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PbrBGAL6 promotes pollen tube growth by influencing apical pectin level in Pyrus bretschneideri

BMC Genomics volume 26, Article number: 321 (2025) Cite this article

Abstract

Background

β-galactosidase (BGAL), which is an important cell wall-degrading enzyme, participates in various biological processes, but its effects on pollen tube growth (PTG) remain unclear.

Results

We identified 12 PbrBGAL genes (named PbrBGAL1–12) in the pear (Pyrus bretschneideri) genome. PbrBGAL members, containing three conserved domains and two enzyme active sites, were grouped into six subclasses. They were distributed in seven chromosomes, with dispersed duplication revealed as the main replication event. PbrBGAL genes contained 1 to 24 exons and 0 to 23 introns, with exon/intron structure mostly conserved within each subclass except for subclass E. Analyses of tissue-specific expression indicated that only PbrBGAL6 was highly expressed specifically in anther and pollen, with decreasing expression levels during PTG. The effective inhibition of PbrBGAL6 expression using antisense oligodeoxynucleotide technology dramatically decreased BGAL enzymatic activity, promoted PTG and increased cytoplasmic leakage and tip widths. Furthermore, suppressing PbrBGAL6 transcription decreased the apical total and methylated pectin contents in pollen tubes by significantly increasing transcription of PbrPME11, PbrPG14, PbrPG20, PbrPG21 and PbrPG24.

Conclusions

We identified 12 PbrBGAL genes in the pear genome, of which PbrBGAL6 precisely modulates the apical pectin content to mediate pear PTG through its effects on PbrPME11 and PbrPGs expression. This study provides direct evidence of the involvement of BGAL in the regulation of polar PTG.

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Background

In flowering plants, self-incompatibility (SI), which is a common mechanism detected in more than 100 families, is controlled by multiple genes that prevent inbreeding depression and promote hybrid vigor, thereby maintaining plant diversity [1]. However, to optimize yield during crop production, SI is overcome via artificial pollination, but this leads to increased costs, especially for pear (Rosaceae) fruit production. Pear has a typical gametophyte SI mechanism in which pistil S-RNase triggers toxic cascade signals that inhibit the pathway necessary for pollen tube growth (PTG), leading to programmed pollen tube cell death and a lack of double fertilization [2]. Therefore, revealing the PTG mechanism may be useful for breaking pear SI to generate high-quality self-compatible germplasm.

PTG involves various processes, including cell wall remodeling and deposition, concentration gradient (ROS, pH, and Ca2+) formation and maintenance, actin cytoskeleton organization and activity, hormone homeostasis, and orderly vesicular trafficking [3]. Among these processes, regulated changes to the cell wall are crucial for maintaining cell wall integrity, which influences PTG [4]. The pollen tube wall primarily consists of methylated pectin (‘soft pectin’) secreted by vesicles in the tip region. Additionally, the shank region contains demethylated pectin (‘hard pectin’), callose, cellulose, and hemicellulose synthesized by related enzyme complexes. [4,5,6]. Previous studies showed that the composition and contents of these cell wall polysaccharides are regulated by various related enzymes that mediate cell wall remodeling and deposition to control PTG, including callose synthase in pear [7] and Arabidopsis thaliana [8], cellulose synthase in pear [9] and tobacco [10], and β-glucanase in lily [11] and peach [12]. Notably, pectin, which is a complex cell wall polysaccharide comprising three main supramolecular domains (homogalacturonan (HG), rhamnoglacturonan I (RG-I), rhamnoglacturonan II (RG-II)) synthesized in Golgi bodies and transported by secretory vesicles [13,14,15], is critical for the precise regulation of structural and mechanical properties during polarized PTG [16]. Additionally, during PTG, HG domain in Golgi bodies is methylated at the C-6 carboxyl group by pectin methyltransferase and exported to the tip, after which they are cross-linked with Ca2+ in the cell wall following a reaction catalyzed by tip-localized pectin methylesterase (PME; EC 3.1.1.11) to form demethylated pectin. This establishes an apical concentration gradient of methylated pectin (i.e., highest methylated pectin level at the tip) that facilitates tip growth, while also increasing shank strength [5, 17, 18]. Meanwhile, polygalacturonase (PG; endo-PG: EC3.2.1.15, exo-PG: EC3.2.1.67) degrades the demethylated HG domain by specifically hydrolyzing the α-(1–4) glycosidic bond to precisely modulate the pectin level [19, 20]. Adjustments to the HG domain esterification state and level requires the coordinated regulation of other enzymes or inhibitors, including pectin acetylesterase (PAE; EC 3.1.1.6), pectate lyases (PL; endo-PLs: EC4.2.2.2, exo-PLs: EC4.2.2.9), pectin lyases (PNL; EC4.2.2.10) [19], and the PME inhibitor [21]. Earlier research on the regulatory effects of pectin on PTG mainly focused on HG-modifying enzymes, with relatively few reports on the rhamnogalacturonan (RG) domain.

RG-I is a heteropolymer composed of alternating α−1,4-linked GalA residues and α−1,2-linked rhamnose that combine primarily with 1,4-β-D-galactose or 1,5-α-L-arabinose residues to form neutral side-chains. RG-II has an HG backbone with side-chains containing various sugars [22]. A β-galactosidase (BGAL) belonging to the glycoside hydrolase 35 family, which is the only exo-β-galactosidase (EC3.2.1.23) in higher plants, catalyzes the removal of β-D-galactosyl residues from the side-chain non-reducing terminal of RG-I in pectin and hemicellulose. Specifically, BGAL is assumed to hydrolyze β-(1,4) (class I BGAL) as well as β-(1,3) and β-(1,6) (class II BGAL) glycosidic bonds to increase cell wall porosity, which enhances the binding of PME, PG, or other hydrolases to pectin to modulate various biological processes [23]. The class I BGAL reportedly influences the ripening and softening of diverse fruits, including peach [24], sweet cherry [25], kiwifruit [26], and mango [27]. Class II BGALs are involved in many developmental stages. For example, they affect hypocotyls and young leaves in radish [28], leaves in spinach [29], and roots in A. thaliana [30]. In addition to degrading pectin side-chains during the above-mentioned processes, BGALs can also regulate peach fruit softening by modifying PG and PME activities [24]. Hence, BGALs serve as enzymes and regulatory factors. However, the possibility BGALs regulate PTG remains unclear.

