The PbrMADS1–PbrMYB169 complex has uniquely emerged to regulate lignification of stone cells in pear

ABSTRACT

Lignified stone cells are a unique feature of pear fruit, significantly affecting fruit texture. Even though some research efforts have already been made, the stone cell formation mechanism is complex, with many aspects yet to be elucidated. Here, through a genome‐wide association analysis of stone cell traits, we identified PbrMADS1, a member of the SEPALLATA3 (SEP3) subfamily, as a candidate gene specifically expressed in stone cells during early fruit development. Functional studies confirmed that PbrMADS1 promotes stone cell formation; however, it does not directly activate lignin‐related genes. Instead, PbrMADS1 interacts with PbrMYB169, enhancing PbrMYB169's binding to AC elements and amplifying downstream gene activation. Notably, homologous MADS1 and MYB169 proteins from closely related species such as apple and loquat do not form a similar complex. Sequence analysis revealed that the protein sequence of PbrMADS1 contains methionine (M) at the 63^rd amino acid position, while apple and loquat homologs carry threonine (T) at the same site. Substituting M with T (PbrMADS1^M63T) weakened its interaction with PbrMYB169 and impaired its function in regulating stone cell formation. This study offers new insights into MADS gene‐mediated stone cell formation and highlights functional divergence within the SEP3 subfamily among apple tribe species of the Rosaceae family.

Genome‐wide association study in pear identified a SEP3‐subfamily transcription factor, PbrMADS1, which interacts with PbrMYB169 to regulate stone cell lignification. The methionine at position 63 in PbrMADS1 was essential for this interaction. This amino acid variation may underlie the functional divergence of homologous SEP3 genes among Rosaceae species.

INTRODUCTION

Pear, a member of the Rosaceae family, is one of the most important temperate fruit crops worldwide, with China leading global production, accounting for 70% of both cultivation area and annual yield (Wu et al., 2013). Unlike other Rosaceae fruits, pears accumulate abundant stone cells, which cause a gritty flesh texture and significantly compromise fruit quality and commercial value (Choi et al., 2007). Stone cells are thick‐walled cells enriched with lignin and cellulose, formed through secondary wall thickening during early stages of fruit development (Tao et al., 2009). The initial formation of stone cell structures begins around 10–15 d after full bloom (DAFB), with the critical synthesis period occurring between 15 and 49 DAFB (Li et al., 2017b; Xue et al., 2019b).

Analysis of stone cell content in 236 Pyrus pyrifolia samples revealed a wide range (2.82% to 29.00%), confirming that stone cell formation is a quantitative trait regulated by multiple genes (Zhang et al., 2020). To explore the genetic basis of this trait, a genome‐wide association study (GWAS) was conducted in 312 P. pyrifolia cultivars, identifying a novel candidate gene, PbrSTONE, located on chromosome 11, which interacts with PbrC3H1 to regulate stone cell formation (Zhang et al., 2021). Subsequently, transcriptome sequencing of fruit flesh from 206 selected cultivars across key developmental stages, combined with gene co‐expression network and eQTL analyses, revealed transcription factors PbrNSC, PbrMYB169, and PbrMYB24 as major positive regulators of stone cell biosynthesis (Xue et al., 2019a, 2023; Wang et al., 2021). Additional transcription factors such as PbrMYB4, PbMYB61, and PbbZIP48 were found to promote lignin synthesis in stone cells (Gong et al., 2023; Zhu et al., 2023; Liu et al., 2025), while others, including PbMYB80, PbMYB308, PbrARF13, and PbARF19, act as inhibitors (Xu et al., 2023a; Zhu et al., 2023; Wang et al., 2024a, 2024b). Together, these studies suggest that stone cell formation is regulated by multiple genes and metabolic pathways. However, the precise regulatory network controlling this process remains largely unresolved.

The MADS‐box transcription factor family plays a crucial role in plant development and has been extensively studied. Based on evolutionary analysis, MADS‐box genes are classified into two major types: Type I and Type II (Alvarez‐Buylla et al., 2000). Among them, Type II MIKC^C‐type genes are the most widely researched and are grouped into 14 main subclades, including AGAMOUS (AG), AGL2 (SEP), AGL6, AGL12, AGL15, AGL17, AP3/PI, GGM13, SVP, AP1 (SQUA), FLC, TM3, and TM8 (Melzer et al., 2010). Early research on MIKC^C‐type genes primarily focused on their roles in floral organ development, leading to the well‐known “ABCDE” model, where AP1, AP3/PI, AG, AG/SHP, and SEP genes correspond to the A, B, C, D, and E functions, respectively (Coen and Meyerowitz, 1991; Pelaz et al., 2000).

More recently, MADS‐box genes have been implicated in fruit ripening and quality regulation. In apple, SEP1/2 subfamily genes MdMADS8, MdMADS9, and MdMADS5 regulate ethylene biosynthesis and fruit ripening (Ireland et al., 2013; Xu et al., 2023c). In banana, SEP3 subfamily genes MaMADS1 and MaMADS2 are essential for fruit ripening, with antisense inhibition or RNAi significantly delaying ripening and altering color development and softening (Elitzur et al., 2016). Similarly, in strawberry, silencing SEP genes FaMADS9 and FaMADS1a disrupts achene development and ripening (Lu et al., 2018; Vallarino et al., 2020). In peach, the SEP gene PrupeSEP1 has been validated to regulate ripening and softening processes (Li et al., 2017a). In pears, research on MADS‐box genes has predominantly focused on flower bud dormancy. Specifically, two transcriptome analyses of flower buds have provided a foundation for identifying dormancy‐related MADS‐box genes (Liu et al., 2012; Bai et al., 2013). Among these, PpDAM1, PpDAM2, and PpyDAM3 have been shown to be involved in the regulation of flower bud dormancy in pear (Li et al., 2019; Yang et al., 2020).

In addition to the MADS‐box genes that regulate reproductive organ development, a few MADS genes are also involved in lignin biosynthesis. In Arabidopsis, the MADS transcription factor AGL15 is involved in lignin synthesis by inhibiting the expression of PRX17 (Cosio et al., 2017), while SHP1 and SHP2 promoted valve margin lignification to regulate fruit dehiscence (Liljegren et al., 2000). In poplar, two TM8 subclade genes, VCM1 and VCM2, were identified as being specifically expressed in the vascular cambium (Zheng et al., 2021). In loquat fruit, the MADS transcription factor EjAGL65 negatively regulates flesh lignification by repressing the transcription of EjMYB8 (Ge et al., 2021), whereas EjAGL15 activates multiple lignin biosynthesis‐related genes to promote lignification (Ge et al., 2023). Moreover, in wheat, the AGL17 subclade MADS transcription factor TaMADS25 has been shown to interact with TaANR1, thereby regulating both lignin synthesis and root development simultaneously (Xu et al., 2023b). In pear, the AGL15 subfamily gene PbMADS49 was recently confirmed to regulate stone cell formation (Meng et al., 2025). Taken together, these findings indicate that members of the AGL15/17, AG/SHP, and TM8 subclades within the MADS‐box family are involved in either promoting or inhibiting lignin formation. However, direct regulation of lignin biosynthesis by members of other subclades has not yet been reported.

In this study, a SEP3 subfamily MADS gene, PbrMADS1, was identified through GWAS and validated as a positive regulator of lignin synthesis in pear fruits, pear callus, and Arabidopsis. Although PbrMADS1 could not directly activate the transcription of lignin biosynthesis genes, it enhanced the activation of downstream targets of PbrMYB169 by forming a protein complex, PbrMADS1–PbrMYB169. Interestingly, neither the protein complex nor the corresponding molecular mechanism has been detected in apple and loquat, two other Maleae species characterized by low stone cell content. This study reveals the specific regulatory network by which the SEP3 gene PbrMADS1 controls stone cell formation, offering new insights into the regulation of fruit quality in Rosaceae plants.

