Pectin dynamics of alfalfa pollen tube elongation in response to low boron based on proteomics

Abstract

Background

Boron is an essential micronutrient for plant reproductive growth, and its key function in plants is mediated through cross-linking with the cell wall pectic polysaccharide rhamnogalacturonan II. As an important forage crop, alfalfa suffers from inhibited pollen tube elongation under low boron conditions, leading to floret abortion and reduced seed set. However, the molecular mechanism by which low boron regulates pectin to affect alfalfa pollen-tube elongation remains unclear.

Results

Under low boron condition, methylesterified pectin shifted from an apical to subapical and shank localization, while de-esterified pectin became predominantly concentrated at the pollen tube apex, indicating a boron-dependent reorganization of pectin distribution. Proteomic analysis of pollen tubes grown under varying boron concentrations revealed significant enrichment of pectin methylesterase activity, polygalacturonase activity, and carbohydrate metabolic processes. Among 56 shared differentially expressed proteins, two pectin methylesterase inhibitors — MsPMEI14 and MsPMEI15 — were identified as candidates potentially associated with this response. Chromosomal localization, phylogenetic, and cis-element analyses suggested that both genes were evolutionarily conserved. Subcellular localization analysis demonstrated that both MsPMEI14 and MsPMEI15 localized to the endoplasmic reticulum and acidic vesicular compartments, indicating that both proteins are secreted to the cell wall via the conventional secretory pathway (CPS), where they regulate pectin methylesterification to support pollen tube elongation.

Conclusions

These findings suggest that low boron-induced inhibition of pollen tube elongation is associated with altered spatial distribution of methylesterified and de-esterified pectins, potentially mediated through the regulatory activity of MsPMEI14 and MsPMEI15 on pectin methylesterase activity. Together, this study provides new insights into how pectin dynamics respond to low boron conditions to regulate pollen tube elongation, and highlights MsPMEI14 and MsPMEI15 as promising candidates for future functional characterization aimed at elucidating the molecular basis of low boron-induced pollen tube growth inhibition.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08976-y.

Highlights

Under low boron condition, the pectin dynamics in different regions of the pollen tube were altered;

Proteomics analysis revealed changes in key cell wall components in response to low boron levels during alfalfa pollen tube elongation;

Genome-wide identification and analysis of alfalfa pectin methylesterase inhibitor;

This work provides potential candidate proteins for increasing alfalfa seed yield under low boron conditions.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-026-08976-y.

Introduction

As a forage crop, alfalfa (Medicago sativa) is cultivated over the largest area and has the widest geographical distribution worldwide. It is predominantly grown in North America, Europe, and Oceania, and is highly valued for its superior nutritional quality [1]. However, floral abortion and low seed set are frequently observed in agricultural production, resulting in reduced seed yield of alfalfa. Therefore, understanding floral fertility is crucial for improving seed yield. Boron is an essential micronutrient for normal plant growth and development [2]. In plant cell walls, boron acts as a crosslinking molecule that forms borate esters with rhamnogalacturonan II (RG-II), thereby increasing wall porosity and elasticity [3]. Previous studies have demonstrated that foliar application of boron fertilizers effectively promotes reproductive development in alfalfa and significantly increases seed yield [4, 5]. Our previous work showed that low-boron conditions inhibit pollen tube elongation and identified a key transcription factor, MsWRKY49, which mediated pollen tube elongation by regulating reactive oxygen species (ROS) homeostasis [6]. However, the molecular mechanisms by which low boron affect seed yield has not yet been fully elucidated.

In angiosperms, pollen germination and pollen tube growth constitute essential steps in sexual reproduction [7]. As the fastest-growing cells in terrestrial plants, pollen tubes undergo rapid tip elongation, which requires an abundant traffic of vesicles containing new cell wall materials and proteins toward the plasma membrane [8]. During pollen tube elongation, the cell wall provides mechanical support, protects the gametes from physical damage, and counteracts turgor pressure, thereby playing a crucial role in ensuring proper pollen tube growth. The pollen tube cell wall is composed of cellulose, callose, pectin, and hemicelluloses. The cell wall at the tip of the pollen tube is contains only a thin layer of pectin, lacking cellulose and callose [9]. Among these components, dynamic changes in pectin are particularly important for polar elongation of the pollen tube. In angiosperms, the pollen tube tip is mainly composed of methylesterified pectin, whereas the subapical and shank regions are enriched in de-esterified pectin [10]. Highly methylesterified pectin at the tip confers extensibility to the cell wall in this region, facilitating pollen tube elongation. Conversely, the accumulation of de-esterified pectin enhances the mechanical stability of the subapical and shank regions, thereby maintaining pollen tube structural integrity [11].