BGAL genes with unique consensus active site sequences (G-G-P-[LIVM](2)-x(2)-Q-x-E-N-E-[FY]) have been identified in A. thaliana (17) [31], Oryza sativa (15) [32], Brassica campestris (27) [33], Ipomoea batatas (17) [34], Linum usitatissimum (43) [35], Gossypium hirsutum (51) [36], Cucumis melo (21) [23], Solanum lycopersicum (17) [37], Pyrus pyrifolia (8) [38], Persea americana (4) [39], Prunus persica (17) [40], Fragaria ananassa (4) [41, 42], Malus domestica (13) [43], suggesting plant BGAL genes belong to a multigene family. On the basis of real-time quantitative PCR (qPCR) results, BGAL genes may be expressed in a tissue-specific manner. For example, in Japanese pear, PpGAL1, -2, -3, and -4 are expressed in fruits, whereas PpGAL5, -6, and -7 can be expressed in both fruits and leaves [38]. Similarly, in avocado, AV-GAL1 is highly expressed exclusively in fruits, while PaGAL2 and PaGAL3 transcripts accumulate in both fruits and leaves [39]. In sweet potato, BGAL-encoding genes are mainly expressed in roots or stems, including Ibbgal3, -5, -6, and -10 in the young stem, Ibbgal4 and Ibbgal13 in the old stem, Ibbgal8 in the fibrous root, and Ibbgal11 in the storage root [34]. Interestingly, many BGAL genes are abundantly expressed in the floral organs of different plants, including AtBGAL7, -11, and -13 in A. thaliana [37], OsBgal10 and OsBgal11 in rice [32], LuBGAL9, -15, 16, -18, -21, and -39 in flax [35], GhBGAL7, -18, -33, and -43 in cotton, CmBGAL2, −3, and −4 in melon [23], Mdβ-Gal6, -7, and -11 in apple [43], and Faβgal1, -2, -3 and -4 in strawberry [41, 42]. Furthermore, Liu et al. (2013) [33] determined that BGAL genes (BcBGAL11, -13, and -15) in Chinese cabbage are specifically expressed in pollen. Rogers et al. (2001) [44] reported that a tobacco BGAL (TP5) affects PTG because it is specifically produced in mature pollen grains [44]. These findings may reflect the functional diversity of BGAL genes, while also suggesting a possible link between BGAL genes and PTG. However, pear BGAL genes have not been identified and their expression patterns in tissues and roles during PTG are unknown.

In this study, we identified 12 PbrBGAL genes in the ‘Dangshansuli’ (Pyrus bretschneideri Rehder) genome and analyzed their chromosomal localization, conserved domains and active sites, structures, and evolutionary relationships using bioinformatics techniques. We revealed their expression patterns in 10 tissues and four pollen tube developmental stages on the basis of qPCR technology. Moreover, PbrBGAL6 was identified as a major gene contributing to PTG using antisense oligodeoxynucleotide (as-ODN) technology. Finally, we investigated whether PbrBGAL6 affects apical methylated pectin by modulating PME and PG transcription. In summary, this study provides direct evidence of the regulatory effects of BGAL on PTG, with potential implications for optimizing the PTG mechanism.

Results

Identification of PbrBGAL family members in Chinese white pear

To clarify the role of BGALs during pear PTG, we first identified 12 PbrBGAL genes in the Chinese white pear genome; these genes were named PbrBGAL1–12 (Table 1) according to their chromosomal positions. The predicted PbrBGAL sequences comprised 569–897 amino acids (aa), with a molecular weight of 64.26–99.56 kDa and an isoelectric point of 6.29–9.29. The PbrBGAL2, −3, −5, −6, −7, −8, −11, and −12 sequences included a signal peptide (Table 1). Four conserved domains were detected in PbrBGAL2, −3, −5, −6, −7, −8, −9, and −12, which was more than the three conserved domains in PbrBGAL1, −10, and −11 and the two conserved domains in PbrBGAL4. All PbrBGALs were predicted to be localized in the cell wall, except for PbrBGAL4 in the cytoplasm (Table 1).

Table 1 Pear BGAL genes identified in this study
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PbrBGAL sequence structure and conservation

PbrBGAL sequence characteristics were revealed by aligning sequences using DNAMAN software. According to the results and the classification by Eda et al. (2016) [45], PbrBGAL sequences can be divided into the following six regions: signal peptide region (PbrBGAL6 as an example: 60 aa, M1–Q60), I (323 aa, V61–A383), II (58 aa, G384–S441), III (28 aa, Q442–E469), IV (167 aa, P470–G636), and C-terminal region (236 aa, L637–H872) (Fig. 1). Additionally, the PbrBGAL sequences contained three conserved domains (typical GH35 conserved sequence (G-G-P-[LIVM](2)-x(2)-Q-x-E-N-E-[FY]), SCOP domain d1b9za2, and GHD domain) and one relatively non-conserved domain (galactose-binding lectin domain) (Fig. 1). Moreover, all PbrBGAL sequences included two enzyme active sites (E217 and E288); however, three important functional sites (W/Y290, W/Q/F293, and V/Y/S/I600) differed among the examined sequences according to previous studies (Fig. 1) [45, 46].