RESULTS

PbrMADS1 gene expression is correlated with the stone cell content of pear

Stone cells are quantitative traits regulated by multiple genes. To identify novel regulators of stone cell formation, we further performed GWAS analysis for this trait and found a strong association signal on one SNP (Chr13:5931787), with a search range extended to ± 150 kb, resulting in the identification of 27 genes within this region (Figure 1A, B). Subsequently, we performed co‐expression analysis between the identified genes and stone cell‐related structural genes (Wang et al., 2021). This analysis was carried out using the RNA‐Seq data that we previously generated for fruit development in five pear cultivars, namely, “Housui,” “Yali,” “Kuerlexiangli,” “Nanguoli,” and “Starkrimson” (Zhang et al., 2016). Pearson's correlation coefficient (PCC) values between gene pairs were filtered using a threshold of > 0.5 or < −0.5. Among the 27 candidate genes, nine showed a strong correlation with stone cell‐related genes, which suggests that these nine genes may be involved in stone cell development (Figure 1C; Table S1). Further expression analysis of the genes revealed that Pbr035643.1 was highly expressed during the early stages of pear fruit development, aligning with the known timing of stone cell formation, which annotated as a MADS transcription factor named PbrMADS1 (Wang et al., 2017). Real‐time quantitative PCR (RT‐qPCR) analysis across eight developmental stages of “Dangshansuli” fruits further confirmed that PbrMADS1 showed higher expression levels during early stages compared to later stages (Figure 1D). Additionally, RNA in situ hybridization showed that PbrMADS1 was specifically expressed within stone cells (Figure 1E). Collectively, the integration of GWAS and transcriptome analyses identified PbrMADS1 as a candidate gene associated with stone cell biosynthesis during early pear fruit development.

Figure 1.

PbrMADS1 is a potential regulator of stone cell formation in pear fruit

(A, B) Manhattan plot of GWAS for stone cell traits on chromosome 13. Manhattan plots and gene structure diagrams of the 1 Mb (A) and 150 kb (B) regions surrounding the significantly associated SNP site (Chr13:5931787). Pbr035643.1 is indicated in red. (C) The gene co‐expression network showed that 27 candidate genes were highly correlated with secondary wall biosynthesis genes. RNA‐seq data were obtained from seven different fruit development stages of five pear cultivars in our previous study (Zhang et al., 2016). (D) The relative expression level of PbrMADS1 in 8 developmental stages of “Dangshansuli” pear fruit was detected by RT‐qPCR. Each value was the mean (± SEM) of three technical repetitions. (E) RNA in situ hybridization showed that PbrMADS1 was specifically expressed in stone cells. “Dangshansuli” pear fruit at 15 DAFB were hybridized with antisense histological sections. Antisense: antisense probe; the scale bar is 200 μm. (F) The phylogenetic tree showed the clustering relationship between PbrMADS1, SEP‐like, and lignin synthesis‐related MADS transcription factors in different species. Different colors represent different branches. PbrMADS1 is highlighted in red, while green circles indicate MADS transcription factors associated with lignin biosynthesis. (G) Subcellular localization of a transgenic Arabidopsis root tip showed that the PbrMADS1–GFP fusion protein was expressed in the nucleus. Scale bars = 25 μm.

PbrMADS1 is a nuclear protein of the SEP3 branch

To predict the functional role of PbrMADS1, a phylogenetic tree was constructed using PbrMADS1 and other SEPALLATA(SEP)‐like MADs proteins. The analysis indicated that PbrMADS1 clustered closely with MaMADS1/2, belonging to the SEP3 branch (Figure 1F). To determine the subcellular localization of PbrMADS1, the CDS sequence of PbrMADS1 was ligated downstream of GFP under the control of the 35S promoter, generating a 35S::GFP‐PbrMADS1 fusion vector. This construct was stably transformed into Arabidopsis. Observation of root tip cells from transgenic Arabidopsis revealed that green fluorescence was specifically localized to the nucleus, demonstrating that PbrMADS1 is a nuclear protein (Figure 1G). These findings demonstrated that PbrMADS1 was a typical nuclear protein and a member of the SEP3 branch within the MADS‐box gene family.

PbrMADS1 promotes lignin biosynthesis in pear fruits and callus

To verify the function of PbrMADS1 in stone cell biosynthesis, PbrMADS1 overexpression and silencing constructs were transiently transformed into “Dangshansuli” pear fruit at 35 DAFB. After 7 d, compared with the control, a markedly stronger lignin staining signal was observed at the sites injected with the PbrMADS1 overexpression construct, whereas lignin staining was noticeably attenuated at the silent sites (Figure 2A). Quantitative analysis showed that overexpression of PbrMADS1 promoted lignin synthesis in pear pulp, while silencing PbrMADS1 produced the opposite effect (Figure 2B). Concomitantly, RT‐qPCR analysis revealed that changes in PbrMADS1 expression levels significantly affected the expression of genes related to lignin biosynthesis (Figure S1). These results suggested that PbrMADS1 promotes lignin accumulation in stone cells of pear fruit.

Figure 2.

The biological function of PbrMADS1 was verified by transient transformation of pear fruit and stable transformation of pear flesh callus and Arabidopsis thaliana

(A) The longitudinal sections of the young fruit of “Dangshansuli” with transient expression of PbrMADS1 were stained with Wiesner's reagent. The darker the color, the higher the lignin content in the stone cells. EV, empty vector; OE, overexpression; and VIGS, virus‐induced gene silencing. Scale bars = 0.5 cm. (B) Lignin content in the infiltrated regions of the fruit flesh depicted in (A) was quantified using the acetyl bromide method. *P < 0.05. Values are mean ± SEM of eight technical replicates. (C) WT and transgenic pear flesh callus were stained with Wiesner's reagent. WT, wild‐type; OE, overexpression; KO: knockout. Scale bars = 1 cm. (D) Lignin content in (C) was quantified using the acetyl bromide method. Each value is mean ± SEM (n ≥ 6 technical replicates). There were significant differences between groups in different letter representations (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test). (E) Cross‐sections of Arabidopsis inflorescence stems were stained with Toluidine blue O, Wiesner's reagent, and Mäule reagent. Scale bars = 200 μm. ve, vessel; xf, xylary fiber; and if, interfascicular fiber cell. (F) The acetyl bromide method was used to determine the lignin contents of Arabidopsis inflorescence stems in 8‐week‐old plants. Each value is mean ± SEM (n ≥ 12 technical replicates). Student's t‐test; *P < 0.05.

To further validate these transient transformation results, PbrMADS1 overexpression and knockout vectors were stably transformed into pear callus. RT‐qPCR analysis showed that PbrMADS‐OE calli had high expression levels, which were selected for phenotypic analysis (Figure S2A). Compared with WT callus, the PbrMADS1‐OE callus showed a stronger lignin staining signal and higher lignin content (Figure 2C, D). CRISPR/Cas9‐mediated PbrMADS1 knockout callus, containing 1 bp or 2 bp deletions at the target site, resulted in frameshift mutations in PbrMADS1 (Figure S2B). The mutant callus was subsequently subcultured and used for phenotype analysis. Wiesner staining revealed that lignin deposition was dramatically reduced in the PbrMADS1 knockout callus (Figure 2C), consistent with a significant reduction in lignin content (Figure 2D). Moreover, RT‐qPCR analysis demonstrated that alteration of PbrMADS1 expression levels correspondingly modulated the expression of lignin biosynthetic genes (Figure S2C). Collectively, these results demonstrated that PbrMADS1 promotes lignin accumulation and regulates lignin biosynthesis‐related genes in pear fruit and callus.