Pectin methylesterases (PMEs) are widely distributed in plants [12]. Numerous studies have demonstrated that PMEs were involved in regulating pollen development and pollen tube elongation [13–15]. Several PME genes exhibiting pollen-specific expression have been identified in Arabidopsis [16]. PME also participates in the biosynthesis of poplar wood (secondary xylem) [17]. Exogenous application of PME caused thickening of the apical cell wall in Nicotiana benthamiana pollen tubes, ultimately inhibiting their elongation [13]. In Arabidopsis, mutation of VGD1, a homologous PME gene, resulted in unstable pollen tube elongation [14]. PME activity is typically regulated by pectin methylesterase inhibitors (PMEIs), which are key regulators of the degree of methylesterification and have significant effects on the mechanical properties of the cell wall. The balance between methylesterified and de-esterified pectins in the pollen tube wall is maintained through the secretion of specific PMEI proteins. Studies showed that AtPMEI2 in Arabidopsis was localized to the apical cell wall of pollen tubes, and its overexpression suppresses PME activity to regulate cellular stability [18]. Ectopic expression of an antisense BoPMEI1 in Arabidopsis resulted in delayed pollen tube growth, leading to male sterility and reduced seed set [7]. However, little is known about whether pectin-related proteins participate in regulating pollen tube elongation under low-boron conditions. With the rapid development of bioinformatics technologies, the PMEI gene family has been systematically identified in multiple plant species. To date, 95 members have been identified in Brassica oleracea [19], 42 in Pyrus bretschneideri [20], 49 in Oryza sativa [21], and 95 in Linum usitatissimum [22]. In Arabidopsis, based on the distribution of conserved PME and PMEI domains, 136 PME/PMEI sequences were classified into three groups: the first group contains only the PME domain, the second group contains both PME and PMEI domains, and the third group contains only the PMEI domain [22]. Functional studies have shown that the PMEI gene family plays important roles in plant growth, development, and responses to abiotic stress. In Camellia sinensis, under both long-day and short-day conditions, CsPMEI2/4 promote early flowering in transgenic Arabidopsis and increase the expression levels of many flowering-related genes [23]; in rice, overexpression of OsPMEI28 leads to a dwarf phenotype and significantly reduced grain size [24]. However, functional studies of the PMEI gene family in alfalfa remain limited, and their specific biological roles require further investigation.

Previous studies have shown that different boron concentrations inhibit pollen tube elongation in alfalfa [6]; in this study, to investigate whether pectin dynamics are involved in the physiological process by which low boron inhibits pollen tube growth, we employed immunofluorescence staining to demonstrate the abnormal accumulation of pectin in pollen tube cell walls under low boron conditions. Based on proteomic sequencing combined with bioinformatic analysis of the MsPMEI gene family, two MsPMEIs (MsPMEI14 and MsPMEI15) were identified as potentially playing important roles in the response to low boron during pollen tube elongation. To further clarify the functional characteristics of the MsPMEI family, we performed a genome-wide comparative analysis, identifying 93 MsPMEI genes in alfalfa and conducting chromosome mapping, phylogenetic tree construction, and cis-element analysis. Subcellular localization experiments demonstrated that both MsPMEI14 and MsPMEI15 localized to the endoplasmic reticulum (ER) and acidic vesicular compartments, indicating that these proteins are delivered to the cell wall via the conventional secretory pathway (CPS). Collectively, our proteomic and genome-wide analyses provide valuable insights into pectin dynamics-mediated regulation of pollen tube elongation under low-boron conditions and establish a foundation for further elucidating the roles of MsPMEIs in the low boron response.

Materials and methods

Plant materials and growth conditions

In this study, the alfalfa (Medicago sativa) seeds of ‘Zhongmu No. 1’ were purchased in 2025 from Jiuquan Daye Seed Co., Ltd., China. The seeds were placed in germination boxes containing moist paper beds and allowed to germinate for 9 days in a growth chamber at 24 °C under a 16 h light/8 h dark photoperiod. The seedlings were then transplanted to a plant house and grown at 22–24 °C with a 16 h light/8 h dark photoperiod and 60% relative humidity. After the plants had grown for 3 months and reached the flowering stage, pollen was collected for in vitro culture.