Fig. 1
figure 1

PbrBGAL amino acid sequence alignment. Solid blue, green, purple, brown, and gray lines represent I, II, III, IV, and C-terminal regions, respectively. Red, faint yellow, cyan, and green shading indicate the GH35 conserved sequence, SCOP domain d1b9za2, GHD domain, and galactose-binding lectin domain, respectively. The sequence in the red box is a signal peptide. Asterisks and triangles indicate enzyme active and functional sites, respectively. Uppercase letters with different colors reflect different similarities (cyan: ≥ 50%, magenta: ≥ 75%, and black: 100%)

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Phylogenetic analysis of BGAL family members

A phylogenetic tree was constructed using the full-length BGAL protein sequences from 13 species. Twelve PbrBGALs were classified into six subclasses (A–C and E–G) according to the classification in Cucumis melo [23], with subclasses A (PbrBGAL1, −5, and −11) and E (PbrBGAL3, −6, −7, and −10) containing the most PbrBGALs. Subclasses B, C, F, and G contained one (PbrBGAL9), two (PbrBGAL2 and PbrBGAL8), one (PbrBGAL12), and one (PbrBGAL4) PbrBGALs, respectively (Fig. 2). Most of the BGALs in the 13 selected species were classified in subclasses A, E, and F (especially subclasses A and E) (Fig. 2).

Fig. 2
figure 2

Phylogenetic tree of BGALs in Chinese white pear and other species. BGALs in twelve species are AtBGALs (Arabidopsis thaliana), BcBGALs (Brassica campestris ssp. chinensis), CmBGALs (Cucumis melo), CsBGALs (Citrus sinensis), LuBGALs (Linum usitatissimum), MdBGALs (Malus domestica), NtTP5 (Nicotiana tabacum), OsBGALs (Oryza sativa), PhBGALs (Petunia hybrida), PpBGALs (Prunus persica), RhBGALs (Rosa hybrid cultivar) and SlTBGs (Solanum lycopersicum). Differentially colored circular rings and branches represent different subclasses. Differentially colored solid dots indicate BGALs in different species. Bootstrap values less than 50 are not provided in this tree

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Genome distribution and structures of PbrBGAL genes in Chinese white pear

The 12 identified PbrBGAL genes were distributed on 7 of 17 Chinese white pear chromosomes. Chromosomes 2, 3, 4, and 10 each contained one gene (PbrBGAL1, PbrBGAL2, PbrBGAL3, and PbrBGAL6, respectively), whereas chromosomes 9 and 11 contained two genes (PbrBGAL4/PbrBGAL5 and PbrBGAL7/PbrBGAL8, respectively) and chromosome 15 contained four genes (PbrBGAL9, PbrBGAL10, PbrBGAL11, and PbrBGAL12) (Fig. 3). We also determined that PbrBGAL genes were involved in three whole-genome duplication events (involving PbrBGAL11 and PbrBGAL1, PbrBGAL11 and PbrBGAL5, and PbrBGAL2 and PbrBGAL8) and 10 dispersed duplication events (including paired duplications involving PbrBGAL3, −6, −7, and −10; duplications involving PbrBGAL4 and PbrBGAL2, −8, −9, and −12). Hence, the latter was the main duplication event (Fig. 3).

Fig. 3
figure 3

Chromosomal localization and replication mode of PbrBGAL genes. Red and blue dashed lines represent gene pairs derived from whole-genome duplication (WGD) and dispersed duplication (DSD) events, respectively

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To clarify the differences in PbrBGAL gene structures in each cluster, we constructed an unrooted phylogenetic tree and analyzed exon/intron structures and motifs. The PbrBGAL genes consisted of 1–24 exons and 0–23 introns. Moreover, the exon/intron structure was essentially conserved in all subclasses, with the exception of subclass E (Fig. 4). Five genes (PbrBGAL2, −3, −6, −8, and −9) encoded all 15 motifs revealed by MEME, but PbrBGAL4 encoded only three motifs. Notably, 15 PbrBGAL genes encoded motif 8 comprising G-G-P-[LIVM](2)-x(2)-Q-x-E-N-E-[FY]. Similar to the exon/intron structure, similar motifs were detected in all subclasses, except for subclass E (Fig. 4).

Fig. 4
figure 4

PbrBGAL structures and encoded motifs in each subclass. Left: phylogenetic tree of PbrBGAL genes in Chinese white pear, with differentially colored branches representing different subclasses. Middle: PbrBGAL intron/exon structures, with Arabic numerals in the top corner indicating the intron phase. Right: motif structures, with each motif consensus sequence provided below the figure

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Identification of PbrBGAL genes involved in pear PTG

We performed a qPCR analysis of the relative expression levels of 12 PbrBGAL genes in 14 pear samples. Only PbrBGAL6 and PbrBGAL7 were highly expressed in pollen tubes. Interestingly, PbrBGAL6 was highly expressed specifically in the anther and pollen, but its expression level tended to decrease during PTG, suggestive of a relationship with PTG (Fig. 5). In addition, PbrBGAL2, −7, −8, and −9 transcripts accumulated to relatively high levels in the style. PbrGAL1, −3, and −4 were highly expressed in petals, while PbrGAL5, −10, and −11 were highly expressed in leaves (Fig. 5). Both PbrBGAL3 and PbrBGAL12 were expressed at relatively low levels in the examined tissues (Fig. 5).