Overexpression of PbrMADS1 promotes lignin biosynthesis in transgenic Arabidopsis plants

To further clarify the role of PbrMADS1 in secondary cell wall (SCW) biosynthesis, three transgenic Arabidopsis lines (OE‐2, OE‐10, and OE‐15) with green fluorescence and high PbrMADS1 expression were selected for phenotypic and molecular analyses (Figure S3A). Morphologically, there were no significant differences in terms of flowering time, floral organ morphology, or silique development between the transgenic line and WT plants (Figure S3C–F). However, RT‐qPCR analysis revealed that overexpression of PbrMADS1 significantly upregulated the expression of lignin biosynthesis genes as well as MYB transcription factors involved in SCW formation, including AtMYB46, AtMYB58, AtMYB63, and AtMYB103 (Figure S3B).

Toluidine Blue O staining of the inflorescence stems showed that the fiber cell layer was notably thicker in the transgenic lines compared with WT (Figure 2E). Furthermore, Wiesner and Mäule staining demonstrated that lignin deposition was significantly enhanced in the interfascicular fibers and xylem tissues of the transgenic plants, and lignin accumulation was also observed in cortical cells, which are typically non‐lignified in WT plants (Figure 2E). Consistent with these histological observations, biochemical assays revealed that the lignin content was significantly higher in the transgenic plants than in WT (Figure 2F). Together, these results confirmed that PbrMADS1 positively regulates lignin biosynthesis and secondary cell wall formation in Arabidopsis.

PbrMADS1 rescues the SCW biosynthesis in the Arabidopsis nst1/nst3 mutant

The Arabidopsis nst1/nst3 mutant displays a phenotype of pendent inflorescence stems, resulting from the loss of SCW formation in interfascicular fiber cells and reduced SCW deposition in vessel cells (Mitsuda et al., 2007; Zhong and Ye, 2015). To investigate whether PbrMADS1 could rescue this phenotype, a construct expressing PbrMADS1 under the control of the AtNST3 promoter was generated and stably transformed into the nst1/nst3 mutant. Analysis of T₃ homozygous lines demonstrated that the expression of PbrMADS1 restored upright growth, thereby rescuing the pendent inflorescence stem phenotype (Figure 3A). Enhanced lignin staining and autofluorescence were observed in the interfascicular fiber cells of both WT and nst1/nst3‐PbrMADS1 plants, in contrast to the weak signals in nst1/nst3 mutants (Figure 3B). Restoration of lignin deposition correlated with the recovery of upright inflorescence stems (Figure 3C). These findings demonstrated that PbrMADS1 promotes lignin biosynthesis and SCW formation, rescuing the nst1/nst3 mutant phenotype.

Figure 3.

Phenotypic and transcriptome data analyses were conducted on Arabidopsis nst1/3 mutants complemented with PbrMADS1

(A) The 4‐week‐old Arabidopsis images showed that PbrMADS1 rescues the phenotype of the pendent inflorescence stem in the nst1/3 mutants. The expression of PbrMADS1 is driven by the AtNST3 promoter. WT, wild type as a control group. (B) The acetyl bromide method was used to determine the lignin contents of Arabidopsis inflorescence stems in 8‐week‐old plants. Each value is mean ± SEM (n ≥ 12 biological replicates). Lowercase letters indicate significant differences (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test). (C) Lignin detection in the inflorescence stem cross‐sections was performed using Wiesner staining, Mäule staining, and UV‐induced autofluorescence. Scale bars = 200 μm. ve, vessel; xf, xylary fiber; and if, interfascicular fiber cell. (D) GO enrichment analysis plot of DEGs. The circles from the outer to the inner represent: GO enrichment terms, number of genes, and color represents the −Log₁₀(P) size, with numbers indicating the total number of genes in that category in the whole genome. The proportion of up‐ and downregulated genes. RichFactor values, with the gray line representing a RichFactor value of 0.19. (E) Volcano plot of DEGs. Genes annotated in yellow, green, and blue represent the pathways of lignin biosynthesis and polymerization, cellulose biosynthesis and assembly, and transcriptional regulation, respectively. (F) Dual‐luciferase assay confirmed that PbrMADS1 activates the promoters of AtSND2 and AtSND3. All values are presented as mean ± SEM (n = 3 technical replicates). Statistical significance was assessed using Student's t‐test for P‐value (ns, not significant).

To elucidate the molecular mechanisms underlying the rescue effect of PbrMADS1, RNA‐seq was performed to compare genome‐wide mRNA expression profiles between nst1/nst3 mutants and PbrMADS1‐complemented lines. RNA was extracted from stems at 1 month after planting, with two biological replicates per group. A high Pearson correlation coefficient among replicates confirmed the reliability of the sequencing results (Figure S4; Table S2). Transcriptome analysis identified 883 differentially expressed genes (DEGs) between nst1/nst3 mutants and lines, with 615 genes upregulated and 268 genes downregulated (Table S3). Gene ontology (GO) enrichment analysis revealed significant enrichment for terms related to cell wall organization and SCW biogenesis (GO: 0009834, GO: 0009832), as well as phenylpropanoid metabolic and biosynthetic processes (GO:0009834, GO:0009699) (Figure 3D; Table S4).

Further analysis indicated that several transcription factors critical for SCW formation, including NST2, MYB46, SND2, SND3, MYB52, MYB103, and MYB85, were upregulated in PbrMADS1‐complemented lines (Figure 3E). To assess whether PbrMADS1 could directly activate these transcription factors, dual‐luciferase assays were performed. The results showed that PbrMADS1 significantly activated the promoters of AtSND2 and AtSND3 (Figure 3F). In pear, the key NAC–MYB transcription factors PbrNSC, PbrMYB24, and PbrMYB169 have been shown to promote stone cell development (Xue et al., 2019a; Wang et al., 2021; Xue et al., 2023). To explore potential downstream targets of PbrMADS1, dual‐luciferase reporter assays were conducted to assess the activation of key MYB, NAC, and lignin biosynthesis genes. The results indicated that PbrMADS1 does not directly activate the transcription of these genes (Figure S5).

PbrMADS1 interacts with PbrMYB169

Previous studies have shown that MADS‐box proteins often function through forming higher‐order protein complexes (Kaufmann et al., 2005). To investigate whether PbrMADS1 interacts with other regulatory proteins, yeast two‐hybrid (Y2H) assays, firefly luciferase complementation (LCI) assays, and bimolecular fluorescence complementation (BiFC) assays were performed.

Y2H assays demonstrated that yeast cells co‐transformed with PbrMADS1‐BD and PbrMYB169‐AD grew normally on selective SD/−Trp−Leu−His−Ade medium, while those containing PbrMADS1‐BD/PbrMYB24‐AD, PbrMADS1‐BD/PbrNSC‐AD, or the negative control did not grow, indicating a specific interaction between PbrMADS1 and PbrMYB169 (Figures 4A, S6A, and S7A). Luciferase complementation imaging and BiFC assays further confirmed this interaction in plants, revealing strong luminescence signals and nuclear localization of the interacting proteins (Figures 4B, C, S6B, C, and S7B, C). Additionally, pull‐down assays using recombinant GST‐PbrMADAS1 and His‐PbrMYB169 proteins expressed in vitro demonstrated that GST‐PbrMADAS1 was able to pull down His‐PbrMYB169, whereas GST alone could not (Figure 4D). The results confirmed the direct interaction between PbrMADS1 and PbrMYB169 both in vitro and in vivo, and demonstrated their colocalization in the nucleus.

Figure 4.