In vitro pollen germination culture

Once the alfalfa plants reached anthesis, pollen was collected from the florets for in vitro culture. Pollen grains were gently removed from the florets using tweezers and dissecting needles and transferred to concave microscope slides, onto which liquid culture medium was added. The liquid medium consisted of 10% sucrose, 0.01% calcium chloride, and boric acid at three concentrations: 0% (B0, boron free), 2 × 10⁻⁴% (B1, low boron), and 0.01% (B2, adequate boron). The slides were placed in germination boxes lined with moist filter paper and incubated at 24 °C in darkness for 2 h, after which pollen tube germination was examined under a DS-Ri2 microscope. Pollen tubes from different boron treatments were then collected, flash-frozen in liquid nitrogen, and stored at − 80 °C.

Immunofluorescence staining of pollen tubes

Pollen was incubated in liquid media containing different boron concentrations for 1 h, followed by fixation in 300 µM m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) for 2 h, and washed three times with PBS buffer (pH 7.4) at room temperature for 5 min each. Samples were blocked with 3% BSA (prepared in PBS) at room temperature for 2 h. The pollen tubes were then washed three times with PBS for 5 min each and incubated overnight at 4 °C in darkness with JIM5 or JIM7 antibodies (Kerafast; 1:10 diluted in PBS containing 0.1% BSA). Afterward, the pollen tubes were washed three times with PBS for 5 min each and incubated for 1 h in darkness with Alexa Fluor 488–conjugated donkey anti-rat IgG (Sangon; 1:100 diluted in PBS containing 0.1% BSA). After three final washes with PBS, samples were observed using a laser confocal microscope.

SRS microscopic imaging

Pollen was cultured in liquid medium with boron concentrations of B1 and B2 for 1 h, then fixed in 4% tissue cell fixative (Solarbio) for 2 h, washed three times with 1× PBS, then dropped onto glass slides and covered with coverslips prior to SRS imaging. The SRS imaging system used was a multimodal nonlinear optical microscope (UltraView, Zhendian (Suzhou) Medical Technology Co., Ltd, Suzhou, China). The stimulated Raman scattering laser beams consisted of a tunable pump beam (680–1300 nm) and a fixed-wavelength Stokes beam (1045 nm, modulated at a resonant frequency of 2.7 MHz). During imaging, the grating-dispersed laser powers were 20 mW at 963 nm and 200 mW at 1045 nm, which were used to excite the stimulated Raman signal of pectin (850 cm⁻¹) in pollen tubes. The microscope was equipped with a UPLSAPO 60×water-immersion objective with a numerical aperture (NA) of 1.2. Three-dimensional spectral scanning was performed over the range of 810–900 cm⁻¹ with a Z-step size of 1 μm and a wavenumber step of 3 cm⁻¹. Each image consisted of 400 × 400 pixels with a field of view of 104 × 104 μm and a spatial resolution of 0.26 μm per pixel. The pixel dwell time was 10 µs, and signals were averaged four times. Spectral images were processed using the K-means clustering algorithm, which iteratively optimizes the partitioning of the dataset into a predefined number of clusters to maximize similarity within clusters and differences between clusters. The different clusters identified by this approach were assigned distinct colors, allowing the spatial localization and distribution of components within each cluster to be distinguished in the images, with image colors corresponding one-to-one with spectral colors. In this experiment, the optimal number of clusters was determined and set to five classes.

DIA-based proteomic analysis

Alfalfa pollen was cultured in liquid germination media with different boron concentrations (B0, B1, and B2) for 2 h, after which the pollen was collected, ground into powder in liquid nitrogen, and transferred into 2 mL centrifuge tubes for proteomic sequencing; each treatment included three biological replicates, with pollen from 100 florets per replicate. The frozen samples dissolved in SDT (containing 100 mM NaCl) with 1/100 volume of DTT (Dithiothreitol). After thorough mixing, the mixture was sonicated on ice for 5 min to ensure complete protein extraction. The lysate was centrifuged, and the supernatant was treated with IAM (Iodoacetylamine) solution and incubated in darkness for 15 min. Proteins were then precipitated by adding four volumes of pre-chilled acetone (− 20 °C) and incubated at − 20 °C for at least 30 min. The mixture was centrifuged at 12,000 g for 15 min at 4 °C, and the resulting pellet was washed with 1 mL of cold acetone (− 20 °C) and air-dried. The dried pellet was fully dissolved in an appropriate volume of Dissolved Buffer (DB buffer), and protein concentrations were determined using the Bradford assay. Proteomic analysis was performed using a Vanquish™ Neo UHPLC–Astral mass spectrometer with a DIA acquisition method to obtain raw data. Raw data were processed using DIA-NN software for database searching, and Gene Ontology (GO) and IPR functional annotation was conducted using InterProScan. Differentially expressed proteins (DEPs) were identified based on fold change ≥ 1.2 and P < 0.05. Identified DEPs were subsequently annotated with GO terms and analyzed for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (https://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD075180.