Fig. 5
figure 5

Tissue-specific expression of 12 PbrBGAL genes in 14 different samples. Ro: roots, St: stems, Le: leaves, Fl: flesh, Pd: pedicels, Ov: ovaries, Re: receptacles, Pt: petals, Sy: styles, An: anthers, Po: mature pollen, 1 h: hydrated pollen, 6 h: growing pollen tubes and 16 h: stopped-growth pollen tubes. Data are presented as the mean ± standard error (n = 3 biological replicates). Different lowercase letters indicate significant differences (P < 0.05)

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Functional analysis of PbrBGAL6

To assess the potential role of PbrBGAL6 during PTG, we first screened primers for an as-ODN experiment. The results indicated that as-ODN4 and as-ODN5 treatments significantly enhanced PTG, with the effects of the latter reaching an extremely significant level (Fig. 6A). Therefore, PbrBGAL6 expression and BGAL enzymatic activity was significantly suppressed in pollen tubes treated with as-ODN5 (as-ODN) (approximately 1.28-fold and 3.2-fold lower than it in the control and s-ODN-treated pollen tubes) (Fig. 6B, 6C), which resulted in a significant increase in pollen tube length (approximately 60 μm) (Fig. 6D, 6E) and apical width (approximately 1.77 μm) (Fig. 6F) as well as cytoplasmic leakage during PTG (Fig. 6D). It may be because effective inhibition of PbrBGAL6 expression affects the apical pectin component to stimulate rapid PTG accompanied by a wide and leaky tip region.

Fig. 6
figure 6

Inhibited PbrBGAL6 expression increases the pear pollen tube length, apical width, and cytoplasmic leakage. A Effect of five as-ODN primer treatments on the pollen tube length. A total of 120 randomly selected pollen tubes per group were used to measure length. B PbrBGAL6 relative expression level. Data are presented as the mean ± standard error (n = 3 biological replicates). C BGAL enzymatic activity of pollen tube. Mean ± standard error (n = 3 biological replicates) are displayed in column diagram. D Representative images of pollen tubes after PbrBGAL6 expression were suppressed. Arabic numerals represent the control, s-ODN, and as-ODN (1, 2, and 3, respectively). The red irregular dashed line and arrow indicate regions with cytoplasmic leakage and pollen tube leakage, respectively; bar = 100 μm. E and F Pollen tube length and apical width. A total of 120 and 48 pollen tubes were used to calculate length and width, respectively. Extremely significant (P < 0.01), significant (P < 0.05), and non-significant differences are indicated by **, *, and n.s., respectively

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PbrBGAL6 affects the apical pectin content in the pear pollen tube

To determine the effect of PbrBGAL6 on PTG, we first measured the total pectin content according to ruthenium red staining. The comparison with the control and s-ODN indicated the apical staining of the as-ODN pollen tube was lighter, with a significant decrease in the average grayscale value (apical 5 μm diameter) (Fig. 7A, 7B). Furthermore, the immunofluorescence of apical methylated pectin was more intense in the as-ODN pollen tube than in the control and s-ODN, which was consistent with the fluorescence intensity (Fig. 7C, 7D). Moreover, inhibited PbrBGAL6 expression clearly decreased the apical ROS content (Fig. 7E, F), but had little effect on actin cytoskeleton depolymerization (Fig. 7G, 7H).

Fig. 7
figure 7

Inhibited PbrBGAL6 expression affects apical pectin, total ROS, and actin cytoskeleton in pollen tubes. A, C, E, and G Representative images of the total apical pectin, methylated pectin, total ROS, and actin cytoskeleton in control, s-ODN and as-ODN, respectively. B, D, F, and H are the quantification of A, C, E, G, respectively. B and F: Average value of grayscale and fluorescence intensity in a circle within tip 5 μm diameter, respectively. D: Fluorescence density from tip to shank on one side of pollen tube. H: Average depolymerization rate of actin cytoskeleton. Bar = 20 μm; 30 (B), 39 (D), 39 (F), and 102 (H) pollen tubes in each group were analyzed. Extremely significant (P < 0.01) and non-significant differences are indicated by ** and n.s., respectively

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We also attempted to determine the reasons for apical pectin changes. Of the three PTG-related PbrPME genes that were analyzed (PbrPME11, −44, and −59), the PbrPME11 expression level was higher (approximately 1.8-fold) in the as-ODN pollen tube than in the control and s-ODN [47] (Fig. 8). We also detected the substantial accumulation of PbrPG14, −20, −21, −22, −24, and −33 transcripts in pollen tubes [48, 49], with PbrPG14 (approximately 1.6-fold), −20 (approximately 1.6-fold), −21 (approximately 1.5-fold), and −24 (approximately 1.7-fold) expression levels that were significantly higher in the as-ODN pollen tubes than in the control and s-ODN (Fig. 8). These results suggest that inhibiting PbrBGAL6 expression may decrease the apical pectin content because of the associated increases in PbrPME11 and PbrPG14, −20, −21, and −24 transcription, thereby temporarily promoting pear PTG.

Fig. 8
figure 8

PbrPME and PbrPG relative expression levels. Data are presented as the mean ± standard error (n = 3 biological replicates). Extremely significant (P < 0.01), significant (P < 0.05), and non-significant differences are indicated by **, *, and n.s., respectively

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Discussion

BGALs contribute to pectin modification-related biological processes through their enzymatic activities. In many species, BGAL families have been screened to identify key members. BGAL genes have been identified in at least 13 species, including 17 AtBGAL genes in A. thaliana [31], 15 OsBgal genes in rice [32], and 27 BcBGAL genes in cabbage [33], 17 Ibbgals in sweetpotato [34], 43 LuBGALs in flax [35], 51 GhBGALs in cotton [36], 21 CmBGALs in melon [23], 17 TBGs in tomato [37], 13 Mdβ-Gals in apple [43], 8 PpGALs in Japanese pear [38], 4 PaGALs in avocado [39], 17 PpBGALs in peach [40] and 4 Faβgals in strawberry [41, 42]. In the current study, we identified 12 PbrBGAL genes in the Chinese white pear genome (Table 1). Interestingly, BGAL gene families are relatively small in Rosaceae species, suggestive of limited duplication (especially in Chinese white pear (Fig. 3), in which only whole-genome duplication and dispersed duplication events were detected). The number of BGAL genes varied greatly among six Rosaceae fruit tree species, possibly because of differences in species and duplication modes. Hence, PbrBGAL genes in Chinese white pear must be comprehensively identified and characterized.