The interaction between PbrMADS1 and PbrMYB169 activated the biosynthesis of stone cells in pear fruit

(A) The yeast two‐hybrid (Y2H) assay showed that PbrMADS1 interacted with PbrMYB169 in yeast cells. EV, empty vector; SD/−T−L: SD medium lacking tryptophan and leucine. SD/−T−L−H−A: SD medium lacking tryptophan, leucine, histidine, and adenine. (B, C) The interaction between PbrMADS1 and PbrMYB169 was tested by luciferase complementation imaging (LCI) (B) and the Bimolecular fluorescence complementation (BiFC) (C) assay in Nicotiana benthamiana leaves. Unfused CLUC, NLUC, YCE, and YNE were used as the control. The red fluorescent protein was the nuclear mark (mcherry) with a scale of 50 μm. The color bar represents the strength of the LUC signal. (D) Pull‐down analysis verified the interaction between PbrMADS1 and PbrMYB169 in vitro. “+” and “−” denote the existence and non‐existence of proteins, respectively. (E) The dual‐luciferase assay in Nicotiana benthamiana leaves confirmed the activation of lignin biosynthesis genes by the interaction between PbrMADS1 and PbrMYB169. The error bars represent the mean ± SEM of three technical replicates. Lowercase letters indicate significant differences (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test). (F) Electrophoretic mobility shift assay (EMSA) was used to validate the binding interactions between PbrMADS1, PbrMYB169, and the AC element within the CCOAMT1 promoter. “+” and “−” denote the presence or absence of protein or DNA probes, respectively. The terms “hot probe,” “cold probe,” and “m” refer to the biotin‐labeled DNA fragment, the unlabeled DNA fragment, and the mutated biotin‐labeled DNA fragment, respectively. (G, I) The longitudinal sections of the young fruit of “Dangshansuli” with transient overexpression (G) or silencing (I) of PbrMADS1 and PbrMYB169 were stained with Wiesner's reagent. The darker the color, the higher the lignin content in the stone cells. EV, empty vector; OE, overexpression; and VIGS, virus‐induced gene silencing. Scale bars = 0.5 cm. (H, J) The acetyl bromide method was used to determine the lignin content in pear flesh around the infiltration sites in (G) and (I). The error bars represent the mean ± SEM of eight technical replicates. Lowercase letters indicate significant differences (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test).

PbrMADS1 enhances the activation effect of PbrMYB169 on lignin biosynthesis structural genes

Previous studies revealed that PbrMYB169 could activate the expression of Pbr4CL1, Pbr4CL4, PbrC3′H, PbrHCT79, PbrCCOMT1, PbrCCR21, and PbrLAC19 (Xue et al., 2019a). Therefore, it was hypothesized that PbrMADS1 might activate lignin biosynthetic genes by forming a protein complex with PbrMYB169. To test this hypothesis, PbrMADS1 and PbrMYB169 were co‐infiltrated with the promoters of lignin biosynthetic genes into Nicotiana benthamiana leaves for dual‐luciferase assays. The results demonstrated that co‐infiltration of PbrMADS1 and PbrMYB169 significantly enhanced the activation of Pbr4CL1, PbrCCOAMT1, and PbrCCR21 compared to the activation by PbrMYB169 alone, with PbrCCOAMT1 showing the most notable increase (Figure 4E).

Furthermore, promoter truncation experiments revealed that the deletion of the AC element prevented PbrMYB169‐mediated activation of PbrCCOAMT1 (Figure S8A, B). Electrophoretic mobility shift assay (EMSA) results confirmed that PbrMYB169 specifically bound to the AC element, whereas PbrMADS1 did not (Figure 4F). Notably, an increase in the abundance of PbrMADS1 progressively enhanced the binding affinity of PbrMYB169 to the AC element (Figure 4F). These results suggested that the interaction between PbrMYB169 and PbrMADS1 reinforced the binding of PbrMYB169 to its target cis‐element, thereby augmenting the activation of lignin biosynthetic genes.

To further elucidate the biological function of the PbrMADS1–PbrMYB169 complex, co‐transformation experiments were performed in pear fruits. Overexpression constructs of PbrMADS1 and PbrMYB169 were simultaneously introduced into “Dangshansuli” pear fruit at 35 DAFB. Wiesner staining showed a stronger lignin signal in fruits co‐expressing PbrMADS1 and PbrMYB169 compared to those overexpressing either gene individually (Figure 4G). Quantitative analysis further confirmed a significant increase in lignin content and the expression of lignin biosynthetic genes in the co‐transformed fruits compared to those that were singly transformed (Figures 4H, S9A). Conversely, simultaneous silencing of PbrMADS1 and PbrMYB169 resulted in a greater reduction in stone cell formation and lignin accumulation compared to the effects observed from silencing either gene individually (Figure 4I, J). Furthermore, compared with the fruit injected with the empty vector, co‐overexpression of PbrMADS1 and silencing of PbrMYB169 significantly reduced both lignin content and the expression levels of lignin biosynthetic genes, but there was no significant difference with silencing PbrMYB169 alone (Figures 4J, S9B). These results indicated that the function of PbrMADS1 is dependent on PbrMYB169, which co‐regulates lignin biosynthesis within the same pathway.

Collectively, these results demonstrated that the interaction between PbrMADS1 and PbrMYB169 enhances the transcriptional activation of lignin biosynthetic genes, thereby promoting lignin deposition and stone cell formation in pear fruits.

Whole‐genome duplication as the primary driver of SEP3 subfamily expansion in Maleae

The above results indicated that PbrMADS1 facilitates lignin biosynthesis through diverse regulatory mechanisms in both Arabidopsis and pear. To investigate the mechanism of action of PbrMADS1, which emerged specifically during the evolutionary process of pear, we initially selected 12 representative species from the Rosaceae family. These included Malus (apple), Pyrus (pear), Eriobotrya (loquat), and Crataegus (hawthorn) from the Maleae, Gillenia (American ipecac) from Gillenieae, Prunus (apricot, plum, peach, cherry) from Amygdaleae, and Fragaria (strawberry) and Rubus (shanmei, raspberry) from Rosoideae (Figure 5A; Table S5). Vitis (grape) was used as the outgroup species, and the MADS‐box gene family members from these 13 species were identified (Figure S10; Table S6).

Figure 5.

Evolutionary, expression, and interaction analyses of SEP genes in Rosaceae species reveal that the PbrMADS1–PbrMYB169 complex has a unique evolutionary trajectory in pear

(A) Phylogenetic relationships of 12 Rosaceae species and the outgroup species grape. Rectangles of the same color represent the same subfamily. “X” denotes the number of chromosomes. (B) Phylogenetic relationships and gene duplication types of the SEP family in 13 species. Different types of SEP gene duplications within the same species are represented by dots of the same color. (C) Ks distribution of syntenic gene pairs from pear, hawthorn, G. trifoliata, and grape. (D) Microsynteny relationships of SEP3 genes in the Maleae subfamily and other Rosaceae species. Curves connect the syntenic genes identified in pear, apple, hawthorn, loquat, G. trifoliata, and peach. The collinearity relationship of PbrMADS1 is marked with a darker curve. (E) The gene expression heatmap shows the expression levels of SEP3 in fruits at different developmental stages of the Maleae and peach. (F, G) Y2H assay showed that MdSEP3–MdMYB169 (F) and EjSEP3.1–EjMYB169 (G) do not interact in yeast cells. EV, empty vector; SD/−T−L: SD medium lacking tryptophan and leucine. SD/−T−L−H−A: SD medium lacking tryptophan, leucine, histidine, and adenine. 3‐AT indicates that 3‐amino‐1,2,4‐triazole is used to inhibit yeast self‐activation activity. Images were captured after 72 h of yeast cell growth on SD medium. (H) LCI assays showed that MdSEP3–MdMYB169 and EjSEP3.1–EjMYB169 do not interact in Nicotiana benthamiana leaves. Images were captured 48 h after Agrobacterium infiltration of Nicotiana benthamiana leaves. The color bar represents the strength of the LUC signal.

A total of 64 genes were found in the SEP subfamily and they were classified into four groups (Group A–Group D), each containing genes from all 13 species (Table S6). This finding indicated that no gene loss occurred in the SEP family throughout the evolutionary history of Rosaceae. However, in contrast to Rosoideae, Amygdaleae, and Gillenieae, Maleae showed extensive gene expansion of SEP genes (Figure 5B). DupGen Finder was utilized to identify the number of genes resulting from tandem duplication (TD), whole‐genome duplication (WGD), proximal duplication (PD), transposed duplication (TRD), and dispersed duplication (DSD) within the SEP subfamilies across the 13 species (Table S7).

