The PuWRKY29-PuMYB62 module responds to salicylic acid to inhibit the synthesis of stone cells in 'Nanguo' pear.

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.

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.

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.

The presence of stone cells in pear fruit, caused by lignified secondary cell walls (SCWs), leads to a grainy texture in the fruit flesh, thereby compromising its overall quality. Lignification is influenced by various environmental signals, including light, however the underlying mechanism are poorly understood. This study reveals that SCW thickening and lignin accumulation in stone cells were regulated by a blue light signal, mediated through the activation of PbNSC by PbbHLH195. The results revealed that the stone cell formation was prompted by supplementary with blue light, with lignin accumulation linked to the upregulation of the NAC STONE CELL PROMOTING FACTOR (PbNSC). PbbHLH195 was identified as a novel molecular hub connecting lignification to blue light signal through its physical interaction with PbCRY1a. The biochemical and functional analysis indicates that PbbHLH195 contributes to stone cell lignification by activating the promoter of PbNSC. Our findings offer novel insights into the mechanisms of lignin biosynthesis in response to blue light, identifying valuable genetic targets for enhancing the fruit quality of pear.

Cryptochrome-mediated blue-light signal contributes to lignin biosynthesis in stone cells in pear fruit

Lignified stone cell content is a key factor used to evaluate fruit quality, influencing the economic value of pear (Pyrus pyrifolia) fruits. However, our understanding of the regulatory networks of stone cell formation is limited due to the complex secondary metabolic pathway. In this study, we used a combination of co-expression network analysis, gene expression profiles, and transcriptome analysis in different pear cultivars with varied stone cell content to identify a hub MYB gene, PbrMYB24. The relative expression of PbrMYB24 in fruit flesh was significantly correlated with the contents of stone cells, lignin, and cellulose. We then verified the function of PbrMYB24 in regulating lignin and cellulose formation via genetic transformation in homologous and heterologous systems. We constructed a high-efficiency verification system for lignin and cellulose biosynthesis genes in pear callus. PbrMYB24 transcriptionally activated multiple target genes involved in stone cell formation. On the one hand, PbrMYB24 activated the transcription of lignin and cellulose biosynthesis genes by binding to different cis-elements [AC-I (ACCTACC) element, AC-II (ACCAACC) element and MYB-binding sites (MBS)]. On the other hand, PbrMYB24 bound directly to the promoters of PbrMYB169 and NAC STONE CELL PROMOTING FACTOR (PbrNSC), activating the gene expression. Moreover, both PbrMYB169 and PbrNSC activated the promoter of PbrMYB24, enhancing gene expression. This study improves our understanding of lignin and cellulose synthesis regulation in pear fruits through identifying a regulator and establishing a regulatory network. This knowledge will be useful for reducing the stone cell content in pears via molecular breeding.

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. The regulation of stone cell formation on transcriptomic, proteomic and metabolomic levels remains largely unknown. In this 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 significantly positive regulation of biological processes, which contributed to the lignification and lignin deposition in stone cells near the fruit core, including sucrose metabolism, phenylalanine, tyrosine, tryptophan, and phenylalanine biosynthesis. We then 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 significantly highly expressed near the fruit core and was then proven to regulate the lignin biosynthesis in stone cells. In conclusion, we provide new insights into understanding the mechanism of lignified stone cell formation near the pear fruit core at multi-omics levels.

PuRBOHF-PuPRX42-like complex activates lignin biosynthesis for stone cell formation in pear fruit.

Stone cells are one of the limiting factors affecting pear fruit quality and commodity value. The formation of stone cell is highly correlated with lignin deposition. However, the molecular mechanism of stone cell formation and regulation is still unclear. Here, we observed that exogenous application of GA significantly inhibited the formation of stone cells and also decreased the content of lignin in 'Nanguo' (Pyrus ussuriensis) pear fruits. The key gene PuPRX73 involved in the lignin synthesis pathway was further identified using RT-PCR, and GA-treatment significantly inhibited the expression of PuPRX73. Overexpression or silencing of PuPRX73 in pear fruits significantly increases or decreases the content of stone cells and lignin. We identified the transcription factors PuMYB91 and PuERF023 using mRNA-seq and their expression was significantly decreased after GA-treatment. Transient overexpression of PuMYB91 and PuERF023 promotes lignin and stone cells content in pear fruits, while silencing of PuMYB91 and PuERF023 led to the opposite results and inhibited the expression of PuPRX73. Yeast one-hybrid (Y1H) and GUS activity analysis revealed that PuMYB91 and PuERF023 directly bind and activate the PuPRX73 promoter, and co-transfection of PuMYB91 and PuERF023 in Nicotiana benthamiana leaves further promoted the promoter activity of PuPRX73. Furthermore, we found that PuMYB91 interacted with PuERF023 in vitro by using Yeast two-hybrid assays (Y2H). In conclusion, our results revealed that exogenous GA-treatment inhibits stone cell production by suppressing the expression of PuMYB91 and PuERF023 in pear fruits.

