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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 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.)

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.)

Pear [Pyrus bretschneideri cv. Dangshan Su] fruit quality is not always satisfactory owing to the presence of stone cells, and lignin is the main component of stone cells in pear fruits. Caffeoyl shikimate esterase (CSE) is a key enzyme in the lignin biosynthesis. Although CSE-like genes have been isolated from a variety of plant species, their orthologs are not characterized in pear. In this study, the CSE gene family (PbCSE) from P. bretschneideri was identified. According to the physiological data and quantitative RT-PCR (qRT-PCR), PbCSE1 was associated with lignin deposition and stone cell formation. The overexpression of PbCSE1 increased the lignin content in pear fruits. Relative to wild-type (WT) Arabidopsis, the overexpression of PbCSE1 delayed growth, increased the lignin deposition and lignin content in stems. Simultaneously, the expression of lignin biosynthetic genes were also increased in pear fruits and Arabidopsis. These results demonstrated that PbCSE1 plays an important role in cell lignification and will provide a potential molecular strategy to improve the quality of pear fruits.

SUMMARY: 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. Significance Statement: We provide new insights into understanding the mechanism of lignified stone cell formation near the pear fruit core at multi‐omics levels. [ABSTRACT FROM AUTHOR]

Light environment is an indispensable factor that regulates multitudinous developmental processes during the whole life cycle of plants, including fruit development. Stone cells which negatively influence pear fruit quality because of their strongly lignified cell wall are also affected by light, however, how light qualities influence lignin biosynthesis in pear remains unclear. Here, the calli of European pear (Pyrus communis L.) treated with different lights were used to explore the changes in phenotype, lignin content, and H 2 O 2 content, coupled with RNA-Seq and quantitative real-time PCR (qRT-PCR) to investigate the possible regulation pathway of light on lignin biosynthesis in stone cells. Results showed that blue light increased the expression of lignin structure genes and promoted lignin accumulation. Besides, four blue light receptors cryptochromes (CRYs) were identified in white pear, named PbCRY1a (Pbr024556.1), PbCRY1b (Pbr001636.3), PbCRY2a (Pbr023037.1), and PbCRY2b (Pbr002655.4). qRT-PCR analysis showed that PbCRY1a is highly expressed in cultivars with a high content of stone cells. Furthermore, the molecular function of PbCRY1a on stone cell formation in pear fruit was demonstrated by genetic transformation of pear calli and Agrobacterium -mediated transient overexpression in pear fruitlets. Co-expression network analyses with RNA-seq data showed that 8 MYB and 5 NAC genes were classified into different co-expression clusters with lignin biosynthesis genes under blue light conditions. These results indicate that CRY-mediated blue-light signal plays an important role in cell wall lignification and promotes the formation of stone cells in pear by regulating downstream genes. • Blue light-irradiation positively regulated lignification in pear calli. • Four blue light receptors cryptochromes (CRYs) were identified in white pear. PbCRY1a is highly expressed in cultivars with a high content of stone cells. • Overexpression of PbCRY1a contributes to lignin biosynthesis in stone cells of pear fruit. • PbMYB103 and PbNAC83 might be the key transcriptional hubs that regulate blue light-induced lignification. [ABSTRACT FROM AUTHOR]

• Endogenous GA 3 was found to be co-localized with lignified cells in pear fruits. • In total, 27 GA oxidase genes were identified in Chinese white pear. • PbGA3ox4 was identified as a key enzyme responsible for the biosynthesis of bioactive GA 3. • Overexpression of PbGA3ox4 in pear calli and Arabidopsis promoted GA 3 biosynthesis and induced lignification. Gibberellin (GA) is one of the most important phytohormones that regulates multiple developmental processes in plants, including fruit quality. Stone cell of pear is the key determinant negatively influencing fruit quality, because of the lignified cell walls. The effect of GA in pear stone cell formation is still unknown and the distribution of GA oxidases (GAoxs) genes in the pear (Pyrus bretschneideri) genome remains uncharacterized. In this study, stone cells and endogenous GA 3 were found to be co-localized in lignified cells by immunofluorescence localization analysis. Then, a comprehensive bioinformatics analysis of the GAox gene family in the pear genome was performed. Genome-wide identification of the PbGAoxs revealed 27 genes encoding PbGAox proteins, which could be divided into four subfamilies (GA20ox, C 19 -GA2ox, GA3ox, and C 20 -GA2ox). Cis-element analysis indicated that PbGAox genes might be involved in plant hormones and light signaling. Evolution and sequence alignment analysis indicated that PbGA3ox4 was responsible for the biosynthesis of bioactive GA 3. The biological function of PbGA3ox4 in promoting GA 3 biosynthesis and cell lignification has been illustrated by overexpression in pear fleshcalli and Arabidopsis. Collectively, this study laid a foundation for the functional analysis of the pear GAox family and provided valuable genetic resources for the improvement of pear fruit quality. [ABSTRACT FROM AUTHOR]