Analysis of gene duplication events revealed that a single copy of SEP3 was present in Rosaceae species that evolved prior to WGD. In contrast, most species possessed two copies of SEP3 (Figure 5B). Specifically, the duplicated SEP3 genes in pear, loquat, and apple were derived from WGD events. However, the second SEP3 copy in apple was not categorized as a true SEP family member due to the loss of the MADS domain. Notably, in hawthorn, the SEP3 duplication was derived from a DSD event rather than WGD.

The distribution of synonymous substitutions per synonymous site (Ks) within syntenic blocks revealed a recent WGD peak (with Ks around 0.16) in pear, loquat, and apple, whereas such a peak was absent in G. trifoliata and grape (Figure 5C). Given that PbrMADS1 belongs to the SEP3 subfamily, a focused intra‐ and intergenomic synteny analysis was performed. A 2:1 syntenic depth ratio of SEP3 was observed when comparing pear with species that diverged prior to the Maleae split (e.g., G. trifoliata and P. persica), while a 1:1 syntenic depth ratio was evident within Maleae (Figure 5D). Furthermore, the Ks values of SEP3 gene pairs in pear and loquat were 0.15 and 0.16, respectively, indicating that their duplication likely resulted from the same recent WGD event. These results indicated that WGD served as the predominant driver for the amplification of SEP3 family members in the apple tribe.

The Met63 residue in PbrMADS1 plays an important role in its interaction with PbrMYB169 for regulating the lignification of stone cells

To gain deeper insights into the role of SEP3 genes in fruit development of the apple tribe, the expression profiles of SEP3 genes were examined in pear, apple, loquat, and peach (neighboring species within Amygdaleae). It was found that peach and apple each possessed a single SEP3 gene, while pear and loquat each have two copies with analogous expression patterns, although one copy showed a substantially higher expression level than the other (Figure 5E). Across these species, PbrMADS1 showed a similar expression pattern to its homolog MdSEP3 in apple, with relatively elevated expression during the fruit‐setting stage. Conversely, EjSEP3.1 in loquat showed elevated expression during the mid‐fruit developmental stage, while SEP3 expression in peach differed entirely, with a peak at the mature stage of fruit development (Figure 5E). These findings suggest that SEP3 genes show diverse expression patterns across species and may be involved in the formation of distinct fruit qualities.

Given that stone cells are a unique trait of pear, with only a few found in loquat and apple, and considering the relatively high expression of SEP3 genes during the early and middle stages of fruit development in all three species, it was hypothesized that the divergence in stone cell traits might not originate at the transcriptional level. Instead, differences in the interaction between PbrMADS1 and PbrMYB169 proteins might underlie this trait variation. Homologous genes of PbrMADS1 and PbrMYB169 from loquat and apple, namely, MdSEP3, EjSEP3.1, MdMYB169, and EjMYB169, were cloned and incorporated into yeast two‐hybrid vectors pGBKT7 (BK) and pGADT7 (AD), respectively. Yeast autoactivation assays showed that MdSEP3‐BK displayed autoactivation with the AD empty vector, which could be suppressed by 50 μM 3‐AT, whereas EjSEP3.1‐BK did not show autoactivation (Figures 5F, G, S11).

Y2H and LCI assays confirmed that no interaction occurred between SEP3 and MYB169 proteins in apple and loquat (Figure 5F–H). However, PbrMADS1 was capable of interacting with both MdMYB169 and EjMYB169 (Figure S12), suggesting that differences among PbrMYB169, MdMYB169, and EjMYB169 were not the primary cause of the divergent interaction patterns. Sequence alignment revealed that while PbrMADS1, MdSEP3, and EjSEP3.1 all share conserved domains and motif structures, there was a notable amino acid difference within the I domain (intervening domain): M in pear corresponded to T in both apple and loquat (Figures 6A, S13).

Figure 6.

The M63 residue of PbrMADS1 is a critical site for its interaction with PbrMYB169 and regulates stone cell formation in pear

(A) The schematic diagram of the SEP3 protein structure and sequence alignment show amino acid sequence differences in the I‐domain among pear, apple, and loquat. MADS, I, K, and C represent the MADS‐domain, the I‐domain, the K‐domain, and the C‐terminal, respectively. (B) Subcellular localization in Nicotiana benthamiana leaves showed that PbrMADS1 and its variants were localized in the nucleus. Scale bars = 50 μm. (C) The Y2H assay showed differences in protein–protein interactions between PbrMADS1–PbrMYB169 and PbrMADS1^M63T–PbrMYB169. Strong protein–protein interactions manifested as growth on SD/−T−L−H−A media and a distinct blue color post X‐α‐gal staining assay, while weak interactions led to comparatively less growth on the same media and merely a light blue color. Images were captured after 72 h of yeast cell growth on SD medium. SD/−T−L−H−A: SD medium lacking tryptophan, leucine, histidine, and adenine. (D, E) The interaction of Pbr MADS1 and its variants with Pbr MYB169 was detected by LCI (D) and LUC activity analyses (E) in Nicotiana benthamiana leaves. The color bar represents the strength of the LUC signal. (F) The dual‐luciferase assay verified the activation effect of co‐expressing PbrMADS1 and its variants with PbrMYB169 on lignin biosynthesis genes. The error bars represent the mean ± SEM of three technical replicates. Lowercase letters indicate significant differences (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test). (G) The longitudinal sections of the young fruit of “Dangshansuli” with transient expression of PbrMADS1 and its variants were stained with Wiesner's reagent. The darker the color, the higher the lignin content in the stone cells. Scale bars = 0.5 cm. (H) Lignin content in the infiltrated regions of the fruit flesh depicted in (G) was quantified using the acetyl bromide method. The error bars represent the mean ± SEM of nine technical replicates. Lowercase letters indicate significant differences (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test). (I) The transgenic pear flesh callus of PbrMADS1 and its variants were stained with Wiesner's reagent. Scale bars = 1 cm. (J) Lignin content in the pear callus depicted in (I) was quantified using the acetyl bromide method. Each value is mean ± SEM (n = 12 technical replicates). There were significant differences between groups in different letter representations (P < 0.05, one‐way ANOVA, Tukey's HSD post hoc test).

To verify the role of this amino acid substitution, site‐directed mutagenesis was performed to replace the M63 residue of PbrMADS1 with T63 (PbrMADS1^M63T). Subcellular localization analysis in Nicotiana benthamiana leaves revealed distinct nuclear GFP signals of PbrMADS1^M63T, indicating that the M63T mutation does not alter the nuclear localization of PbrMADS1 (Figure 6B). Subsequently, Y2H assays demonstrated that yeast co‐transformed with WT PbrMADS1 and PbrMYB169 showed robust growth and strong blue coloration after 24 h on selective medium (SD/−Trp−Leu−His−Ade). In contrast, yeast expressing the PbrMADS1^M63T mutant failed to grow and showed a faint blue color even after 72 h (Figure 6C). Luciferase complementation imaging experiments further confirmed this change in interaction intensity in plants, revealing that the M63T mutation of PbrMADS1 significantly weakened its interaction with PbrMYB169 (Figure 6D, E). Furthermore, dual‐luciferase assays demonstrated that PbrMADS1^M63T failed to enhance the transcriptional activation of downstream genes by PbrMYB169, confirming that the M63 residue is critical for their interaction (Figure 6F). These results suggest that the amino acid substitution at position 63 is a key determinant of this interaction between MADS1 and MYB169 proteins.

To further investigate whether the M63T mutation affects the biological function of PbrMADS1 in stone cell formation, we performed transient overexpression assays in pear fruits. The results showed that the lignin content and the expression levels of key lignin biosynthesis genes were significantly reduced in fruits overexpressing PbrMADS1 ^M63T , compared with overexpressing PbrMADS1 (Figures 6G, H, S14A). These findings were further supported by stable transformation assays in pear callus, where similar reductions in lignin content and gene expression were observed in lines overexpressing PbrMADS1 ^M63T (Figures 6I, J, S14B). These results demonstrate that the M63T mutation significantly impairs the ability of PbrMADS1 to regulate lignification of stone cells.