A systematic sequence alignment showed that 12 PbrBGAL sequences contained a typical GH35 conserved sequence and two enzyme active sites (E217 and E288) (Fig. 1), which is in accordance with published results for Japanese pear [38], avocado [39], peach [40], and tomato [37]. Thus, these proteins may have GH35 family hydrolytic enzyme activities. However, PpGAL1 and −4 in Japanese pear [38] and AV-GAL1 in avocado [39] lack galactose-binding lectin domain substrates, which is consistent with our findings for PbrBGAL1, −10, and −11 (Fig. 1). The same phenomenon was reported for PpBGAL6 and −7 in peach [40] and CmBGAL2, −3, −4, −12, −13, and −16 in melon [23]. These results imply that PbrBGALs may bind to different substrates to perform various functions. The functional sites of tomato TBG4 (N282, N459, and V548; unlabeled in Fig. 1) [37] and S. pneumoniae BGAL (W240, W243, and Y455) were not conserved in PbrBGALs, which further reflects the functional divergence among PbrBGALs. This is likely directly associated with gene duplication [50]. Moreover, two other conserved domains (SCOP and GHD) were detected, but their functions will need to be experimentally verified.

A total of 119 BGAL genes from 14 species (including Chinese white pear) were classified into seven subclasses (A–G) that differed regarding function. As expected, all subclass E BGAL genes were highly expressed in flowers (Fig. 2). According to expression characteristics and earlier research by Pan et al. (2022) [23], we subdivided subclass E into part 1 [from OsBgal10 (NM 001403689.1) to BcBGAL13-2 (Bra013052)] and part 2 [from OsBgal6 (P0636F09.15) to AtBGAL6 (AT5G63800)] (Fig. 2). Genes in part 1 were mainly specifically expressed in flowers, pollen, and pollen tubes, including MdBGAL11 (homolog of PbrBGAL3 and −6) [43], NTP5 (participates in PTG) [44], BcBGAL11, −13, and −14 [33], and AtBGAL11, −13, and −14 [37]. Although genes in part 2 were highly expressed in flowers, they were also expressed in other tissues (e.g., stems, leaves, and fruits), including MdBGAL5 and −7, CmBGAL14, and BcBGAL6. Therefore, compared with the part 2 genes PbrBGAL7 and −10, the part 1 genes PbrBGAL3 and−6 are more likely to affect pear PTG. These findings may also help to explain the diversity in intron/exon structures and motifs among subclass E BGAL genes (Fig. 4). Moreover, subclass A BGAL genes may primarily contribute to fruit ripening and softening, shedding, and stem and leaf development. For example, MdBGAL1 and MdBGAL2, which are homologs of PbrBGAL11 and PbrBGAL1, are highly expressed during apple fruit ripening and softening [43]. The antisense-based inhibition of tomato SlTBG4 expression can delay fruit softening (40% increase in firmness) [51]. In addition, RhBGAL1 and CmBGAL may be involved in the abscission of flowers and young fruits [52, 53]. MdBGAL9, which is homologous to PbrBGAL5, is highly expressed in stems and leaves, and probably contributes to their development [43]. Both AtBGAL2 [37] and BcBGAL1-2 [33] may have similar functions. Accordingly, PbrBGAL1, −5, and −11 in subclass A may influence fruit ripening and softening or stem and leaf development. Similarly, we also deduced that subclasses F, B/C/D, and G may be mainly related to the development of leaves/flowers/fruits, stems/leaves/fruits, and all tissues, respectively. These results suggest that PbrBGAL3 and PbrBGAL6 may regulate pear PTG.

To verify the results of the above-mentioned bioinformatics analysis, we completed a qPCR analysis of the tissue expression patterns of 12 PbrBGAL genes. PbrBGAL6 was abundantly expressed in the anther, pollen, and pollen tube; this gene probably plays a negative regulatory role during PTG (Fig. 5). In other species, more BGAL genes are specifically expressed in the stamen. For example, in cotton, GhBGAL7, -17, -18, -33, and -43 are specifically expressed in the anther and filament [36]. Similar results were obtained for Chinese cabbage genes BcBGAL11, -13, and -15 and rice genes OsBgal10 and −11 [32, 33]. Moneo-Sánchez et al. (2018) [54] detected relatively few phenotypic changes in β-galactosidase subfamily a1 mutants, indicating that A. thaliana BGAL genes may have redundant functions [54]. The functional redundancy among these genes may be sufficient for regulating stamen-related processes. However, the functional redundancy of BGAL genes may be difficult to assess in pear stamens and pollen tubes because of the substantial transcription of only one gene, suggesting PbrBGAL6 is probably crucial for pear PTG.