Identification of MsPMEI gene family members

To comprehensively identify the MsPMEI gene family in alfalfa, we retrieved the genome information of the cultivar ‘Zhongmu No.1’ from the Figshare website. To improve the accuracy of this identification process, we downloaded 114 AtPMEI protein sequences from Arabidopsis via the TAIR database and used them as query sequences, all 114 AtPMEI proteins have been reported to contain the conserved PMEI domain [22]. Candidate MsPMEI proteins in alfalfa were identified using BLASTP with E-value cutoff of 1.0 × 10⁻¹⁰. The identified alfalfa MsPMEI protein sequences were aligned using HMMER3.0 based on a hidden Markov model with an E-value threshold of 0.001. The presence of the predicted PMEI domain within these sequences was verified using the Pfam database with the HMM profile (PF04043). The final set of 93 identified MsPMEI genes were designated MsPMEI1 to MsPMEI93 according to their order in the genome assembly. These genes were named MsPMEI1 to MsPMEI93 based on their physical locations across the eight chromosomes. Visualization was performed using TBtools.

Phylogenetic tree construction and cis-element analysis

Multiple sequence alignment was performed using MEGA11 on 93 MsPMEI proteins from alfalfa and 114 AtPMEI proteins from Arabidopsis to investigate their evolutionary relationships. Phylogenetic trees were constructed using the Neighbor-Joining (NJ) method, and the reliability of the inferred topology was assessed with 1,000 bootstrap replicates (https://www.megasoftware.net/). The phylogenetic tree was visualized using the online tool Evolview (evolclient.evolgenius.info). The 2,000 bp nucleotide sequences upstream of the start codon of 93 MsPMEI genes were extracted from the alfalfa genome and submitted to the PlantCARE database (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/) for cis-regulatory element prediction. The predicted regulatory elements were classified according to their putative regulatory functions. Finally, these cis-regulatory elements were visualized using TBtools.

Subcellular localization

Specific primers were designed using SnapGene, with SpeI and KpnI restriction sites introduced, and synthesized by Sangon Biotech (Beijing, China) (MsPMEI14: F- ggtcgacatttaaatactagtATGGATTCCAGGTTCAATAAAAGC, R- gcccttgctcaccatggtaccAAACTTATGGTTATTAACTAAAGTGTTCATG; MsPMEI15: F-ggtcgacatttaaatactagtATGATGGATTCTAGGTTCAATAAAAGC, R-gcccttgctcaccatggtaccAATCTTGTGGTTATTAACTAAAGTGTTCAT). The coding sequences of MsPMEI14 and MsPMEI15 were amplified from whole-plant alfalfa cDNA using Phanta Max High-Fidelity DNA Polymerase (Vazyme, Nanjing, China). The purified PCR fragments were inserted into the linearized pSuper1300-GFP vector using a gel extraction kit and the ClonExpress II One-Step Cloning Kit (Vazyme, Nanjing, China), followed by sequence verification. Sequencing results were aligned with the MsPMEI14 and MsPMEI15 gene sequences using SnapGene, and plasmids were extracted from bacterial cultures containing correct inserts. The confirmed plasmids were transformed into Agrobacterium tumefaciens strain GV3101 by heat shock, and the transformed cells were subsequently infiltrated into 4-week-old N. benthamiana leaves. After incubation for 40–60 h, signals were observed using a confocal laser scanning microscope (Zeiss 880).

Results

Effect of low-boron treatment on pectin dynamics in alfalfa pollen tubes

To investigate whether pectin dynamics are involved in the process of low-boron-induced inhibition of pollen tube elongation, we used the pectin-specific antibodies JIM5 and JIM7 to perform immunofluorescence labeling of pollen tubes grown under different boron concentrations. Microscopic observation revealed that under the B2 (adequate boron), JIM5-labeled de-esterified pectin was mainly distributed along the shank region of the pollen tube, whereas under the B1 (low boron), it became concentrated at the tip and diminished in the shank region. For JIM7-labeled methylesterified pectin, the B2 condition led to its accumulation at the pollen tube tip and lower abundance in the shank, whereas the low-boron condition resulted in even distribution along the entire tube (Fig. 1A-B).