DISSCUSION

Divergence in the biological function of SEP subfamily genes regulates fruit traits

Within the Rosaceae family, fruit diversity arises from the differential enlargement of specific floral tissues in response to pollination and fertilization signals, resulting in a wide variety of fruit types. For example, drupes such as peach, plum, and cherry develop fleshy fruits encased by a rigid endocarp that protects the seed. In contrast, pomes, represented by apple and pear, develop from the hypanthium. Drupetums like raspberry consist of multiple small drupe‐like fruits, while achenes, which are dry fruits with a solitary seed, and achenetums such as strawberry, which have multiple achenes on a fleshy receptacle, further add to this diversity (Liu et al., 2020). Among these, peach is considered a standard botanical fruit, with its pulp originating from the ovary wall. In contrast, apples and strawberries are classified as accessory fruits, with the former developing from the hypanthium and the latter developing from the receptacle. The distinct capacity of floral tissues to differentiate into fruits greatly contributes to the fruit diversity observed within Rosaceae.

WGD events, followed by preferential gene retention and subsequent divergence, have been proposed as major contributors to angiosperm diversification. In particular, the duplication of MADS‐box genes from an ancient WGD event in the common ancestor of angiosperms is thought to be accountable for the origin of flowers and other evolutionary innovative traits such as fruits (Van de Peer et al., 2009; Soltis and Soltis, 2016). Consistently, whole‐genome sequencing has revealed a relatively recent WGD event that occurred approximately 50 million years ago within the Maleae tribe (Velasco et al., 2010). To investigate the duplication history of SEP subfamily genes, analyses including DupGen Finder, the calculation of synonymous substitution rates (Ks), and intra‐ and intergenomic syntenic analysis were conducted across 12 Rosaceae species and Vitis (grape, as an outgroup). The results indicate that WGD was the dominant driving factor behind the expansion of the SEP subfamily in the apple tribe. It was further hypothesized that this WGD event contributed to the evolution of pome fruit, a defining characteristic of the Maleae tribe.

Previous studies have shown that SEP1/2/4‐ and FBP9‐related genes promote the formation of fleshy fruit tissues in apple. Antisense suppression of SEP1/2/4‐like genes (MdMADS8 and MdMADS9) and the FBP9‐like gene MdMADS7 resulted in sepaloid petals and a significant reduction in apple fruit flesh within the cortex layer (Ireland et al., 2013). This decrease may partly be due to a reduction in MdMADS6 expression levels observed in antisense plants. Interestingly, while MdMADS7 and MdMADS6 show low expression levels during the early stages of fruit development in WT apples, their expression gradually increases as the fruit matures (Ireland et al., 2013). Together, the expression patterns and phenotypes suggest that SEP1/2/4 and FBP9 MADS‐box genes positively regulate the development of fruit flesh in apple. Unlike previously discussed floral identity genes from the C and A classes, SEP1/2/4 and FBP9 appear to act directly on fruit development. However, the positive roles of these genes in fruit development in other Maleae species have not yet been determined.

In the present study, phylogenetic analysis positioned PbrMADS1 within the SEP3 subfamily, identifying it as an E‐function gene. Orthologs in the PbrMADS1 cluster, such as FveSEP3, ZjSEP3, and MaMADS1/2, are known to regulate floral organ formation, flowering time, and fruit ripening (Elitzur et al., 2016; Gao et al., 2021; Pi et al., 2021). Although MdMADS18/118 in apple are also part of the SEP3 branch, their functions remain unknown (Tanaka and Wada, 2022). Here, this study reveals that overexpression of the SEP3 subfamily gene PbrMADS1 promotes lignin biosynthesis in pear fruit, callus tissue, and Arabidopsis, expanding the known functional scope of SEP subfamily genes. These findings not only demonstrate functional divergence within the SEP3 subfamily but also provide a theoretical foundation for exploring the functions of homologous genes in other species. Furthermore, it is speculated that the additional MADS‐box gene copies generated by WGD may have facilitated the functional innovations required for the evolution of the pome fruit.

The PbrMADS1–PbrMYB169 module shows a species‐specific regulatory mechanism in pear stone cell formation

SEP3 subfamily MADS‐box genes regulate NAC and MYB transcription factors by directly binding to CArG elements in promoter regions. For instance, the SEP3 genes MaMADS1 and EjSEP3 bind to the CarG motif in the promoters of MaNAC083 and LHY, respectively, thereby regulating banana ripening and jujube flowering time (Gao et al., 2021; Wei et al., 2023). In the present study, complementary experiments using Arabidopsis mutants and dual‐luciferase assays revealed that PbrMADS1 activates the expression of the Arabidopsis NAC transcription factors AtSND2 and AtSND3. However, in pear, PbrMADS1 did not activate the expression of key NAC–MYB transcription factors or lignin biosynthesis genes.

This discrepancy may stem from the ability of MADS‐box proteins to form protein complexes, thereby facilitating binding to downstream targets through interactions with other regulatory factors. For instance, in kiwifruit, the SEP3 gene AcMADS68 does not directly activate anthocyanin biosynthesis genes but instead interacts with AcMYBF110 to co‐regulate anthocyanin biosynthesis (Liu et al., 2023). Similarly, in the current study, PbrMADS1 was found to interact with PbrMYB169 within the nucleus, forming a PbrMADS1–PbrMYB169 protein complex. Dual‐luciferase assays, EMSA, and transient transformation of pear fruit confirmed that this complex enhances the binding ability of PbrMYB169 to the AC element, thereby amplifying its activation of lignin biosynthesis genes and promoting stone cell formation. These findings demonstrate that SEP3 subfamily genes show diverse modes of action in regulating fruit quality and developmental processes.

To further investigate the evolutionary basis of these functional differences, SEP subfamily members were identified across 12 Rosaceae species. Phylogenetic analysis revealed that a recent WGD event acted as the major driver of SEP3 family expansion within the apple tribe, resulting in a 2:1 syntenic depth ratio compared to non‐apple tribe species. Expression profiling showed that SEP3 genes in pear, loquat, and apple show higher expression during the early to middle stages of fruit development. Nevertheless, the PbrMADS1–PbrMYB169 interaction mechanism appeared to be specific to pear, as no interaction was detected between homologous SEP3 and MYB genes in apple and loquat.

The structural basis for this species‐specific interaction was further explored. Previous studies have demonstrated that the I‐domain of SEP3 proteins plays a crucial role in determining DNA binding specificity and dimerization (Lai et al., 2021). Amino acid sequence alignment revealed that, in pear, the key residue at position 63 is M (M63), whereas in apple and loquat, this residue is T (T63). Structural studies have suggested that the loop formed by residues 58–62, along with Tyr70 and Met63, constitutes a crucial region influencing the interaction specificity of MIKC‐type MADS proteins (Thoris et al., 2024). Alterations such as loop length extension or substitution of Met63 have been shown to reduce the interaction affinity between SOC1 and other MADS proteins. In SEP3, shortening of the loop was previously linked to changes in interaction strength with specific partners, although the precise role of M63 had remained unclear.

The current study revealed that the M63 residue of PbrMADS1 is critical for its interaction with PbrMYB169. Substitution of M63 with T63 (M63T mutation) abolished the ability of PbrMADS1 to enhance the transactivation activity of PbrMYB169 on downstream lignin biosynthesis genes and impaired its function in regulating stone cell formation. These findings provide direct evidence that a single amino acid substitution can lead to species‐specific differences in SEP3‐mediated regulatory mechanisms.