We also observed that inhibiting PbrBGAL6 expression caused BGAL enzymatic activity decrease to rapidly enhance PTG and increase the apical width, which was accompanied by gradual cytoplasm leakage (Fig. 6). A previous study revealed that BGALs can negatively regulate the pectin content to increase cell wall porosity by depolymerizing pectin side-chains involving β-(1,4)/-(1,3)/-(1,6)-glycosidic bonds [23]. This may explain how PbrBGAL6 contributes to pear pollen tube elongation and leakage. However, our results indicated the total and methylated apical pectin content decreased after PbrBGAL6 expression was suppressed (Fig. 7 A-D). We previously observed that changes in PpBGAL10 and PpBGAL16 expression affect PpPME3 and PpPG21 transcription, which influences peach fruit softening [24]. We speculate that in addition to its enzymatic activity, PbrBGAL6 may also affect the expression of genes encoding pectin-modifying enzymes (PbrPME and PbrPG) to help regulate the above-mentioned phenotype. Therefore, the expression of the main PbrPME genes (PbrPME11, PbrPME44, and PbrPME59) [47] and PbrPG genes [49] related to pear PTG were analyzed. The results showed that the transcription of PbrPME11 (approximately 1.8-fold) and PbrPG14 (approximately 1.6-fold), −20 (approximately 1.6-fold), −21 (approximately 1.5-fold), and −24 (approximately 1.7-fold) increased significantly in control and as-ODN pollen tubes (Fig. 8). PbrPMEs may be associated with an increase in demethylated pectin (hard pectin) and a decrease in the amount of methylated apical pectin (soft pectin). An increase in the hard pectin content can inhibit PTG [18]; however, PbrPGs specifically break the α-(1–4) glycosidic bond of hard pectin, leading to the degradation of HG pectin [19, 20]. Additionally, more PbrPG genes (PbrPG14, -20, -21, and -24) had increased expression levels than PbrPME genes (PbrPME11) (Fig. 8), which may ensure that for hard pectin, the degradation rate is greater than the synthesis rate. The PbrBGAL6 may lead to some pectin degradation, ultimately decreasing apical pectin levels in pollen tubes. These results suggest that the PbrBGAL6 enzymatic activity (direct degradation of RG1 pectin) and regulatory effects (indirect degradation of HG pectin via increased PME and PG production) decrease the apical pectin content, leading to transient overgrowth accompanied by cytoplasmic leakage in the pear pollen tube. According to previous speculation [24], the possibility that PbrBGAL6 can produce small sugars (including galactose) by degrading pectin and that sugars act as signaling molecules to promote the synthesis of hormones, such as ethylene [55], or the expression of transcription factor genes (including SEP1 and PbrbZIP77) [16, 56] remains the key to explaining the regulatory effects of PbrBGAL6 on PbrPME11, PbrPG14, -20, -21, and -24 expression. Moreover, ROS may serve as signaling molecules [57], oxidants affecting cell wall cross-linking, and cell wall-loosening compounds [58] to participate in PTG. High ROS concentrations can result in cell wall rigidification that prevents cell expansion [59]. Considering our results, inhibiting PbrBGAL6 expression can decrease the apical ROS level (Fig. 7E, 7F), suggestive of the loosening of the apical cell wall. Cell wall loosening within a suitable range due to an appropriate decrease in the apical ROS content may promote polar cell growth; however, excessive loosening can cause the cell to rupture, thereby limiting growth [60]. In this study, PbrBGAL6 was revealed to likely influence changes in apical ROS levels within an appropriate range to promote transient PTG. Therefore, the pollen tube phenotype due to inhibited PbrBGAL6 expression may also be related to the apical ROS content. The relationship between apical ROS and pectin content changes will need to be explored in future investigations.

Conclusions

In summary, we identified 12 PbrBGAL genes (PbrBGAL1–12 divided into six subclasses) in the Chinese white pear genome. Bioinformatics analyses revealed the functional specificity of each PbrBGAL subclass, with PbrBGAL6 in subclass E potentially involved in PTG. The results of an as-ODN assay indicated that PbrBGAL6 can negatively regulate pear PTG, while positively regulating the pollen tube apical pectin content by decreasing the expression of PbrPME11 and PbrPG14, -20, -21, and -24. Our results provide direct evidence of the regulatory effects of BGAL on PTG.

Methods

Bioinformatic analysis

Pear PbrBGAL genes were identified using a three-step process. First, AtBGAL and CmBGAL sequences in A. thaliana and C. melo, respectively, were selected as queries to screen the Pear Genomics Database (https://peargenome.njau.edu.cn/) [61]. Second, we searched the Pear Genomics Database using Hidden Markov Model profiles of the Glyco_hydro_35 domain (Accession no. PF01301) [23] from the Pfam database (https://pfam.xfam.org/). Third, candidate PbrBGAL genes obtained in the first two steps were screened for the presence of the Glyco_hydro_35 domain using the online SMART database (https://smart.embl.de/index 2.cgi). General information regarding PbrBGAL1–12 was obtained from the Pear Genomics Database, including the gene description, length of the encoded amino acid sequence, and genome position and direction. In addition, the molecular weight/theoretical isoelectric point and signal peptide of each PbrBGAL were predicted using the online software ExPASY (https://web.expasy.org/protparam/) and SignalP v3.0 (https://www.cbs.dtu.dk/services/SignalP/index.php), respectively. Moreover, PbrBGAL subcellular localizations were determined using the online software Cell-PLoc 2.0 (http://www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/). For multiple sequence alignments, DNAMAN6.0 (Lynnon Biosoft, San Ramon, CA, USA) and WebLogo (https://weblogo.berkeley.edu/logo.cgi) were used, with default parameters and consensus sequences, respectively. A phylogenetic tree was constructed using MEGA6.0 software. Specifically, a neighbor-joining method with 1,000 bootstrap replicates to evaluate edge support was applied [62]. MapDraw was used to clarify the chromosomal localization of PbrBGAL genes [63]. Information regarding the replication of these genes was previously reported by Qiao et al. (2019) [64]. Exon/intron structures and motifs were analyzed using the online programs GSDS (https://gsds.cbi.pku.edu.cn/) and MEME, respectively (https://meme-suite.org/tools /meme).