The deposition of lignin in flesh parenchyma cells for pear stone cells, and excessive stone cells reduce the taste and quality of the fruit. The effect of metaxenia on the quality of fruit has been heavily studied, but the effect of metaxenia on stone cell formation has not been fully elucidated to date. This study used P. bretschneideri (Chinese white pear) cv. ‘Yali’ (high-stone cell content) and P. pyrifolia (Sand pear) cv. ‘Cuiguan’ (low-stone cell content) as pollination trees to pollinate P. bretschneideri cv. ‘Lianglizaosu’ separately to fill this gap in the literature. The results of quantitative determination, histochemical staining and electron microscopy indicated that the content of stone cells and lignin in YL fruit (‘Yali’ (pollen parent) × ‘Lianglizaosu’ (seed parent)) was significantly higher than that in CL fruit (‘Cuiguan’ (pollen parent) × ‘Lianglizaosu’ (seed parent)). The transcriptome sequencing results that were obtained from the three developmental stages of the two types of hybrid fruits indicated that a large number of differentially expressed genes (DEGs) related to auxin signal transduction (AUX/IAAs and ARFs), lignin biosynthesis, and lignin metabolism regulation (MYBs, LIMs, and KNOXs) between the CL and YL fruits at the early stage of fruit development. Therefore, metaxenia might change the signal transduction process of auxin in pear fruit, thereby regulating the expression of transcription factors (TFs) related to lignin metabolism, and ultimately affecting lignin deposition and stone cell development. In addition, we performed functional verification of a differentially expressed gene, PbC4H2 (cinnamate 4-hydroxylase). Heterologous expression of PbC4H2 in the c4h mutant not only restored its collapsed cell wall, but also significantly increased the lignin content in the inflorescence stem. The results of our research help to elucidate the metaxenia-mediated regulation of pear stone cell development and clarify the function of PbC4H2 in cell wall development and lignin synthesis, which establishes a foundation for subsequent molecular breeding.

PbARF19-mediated auxin signaling regulates lignification in pear fruit stone cells

Gap-free genome assemblies of two Pyrus bretschneideri cultivars and GWAS analyses identify a CCCH zinc finger protein as a key regulator of stone cell formation in pear fruit.

BackgroundStone cells in fruits of pear (Pyrus pyrifolia) negatively influence fruit quality because their lignified cell walls impart a coarse and granular texture to the fruit flesh.ResultsWe generate RNA-seq data from the developing fruits of 206 pear cultivars with a wide range of stone cell contents and use a systems genetics approach to integrate co-expression networks and expression quantitative trait loci (eQTLs) to characterize the regulatory mechanisms controlling lignocellulose formation in the stone cells of pear fruits. Our data with a total of 35,897 expressed genes and 974,404 SNPs support the identification of seven stone cell formation modules and the detection of 139,515 eQTLs for 3229 genes in these modules. Focusing on regulatory factors and using a co-expression network comprising 39 structural genes, we identify PbrNSC as a candidate regulator of stone cell formation. We then verify the function of PbrNSC in regulating lignocellulose formation using both pear fruit and Arabidopsis plants and further show that PbrNSC can transcriptionally activate multiple target genes involved in secondary cell wall formation.ConclusionsThis study generates a large resource for studying stone cell formation and provides insights into gene regulatory networks controlling the formation of stone cell and lignocellulose.

The content and size of stone cell clusters affects the quality of pear fruit, and monolignol polymerization and deposition in the cell walls constitute a required step for stone cell formation. Laccase (LAC) is the key enzyme responsible for the polymerization of monolignols. However, there are no reports on the LAC family in pear (Pyrus bretschneideri), and the identity of the members responsible for lignin synthesis has not been clarified. Here, 41 LACs were identified in the whole genome of pear. All Pyrus bretschneideri LACs (PbLACs) were distributed on 13 chromosomes and divided into four phylogenetic groups (I-IV). In addition, 16 segmental duplication events were found, implying that segmental duplication was a primary reason for the expansion of the PbLAC family. LACs from the genomes of three Rosaceae species (Prunus mummer, Prunus persica, and Fragaria vesca) were also identified, and an interspecies collinearity analysis was performed. The phylogenetic analysis, sequence alignments and spatiotemporal expression pattern analysis suggested that PbLAC1, 5, 6, 29, 36 and 38 were likely associated with lignin synthesis and stone cell formation in fruit. The two target genes of Pyr-miR1890 (a microRNA identified from pear fruit that is associated with lignin and stone cell accumulation), PbLAC1 and PbLAC14, were selected for genetic transformation. Interfamily transfer of PbLAC1 into Arabidopsis resulted in a significant increase (approximately 17%) in the lignin content and thicker cell walls in interfascicular fibre and xylem cells, which demonstrated that PbLAC1 is involved in lignin biosynthesis and cell wall development. However, the lignin content and cell wall thickness were not changed significantly in the PbLAC14-overexpressing transgenic Arabidopsis plants. This study revealed the function of PbLAC1 in lignin synthesis and provides important insights into the characteristics and evolution of the PbLAC family.

[This corrects the article DOI: 10.1371/journal.pone.0210892.].