In summary, this study discovered a novel, species‐specific PbrMADS1–PbrMYB169 regulatory module that promotes stone cell formation in pear fruit. It was further demonstrated that the M63 residue of PbrMADS1 is essential for this interaction. Differences at this amino acid position may contribute to the functional divergence and specialization of SEP3 gene subfamily genes across Rosaceae species.

MATERIALS AND METHODS

Genome‐wide association studies

The SNP genotypes and phenotypic data of 312 sand pear samples were obtained from previous studies (Zhang et al., 2021). The genome‐wide association analysis for stone cell content was performed using the mixed linear model of GEMMA software (Genome‐wide Efficient Mixed Model Association algorithm) (Zoubarev et al., 2012).

Co‐expression analysis

The RNA‐Seq data of five pear cultivars, namely, “Housui,” “Yali,” “Kuerlexiangli,” “Nanguoli,” and “Starkrimson,” were obtained from our previous study (Zhang et al., 2016). Pearson's correlation coefficient (PCC) values were used as a measurement of expression similarity between gene pairs, and filtered using Excel software (parameter was set as > 0.5 or < −0.5). Visualizations of the data were carried out using Cytoscape software (Shannon et al., 2003).

Plant materials

In the study, the different tissues and fruits at different developmental stages of “Dangshansuli” pear were harvested from Xuzhou, Jiangsu Province, China. Under sterile dark conditions, the pear callus grew on solid Murashige and Skoog (MS) medium, usually subcultured every 15 d. The double mutant Arabidopsis thaliana (CS67921) with T‐DNA insertion of NST1 and NST3 was derived from Arabidopsis thaliana information resources. Arabidopsis and Nicotiana benthamiana plants were grown in a greenhouse at 22°C with a 16 h light/8 h dark photoperiod and a light intensity of 10,000 lux.

RNA extraction and gene expression analysis

Total RNA was extracted using the polysaccharide polyphenol plant RNA extraction kit (FUJI, Chengdu, Sichuan Province, China) and the first‐strand cDNA was synthesized by reverse transcription using the RevertAid^TM First Strand cDNA Synthesis Kit (Transgen, Beijing, China). LightCycler 480 SYBR Green I Master (Roche, Basel, Switzerland) was used for RT‐qPCR analysis. The primers were designed using software Primer 5, and the primer sequences are shown in Table S8. AtActin and PbrGAPDH were the internal reference genes of Arabidopsis thaliana and pear samples, respectively.

HISAT (v2.2.1) was used to map clean reads to the reference genome (Kim et al., 2015). The transcripts were quantified using featureCounts (Liao et al., 2014) and the lengths of the transcripts in the sample were normalized to FPKM (fragments per kilobase of exon per million fragments mapped) values. Differential gene expression gene analysis was performed using DESeq. 2 (Love et al., 2014), with an FDR (false discovery rate) cut‐off of 0.05 and Log₂ fold change| cut‐off of 1. GO and KEGG enrichment analyses of differential genes were performed using the R‐package ClusterProfiler (v4.8.3) (Wu et al., 2021).

RNA in situ hybridization

Tissues of early developmental stages of “Dangshansuli” pear fruit were collected and dehydrated through an ethanol gradient, cleared in xylene, embedded in paraffin, and sectioned to a thickness of 10 μm. An RNA probe for PbrMADS1 was designed and labeled with biotin. After deparaffinization, the tissue sections were hybridized with the biotin‐labeled RNA probe. Finally, the spatial expression pattern of PbrMADS1 was detected via a colorimetric reaction.

Gene cloning and sequence analysis of PbrMADS1

The specific primers of PbrMADS1 were designed by CE Design v1.03, and its coding sequence was cloned from the cDNA of “Dangshansuli” flesh at 35 DAFB. The PCR product of PbrMADS1 was recovered after gel electrophoresis and sub‐cloned into pDONR221, and then inserted into the binary vector pK7WGF2 using Gateway® Technology.

The neighbor‐joining method in MEGA 5 was used to construct the phylogenetic tree of the SEP subfamily. Pear MADS protein sequences were downloaded from the Pear Genomics Database (Chen et al., 2023), and other related MADS protein sequences were downloaded from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov) or public databases. Multiple sequence alignment was performed using Clustal X with default parameters and manually adjusted in Jalview.

Arabidopsis transformation and subcellular localization

The Agrobacterium tumefaciens containing the PbrMADS1 overexpression vector was stably transformed into 4‐week‐old Col‐0 Arabidopsis thaliana using the floral dip method (Clough and Bent, 1998). RNA extraction and RT‐qPCR analysis were performed on T₁ generation Arabidopsis thaliana plants screened by hygromycin to identify positive transgenic lines. The green fluorescence signal in the root tip tissue of T₂ transgenic Arabidopsis seedlings was observed using a Zeiss LSM880 spectral confocal microscope (Zeiss, Oberkochen, Baden‐Württemberg, Germany).

Transient gene expression in pear fruit flesh

The GV3101 containing the overexpression (35S‐GFP‐PbrMADS1) and silencing (PbrMADS1‐TRV2) constructs of PbrMADS1 were suspended in infiltration buffer (10 mM MgCl₂; 200 μM acetosyringone; 10 mM MES), and then injected into the “Dangshansuli” fruit flesh at 35 DAFB. For specific steps, refer to the previously described method (Xue et al., 2019b).

Genetic transformation of pear callus

Pear calli transformations were performed according to the methods of Bai et al. (2019). For PbrMADS1 knockout, the CRISPR/Cas9–PbrMADS1 vector was constructed using a Golden Gate cloning and three‐way Gateway cloning system according to the previously described method (Ming et al., 2022). A single guide RNA sequence is shown in Table S8. The screened positive callus was induced for lignin synthesis in pear callus for phenotypic detection according to the previously described method (Xue et al., 2023).

Histological analysis

For optical microscope analysis, sections were prepared from the bases of 8‐week‐old T₃ generation Arabidopsis thaliana inflorescence stems. Samples were sectioned to a thickness of 100 μm using a vibratome (Leica VT1000S, Wetzlar, Hesse, Germany). The sections were stained with toluidine blue, Wiesner, and Mäule stains, respectively. Subsequently, lignin accumulation and cellular morphology in the sections were observed using a Nikon Ni‐U microscope (Nikon, Tokyo, Japan).

Analyses of lignin contents

The inflorescence stems of Arabidopsis plants, pear flesh, and pear callus tissues were dried to a constant weight. The crude cell wall components of the dried samples were extracted in turn in phosphate buffer, methanol, chloroform, acetone, and dimethyl sulfoxide. The lignin content was determined using the acetyl bromide method (Van Acker et al., 2013).

Yeast two‐hybrid (Y2H) assay

The coding sequences of PbrMADS1, MdSEP3, EjSEP3.1 and PbrMYB169, MdMYB169, and EjMYB169 were individually cloned into the pGBKT7 and pGADT7 vectors, respectively. According to the Matchmaker TM GAL4 Two‐Hybrid System (Clontech, Mountain View, California, USA) manual, the two recombinant plasmids were co‐transformed into yeast strain AH109. The normally growing yeast colonies were transferred to SD/−Trp−Leu−His−Ade medium and incubated at 28°C for approximately 2–3 d to observe the growth of yeast colonies.

Bimolecular fluorescence complementation (BiFC) assay

PbrMADS1 and PbrMYB169 were cloned into the pSPYCE173 and pSPYNE173 vectors, generating the YCE and YNE recombinant plasmids, respectively. Subsequently, both recombinant plasmids were transiently transformed into 4‐week‐old Nicotiana benthamiana leaves through a needle‐free injection. After infiltration for 3 d, suitable leaf samples were obtained and pressed for observation. The fluorescence signals in the leaf were examined using a confocal microscope (Zeiss, Oberkochen, Baden‐Württemberg, Germany).