Plant materials

Ten different tissues (roots, stems, leaves, fruit flesh, pedicels, ovary, receptacles, petals, styles, and anthers) were obtained from ‘Dangshansuli’ trees growing at the Fruit Experimental Field of Anhui Science and Technology University and then immediately frozen in liquid nitrogen prior to isolating RNA. Uncracked mature anthers were incubated at 25 ± 1 °C to produce pollen. Dried pollen grains were wrapped in sulfuric acid paper and then stored at − 20 °C in a silicone desiccant. Pollen was cultured in pear pollen medium containing 0.4 mM Ca(NO3)2, 1.5 mM H3BO3, 5 mM 2-(N-morpholino) ethanesulfonic acid hydrate, and 292.1 mM sucrose (pH adjusted to 5.8 using NaOH) at 25 °C with shaking (120 rpm). Pollen and pollen tubes were collected by centrifugation (400 g at 25 °C) at four time points during the culture period as described by Zhou et al.(2016) [48]: 0 h (mature pollen), 1 h (hydrated pollen), 6 h (growing pollen tubes), and 16 h (no detectable pollen tube growth). All four samples were quickly stored at − 80 °C prior to the subsequent RNA extraction.

Real‑time quantitative PCR (qPCR) assays

RNA and cDNA preparation: Total RNA was isolated from each sample using a FastPure Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China). RNA quality was assessed by 1.2% agarose gel electrophoresis, whereas RNA integrity was evaluated using a NanoDrop 2000 ultraviolet spectrophotometer (Thermo Fisher Scientific, Waltham, USA). An EasyScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit (TransGen, Beijing, China) was used to reverse transcribe high-quality RNA to cDNA. Primer preparation: Gene-specific primers (Additional file 1) were designed using Primer Premier 6.0 (PREMIER Biosoft International, San Francisco, USA) and synthesized (PAGE purification type) by Sangon Biotech (Shanghai) Co., Ltd (Sangon, Shanghai, China). qPCR assay: Samples comprising 1 μL 10 μM sense and anti-sense primers, 1 μL 100 ng/μL cDNA, 2 μL double-distilled H2O, and 5 μL 2 × SYBR Premix Ex Taq II (Vazyme) were prepared for a qPCR analysis, which was completed using a LightCycler 480 II system (Roche, Basel, Switzerland) and the following program: 95 °C for 1 min; 40 cycles of 95 °C for 10 s, annealing temperature for 30 s, and 72 °C for 30 s; 40 cycles to construct a melting curve. Relative gene expression levels were calculated according to the 2−ΔΔCt method [65], with a pear TUB gene (Pbr042345.1) serving as a reference control. The qPCR assay was repeated at least three times.

Antisense oligodeoxynucleotide (as‑ODN) assays

A published procedure that was modified slightly was used to complete as-ODN assays (Chen et al. 2018) [66]. Primer preparation: PbrBGAL6 as-ODN primers were designed using the RNAfold web server (https://rna.tbi.univie.ac.at/cgi-bin/RNAWebSuit e/RNAfold.cgi). Candidates were screened by evaluating their match to target regions using Snap Gene 2.4.3 (https://www.snapg ene.com). Target primers for as-ODNs and the corresponding sense-ODNs (s-ODNs) were synthesized using phosphorothioate oligos that were purified via high-performance liquid chromatography (Additional file 1). As-ODN assays: ODN primers and a lipofectamine 3000 transfection reagent (Thermo Fisher Scientific) were mixed in the above-mentioned pear pollen culture medium for 15 min at room temperature. The mixture was then co-incubated with pollen tubes (approximately 50 μm) in the culture medium (final ODN primer concentration: 20 μM) for 1.5 h at 25 °C with shaking (120 rpm). After rinsing three times with the culture medium, pollen tubes were examined and photographed using an Olympus IX73 inverted fluorescence microscope (Olympus, Tokyo, Japan). The length and apical width were measured for at least 40 and 16 pollen tubes, respectively, for each replicate of each sample using Image-ProPlus 6.0 software (Media Cybernetics, Rockville, USA). Experiments were repeated three times.

β-galactosidase activity

BGAL activity of pollen tube was determined by β-Galactosidase (β-GAL) Activity Assay Kit (Boxbio, Beijing, China) according to instruction. The pollen tube samples of as-ODN assay were collected (approximately 0.15 g) to crush using pestle in liquid nitrogen. Ground samples were added to 1 ml extraction buffer, and then centrifuged at 15,000 g 4 °C for 20 min. The 50 μL supernatant was orderly mixed with reagent one (200 μL) and reagent two (250 μ L) to incubate for 30 min at 37 °C. Next, the reagent three (1 mL) was added to above mixture, and let it stand at room temperature for 2 min. As BGAL decomposed p-nitrophenyl-β-D-galactopyranoside to p-nitrophenol, which has the maximum absorption at 400 nm, a microplate reader (Thermo Fisher Scientific) was used to measure the absorbance. The production of 1 nmol of p-nitrophenol per gram tissue per hour under 37 °C was defined as one enzyme activity unit (U). Three replicates for each sample.

Detection of pectin at the pear pollen tube apex

Total apical pectin was stained using ruthenium red as previously described [67]. Briefly, 0.05% ruthenium red (Coolaber, Beijing, China) (final concentration) was added to the above-mentioned culture medium containing approximately 50 μm pollen tubes for a 1-h co-incubation at 25 °C with shaking (120 rpm). Pollen tubes were washed three times with culture medium, after which they were examined and photographed using an Olympus IX73 inverted fluorescence microscope (Olympus). Image-ProPlus 6.0 software (Media Cybernetics) was used to calculate the apical grayscale value.