Fig. 1.

Distribution of methylesterified and de-esterified pectin in pollen tubes under different boron concentrations. A Immunofluorescence staining showing the localization of JIM5 and JIM7 in pollen tubes treated with different boron concentrations. Scale bars = 10 μm. B Average relative fluorescence intensity profiles the pollen tube for JIM5 and JIM7 in B1 and B2 treated pollen tubes. Red and blue curves represent the mean relative fluorescence intensity profiles along the pollen tube tip under B1 and B2 conditions, respectively. At least 20 pollen tubes were analyzed in three independent experiments. Shaded areas indicate the 95% confidence interval. C Stimulated Raman scattering microscopy showing the distribution of methylesterified pectin in pollen tubes under low-boron conditions. Five pseudocolors (classes 1–5) denote distinct regions along the pollen tube. The x-axis represents wavenumber and the y-axis represents Raman intensity; lower wavenumbers correspond to a higher degree of pectin methylesterification. Scale bars = 10 μm

Stimulated Raman scattering microscopy is an emerging label-free imaging technique with high chemical selectivity and specificity. Using SRS, we mapped the methylesterification status of pectin in pollen tubes and observed marked regional differences under different boron concentrations. Under the B2 condition, marked differences in peak intensity were observed in the range of 800–880 cm⁻¹, with the curve representing the pollen tube tip showing a stronger peak at 820 cm⁻¹. This indicated that, under the B2 condition, the pollen tube tip exhibited a higher level of methylesterified pectin and lower Raman wavenumbers. For the shank, the peak shifted toward higher wavenumbers, indicating a gradual decrease in methylesterified. In contrast, under B1 conditions, the pollen tube tip showed a lower level of methylesterified pectin and higher Raman wavenumbers, while from the tip to the shank region, the degree of methylesterification gradually increased and the Raman wavenumbers decreased (Fig. 1C), which was consistent with the immunofluorescence results. These findings suggest that low-boron conditions promote the accumulation of de-esterified pectin in the pollen tube tip, which stiffens the wall and thereby inhibits pollen-tube elongation.

Analysis of differentially expressed proteins in response to low-boron treatment

To identify the key proteins responsible for low-boron induced inhibition of pollen-tube elongation in alfalfa, we extracted proteins from pollen tubes treated with different boron concentrations and subjected them to DIA mass spectrometry. Identified proteins were annotated using common functional databases, resulting in 70,689 peptides and 8,502 proteins. Differentially expressed proteins (DEPs) were identified by performing t-tests on the relative quantification of each protein between sample groups, calculating P-values. Proteins with fold change > 1.2 and P < 0.05 were considered upregulated, whereas those with fold change < 0.85 and P < 0.05 were considered downregulated. Principal component analysis (PCA) was performed to assess similarities and variations between protein expression profiles across groups. Samples from the same treatment clustered closely together, the proteomic data showed high reproducibility (Fig. 2A). A total of 1506, 1130, and 517 DEPs were identified from B0_vs_B1, B0_vs_B2, and B1_vs_B2 comparisons, respectively. In the B0_vs_B1 treatment groups, 897 differentially expressed proteins were significantly upregulated, and 609 were significantly downregulated. There were 698 significantly upregulated and 432 significantly downregulated proteins between B0_vs_B2. The comparison between B1_vs_B2 showed the fewest differentially expressed proteins, including 198 upregulated and 319 downregulated (Fig. 2B). Venn diagram analysis indicated that 56 DEPs were common among all three treatment groups (Fig. 2C), suggesting a shared core response mechanism.

Fig. 2.

Proteomic analysis of pollen tubes treated with different boron concentrations. A PCA of proteomic profiles from pollen tube samples treated with different boron concentrations. B Numbers of up- and down-regulated DEPs identified in each pairwise comparison group. C Venn diagram showing shared and unique DEPs across different comparison groups