Luminescence complementation imaging (LCI) assay

The coding sequences of PbrMYB169, MdMYB169, and EjMYB169 were inserted into the pCAMBIA‐nLUC vector, while PbrMADS1, MdSEP3, and EjSEP3.1 were inserted into the pCAMBIA‐cLUC vector. Subsequently, an equal volume of Agrobacterium carrying these plasmids was mixed and co‐injected into Nicotiana benthamiana leaves. Luminescence images were captured using a cooled plant imaging system (Princeton Instruments, Trenton, New Jersey, USA) to visualize the interaction between PbrMYB169 and PbrMADS1 based on the complementation of the split luciferase.

Pull‐down assay

The coding sequences of PbrMYB169 and PbrMADS1 were cloned into the pCold TF vector with a His tag and the pGEX‐4T vector with a GST tag, respectively. Initially, the GST‐tagged recombinant protein was added to a GSTSefinose™ resin column and allowed to bind at 4°C for 4 h. Subsequently, the purified His‐tagged recombinant protein was added and incubated at 4°C for an additional 4 h. Finally, Western blot analysis was performed using an anti‐His tag antibody to detect the interaction between Input and GST pull‐down samples.

Dual‐luciferase reporter assay

The promoters of the cloned lignin biosynthesis genes were ligated with the pGreenII 0800‐LUC vector to serve as the reporter vectors. Overexpression vectors of PbrMADS1 and PbrMYB169 were used as effector vectors. The Agrobacterium containing the effector vector and the reporter vector was injected into 4‐week‐old Nicotiana benthamiana leaves at a ratio of 9:1. The activities of LUC and REN were detected using a reagent kit (Promega, Madison, Wisconsin, USA) and a luminometer (Molecular Devices, Sunnyvale, California, USA). The transcriptional activation ability of candidate genes on downstream genes was indicated by the LUC/REN ratio.

Electrophoretic mobility shift assay (EMSA)

The coding sequences of PbrMADS1 and PbrMYB169 were inserted into the pCold TF vector to generate recombinant His‐PbrMADS1 and His‐PbrMYB169 expression vectors. A synthetic DNA probe containing the AC element was synthesized and biotin‐labeled at the 3’ end. The biotin‐labeled DNA probe was incubated with His‐PbrMADS1 and His‐PbrMYB169 protein in binding buffer for 30 min. The biotin‐labeled probe was detected using the chemiluminescence EMSA kit (Beyotime, Shanghai, China).

Comparative genomic analysis

RaxML (v8.2.12) (model PROTGAMMAILGF) was used to construct a maximum likelihood (ML) phylogenetic tree based on 13 species' single‐copy orthologous genes (Stamatakis, 2014). Based on the topological structure of the phylogenetic tree and the fossil calibration from TIMETREE (http://www.timetree.org/), mcmctree in the PAML package (v4.10.7) was used to estimate divergence times (Yang, 2007). Subsequently, the synonymous mutation rate (Ks) was calculated using ParaAT (v2.0) and density mapping was performed using ggplot2 (v2.2.1) (Zhang et al., 2012).

MADS family identification and phylogenetic analysis

To identify MADS family members in the 13 species, HMM searches were first performed on the protein sequences of the 13 species using HMMER software (v3.4) and the MADS domain HMM profile (PF00319) with an E‐value threshold of 1e−1 (Eddy, 2011). The candidate members were then submitted to the NCBI CDD (Conserved Domain Database, http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to confirm the presence of the MADS domain (Marchler‐Bauer et al., 2015). A total of 1,002 MADS family members were identified. The sequences were aligned using Muscle (v3.8). Finally, a phylogenetic tree was constructed based on the maximum likelihood method using IQtree (v2.1.4) (Nguyen et al., 2015).

Overlap PCR

The M63T mutation in PbrMADS1 was introduced using an overlap PCR approach. Specific primers containing the desired mutation were designed, and the sequences are shown in Table S8. Two separate fragments (Fragment 1 and Fragment 2) with overlapping ends were amplified using Phanta Max Super‐Fidelity DNA Polymerase (Vazyme, Nanjing, Jiangsu Province, China) and subsequently purified. Approximately 100 ng of each purified fragment was mixed and used as the template for an overlap PCR reaction to generate the full‐length mutated sequence. The final product was then amplified using the flanking primers specific to PbrMADS1.

Statistical analysis

Statistical analyses were performed using GraphPad Prism software. Comparisons between two sample groups were conducted using Student's t‐test. For multiple comparisons, significance was assessed using analysis of variance (ANOVA), followed by Tukey's HSD post hoc test.

Accession numbers

The sequence data mentioned in this study can be accessed in the Pear Genomics Database (Chen et al., 2023), or NCBI (https://www.ncbi.nlm.nih.gov/). The Arabidopsis thaliana RNA sequencing data generated in this study have been deposited in the NCBI Sequence Read Archive (SRA) under BioProject number PRJNA1204165.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

C.X. and J.W. conceived and designed the experiments. Y.S.X., C.X., P.F.Z., Y.Y.H., and M.D. performed the experiments. S.L.C., R.Z.W., and M.Y.Z. performed the data analysis. Y.S.X., S.L.C., J.W., and C.X. wrote and revised the manuscript. All authors have read and approved the contents of this paper.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.70071/suppinfo

Figure S1. Analysis of the expression levels of PbrMADS1 and lignin biosynthesis genes in transiently transformed young pear fruit

Figure S2. Identification of transgenic pear callus and analysis of lignin biosynthesis gene expression levels

Figure S3. Phenotypic analysis and lignin synthesis gene expression in WT and PbrMADS1 overexpressing Arabidopsis

Figure S4. The Pearson correlation coefficient was calculated to assess the similarity between RNA‐seq data of nst1/nst3 mutants and the PbrMADS1 complementation lines

Figure S5. Dual‐luciferase assay confirmed that PbrMADS1 had no activation effect on lignin biosynthesis genes

Figure S6. PbrMADS1 has no interaction with PbrNSC in yeast and Nicotiana benthamiana leaves

Figure S7. PbrMADS1 has no interaction with PbrMYB24 in yeast and Nicotiana benthamiana leaves

Figure S8. Dual‐luciferase assays assessed the activation of the Pbr4CL1 and PbrCCOAMT1 promoters and their AC element‐deleted variants by PbrMYB169

Figure S9. Analysis of the expression levels of PbrMADS1, PbrMYB169, and lignin biosynthesis genes in transiently transformed pear fruit

Figure S10. The phylogenetic tree showed the clustering relationship of MADS family genes in 13 different species

Figure S11. Yeast two‐hybrid assay confirmed that MdSEP3 shows transcriptional self‐activation activity

Figure S12. Yeast two‐hybrid assays were performed to validate the interaction between PbrMADS1 and MdMYB169 as well as EjMYB169

Figure S13. Amino acid sequence alignment and analysis of PbrMADS1 and PbrMYB169, along with their homologous genes in apple and loquat

Figure S14. Analysis of the expression levels of PbrMADS1 and lignin biosynthesis genes in transiently transformed pear fruit and stable transformation of pear callus

Table S1. RPKM value for GWAS candidate genes and stone cell‐related structural genes in five pear cultivars

Table S2. RNA‐seq analysis for the nst1/nst3 mutant and PbrMADS1 complementary lines

Table S3. Differentially expressed genes for the nst1/nst3 mutant and PbrMADS1 complementary lines

Table S4. GO pathway enrichment analysis of DEGs for the nst1/nst3 mutant and PbrMADS1 complementary lines

Table S5. Summary of 13 genomes used in this study

Table S6. The members of the MADS gene family were identified from 12 representative species of Rosaceae and grape

Table S7. The duplication events were identified from 12 representative species of Rosaceae and grape

Table S8. Primer information

Biographies

Xue, Y. , Chen, S. , Hao, Y. , Shan, M. , Zheng, P. , Wang, R. , Zhang, M. , Wu, J. , and Xue, C . (2026). The PbrMADS1–PbrMYB169 complex has uniquely emerged to regulate lignification of stone cells in pear. J. Integr. Plant Biol. 68: 239–256.