Immunofluorescence technology was used to detect methylated pectin as described by Tang et al. (2023) [16]. Treated pollen tubes were fixed in 4% paraformaldehyde at 25 °C for at least 30 min, after which they were rinsed three times with phosphate-buffered saline (PBS) and blocked with 1% BSA for 30 min. They were subsequently washed three times with 0.1% BSA and then incubated with 0.1% diluted LM20 polyclonal antibody (1:10) overnight. Next, pollen tubes were rinsed three times with 0.1% BSA and then mixed with 0.1% diluted FITC (goat anti-rat; 1:50) for at least 45 min. Pollen tubes were examined using an Olympus IX73 inverted fluorescence microscope (Olympus). ZEN software (Zeiss, Oberkochen, Germany) was used to analyze the fluorescence intensity on one side extending from the tip to the shank along the cell wall.

Staining of total apical ROS and the actin cytoskeleton in pear pollen tubes

The apical ROS content of pollen tubes was determined using CM-H2DCFDA (20 μM final concentration) [62]. Pollen tubes after the as-ODN experiment were immediately washed with culture medium and then co-incubated with CM-H2DCFDA for 20 min at 25 °C in darkness. Pollen tubes were rinsed with culture medium at least three times and then examined using an Olympus IX73 inverted fluorescence microscope (Olympus).

The actin cytoskeleton was examined as described by Chen et al. (2018) [66]. Pollen tubes were fixed and rinsed following a three-step process as described for the immunofluorescence assay. Pollen tubes were permeabilized using 0.5% Triton X-100 at 25 °C for 10 min. After washing three times with PBS, the actin cytoskeleton was stained using 1% phalloidin for 20 min at 25 °C and then examined using an Olympus IX73 inverted fluorescence microscope (Olympus). The average apical fluorescence density of ROS and the actin cytoskeleton in a region with a 5 μm diameter was measured using ZEN software. The experiment was repeated three times.

Statistical analysis

GraphPad Prism 8.0.1 (GraphPad, San Diego, USA) was used to generate figures presenting mean values and standard errors. Post hoc Tukey’s tests for a one-way ANOVA (P < 0.05) and Student’s t-test (P < 0.05) were completed using IBM SPSS Statistics 22 software (IBM, Armonk, USA) to determine the significance of differences between multiple samples and between two samples, respectively.

Data availability

The datasets analyzed during the current study are available in the Pear Genome Project, CuGenDB, GenBank and TAIR repository, the gene accession numbers of Pyrus bretschneideri from Pear Genome Project (https://peargenome.njau.edu.cn/) are shown in Table 1, The gene accession numbers of Arabidopsis thaliana from TAIR (http://www.arabidopsis.org/), of Solanum lycopersicum and Prunus persica from Phytozome v13 (https://phytozome.jgi.doe.gov), of Cucumis melo from CuGenDB (http://cucurbitgenomics.org/), of Brassica campestris ssp. chinensis, Citrus sinensis, Linum usitatissimum, Malus domestica, Nicotiana tabacum, Oryza sativa, Petunia hybrida and Rosa hybrid cultivar from GenBank (https://www.ncbi.nlm.nih.gov/genbank/) respectively are shown in Additional file 2.

Abbreviations

BGAL:

β-Galactosidase

SI:

Self incompatibility

PTG:

Pollen tube growth

GH35:

Glycosyl hydrolase 35

Chr:

Chromosome

Mw:

Molecular weight

pI:

Theoretical isoelectric point

WDG:

Whole-genome duplication

DSD:

Dispersed duplication

PME:

Pectin methylesterase

PG:

Polygalacturonase

HG:

Homogalacturonan

RG-I:

Rhamnogalacturonan I

RG-II:

Rhamnogalacturonan II

PAE:

Pectin acetylesterase

PL:

Pectate lyases

PMEI:

PME inhibitor

qPCR:

Real-time quantitative PCR

as-ODN:

Antisense oligodeoxynucleotide

s-ODN:

Sense oligodeoxynucleotide

PBS:

Phosphate buffer solution

BSA:

Bovine serum albumin

FITC:

Fluorescein isothiocyanate

ROS:

Reactive oxygen species

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Acknowledgements

We thank Xiaomin Lu for editing the English text of a draft of this manuscript.

Funding

This study was supported by grants from Youth Fund of the National Natural Science Foundation of China (Grant number 32302615), Natural Science Research Project of Anhui Educational Committee, China (Grant number 2022AH051616), and College Students’ Innovative Entrepreneurial Training Plan Program, China (Grant number S202310879169).

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Authors and Affiliations

  1. Department of Horticulture, College of Agriculture, Anhui Science and Technology University, Chuzhou, 233100, China

    Yusheng Xu, Lan Xu, Hao Wang, Yuqian Wang, Xueping Zhang, Kaijing Zhang, Yihu Sui, Jingjing Qian, Shuangshuang Jia, Ming Qian & Guangrong Cui

  2. College of Horticulture, Nanjing Agricultural University, Nanjing, 210032, China

    Mingliang Zhang

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  1. Yusheng Xu
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  2. Lan Xu
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Contributions

YX, SJ, GC and MQ designed the experiments. YX, LX, MZ, HW and YW took all pear samples. YX, LX, MQ, YW, HW and XZ performed all the molecular biology experiments. YX, KZ, YS, JQ and MQ performed bioinformatics analysis. YX, LX, MZ and MQ analyzed all the data. YX, MQ and GC wrote the manuscript. YX and MQ revised the whole manuscript.

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Correspondence to Ming Qian or Guangrong Cui.

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Xu, Y., Xu, L., Zhang, M. et al. PbrBGAL6 promotes pollen tube growth by influencing apical pectin level in Pyrus bretschneideri. BMC Genomics 26, 321 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12864-025-11429-9

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BMC Genomics

ISSN: 1471-2164

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