gene was found to associate with low levels of fruit lignin, after analysis of the genome sequences of sixty pear varieties. This SNP created a TCA element that responded to salicylic acid to induce gene expression as confirmed using a cell‐based assay system. Furthermore, stable overexpression of PbrmiR397a in transgenic tobacco plants reduced the expression of target LAC genes and decreased the content of lignin but did not change the ratio of syringyl‐ and guaiacyl‐lignin monomers. Consistent with reduction in lignin content, the transgenic plants showed fewer numbers of vessel elements and thinner secondary walls in the remaining elements compared to wild‐type control plants. This study has advanced our understanding of the regulation of lignin biosynthesis and provided useful molecular genetic information for improving pear fruit quality.

Summary MYB comprises a large family of transcription factors that play significant roles in plant development and stress response in plants. However, knowledge concerning the functions of MYBs and the target genes remains poorly understood. Here, we report the identification and functional characterization of a novel stress‐responsive MYB gene from Pyrus betulaefolia. The MYB gene, designated as PbrMYB21, belongs to the R2R3‐type and shares high degree of sequence similarity to MdMYB21. The transcript levels of PbrMYB21 were up‐regulated under various abiotic stresses, particularly dehydration. PbrMYB21 was localized in the nucleus with transactivation activity. Overexpression of PbrMYB21 in tobacco conferred enhanced tolerance to dehydration and drought stresses, whereas down‐regulation of PbrMYB21 in Pyrus betulaefolia by virus‐induced gene silencing (VIGS) resulted in elevated drought sensitivity. Transgenic tobacco exhibited higher expression levels of ADC (arginine decarboxylase) and accumulated larger amount of polyamine in comparison with wild type (WT). VIGS of PbrMYB21 in Pyrus betulaefolia down‐regulated ADC abundance and decreased polyamine level, accompanied by compromised drought tolerance. The promoter region of PbrADC contains one MYB‐recognizing cis‐element, which was shown to be interacted with PbrMYB21, indicating the ADC may be a target gene of PbrMYB21. Take together, these results demonstrated that PbrMYB21 plays a positive role in drought tolerance, which may be, at least in part, due to the modulation of polyamine synthesis by regulating the ADC expression.