To investigate the systematic impact of different boron levels on pollen tube metabolic networks, KEGG pathway enrichment analysis was performed on DEPs identified between experimental groups. The results showed that in B0_vs_B1, pathways such as pentose-glucuronate interconversions, linoleic acid metabolism, glutathione metabolism, and flavonoid biosynthesis were significantly enriched (Fig. S1A). For B0_vs_B2, pathways including indole alkaloid biosynthesis, flavonoid biosynthesis, pentose-glucuronate interconversions, anthocyanin biosynthesis, and glutathione metabolism were significantly enriched (Fig. S1B). In the B1_vs_B2 group, pathways such as indole alkaloid biosynthesis, sphingolipid metabolism, and plant hormone signal transduction were significantly enriched (Fig. S1C). GO enrichment analysis of DEPs was performed to reveal significant differences between experimental groups. In B0_vs_B1, 161 GO terms were significantly enriched. Notable enriched terms included cell wall organization or biogenesis (GO:0071554), metabolic process (GO:0008152), carbohydrate metabolic process (GO:0005975), enzyme inhibitor activity (GO:0004857), antioxidant activity (GO:0016209), response to abiotic stimulus, pectinesterase activity (GO:0030599), polygalacturonase activity (GO:0004650), and peroxidase activity (GO:0004601). In B0_vs_B2, 145 GO terms were significantly enriched, highlighting processes such as cell wall modification (GO:0042545), response to abiotic stimulus (GO:0009628), cellular component organization (GO:0016043), enzyme inhibitor activity (GO:0004857), pectinesterase activity (GO:0030599), and polygalacturonase activity (GO:0004650). Finally, in B1_vs_B2, 105 GO terms were significantly enriched, including regulation of gene expression (GO:0010468) and transcription corepressor activity (GO:0003714) (Fig. S2).

Analysis of signaling pathways associated with cell wall pectinases

Boron acid, an essential micronutrient for plant growth and development, primarily interacts with rhamnogalacturonan-II (RG-II) to form borate diesters, thereby increasing cell wall porosity and extensibility. Based on previous immunofluorescence staining results demonstrating abnormal accumulation of pectin at the pollen tube tip under low-boron conditions, we propose that pectin, as a key component of the cell wall, plays a critical role in regulating pollen tube elongation under low-boron conditions. We then performed KEGG enrichment analysis and volcano plot–based differential protein assessment on the 56 DEPs shared across all three treatment groups. In the B0_vs_B1 and B0_vs_B2 comparisons, processes such as pectinesterase inhibitor activity, pectinesterase activity, pectin catabolic processes, and cell wall modification were significantly enriched (Fig. 3A, B). In the B1_vs_B2 comparison, processes associated with hydrogen peroxide and the response to ROS were significantly enriched (Fig. 3C). Protein domains represent fundamental units of function and evolution, and enrichment of a given domain directly reflects a specific biochemical activity. Domain enrichment analysis of the 56 shared DEPs revealed significant enrichment of domains related to pectinesterase inhibitors, pectinesterases, pectinase-fold structures, and HSP20-like chaperones (Fig. S3). Collectively, these findings suggest that dynamic remodeling of the pollen tube cell wall under low-boron conditions may represent a primary factor contributing to the inhibition of pollen tube elongation. To identify pectin-related proteins potentially involved in pollen tube elongation under low boron, volcano plots were constructed based on the shared DEPs. The results showed that two pectin methylesterase inhibitor-related proteins (MsG0180004784.01 and MsG0180004785.01) were found among the downregulated differentially expressed proteins (Fig. 3D), suggesting that PMEI-related protein expression increases with rising boron concentration. These results further indicate that PMEI proteins play a key role in the pollen tube response to low boron.

Fig. 3.

KEGG functional enrichment and volcano plot analyses of the 56 DEPs shared among the three treatment groups. A–C KEGG enrichment analysis of the 56 shared DEPs identified in the B0_vs_B1, B0_vs_B2, and B1_vs_B2 comparisons. D Volcano plot analysis of the 56 shared DEPs in the B0_vs_B2 comparison. Red dots represent downregulated DEPs, blue dots indicate upregulated DEPs, while black dots indicate no significant difference

Identification of PMEI gene family members in alfalfa

Proteomic analysis identified two PMEI proteins exhibiting pronounced differences in abundance across treatments with different boron concentrations, suggesting that they may play crucial roles in regulating pollen tube cell wall mechanics under boron stress. Therefore, we performed a genome-wide identification and characterization of the PMEI gene family in alfalfa to determine the phylogenetic positions and genomic features of the two MsPMEI genes, and to infer their potential biological functions. A comprehensive survey of MsPMEI gene distribution was conducted using HMM profiles and BlastP searches with 114 AtPMEI protein sequences from Arabidopsis as queries, resulting in the identification of 93 MsPMEI proteins, which were further validated by the presence of the conserved PMEI domain (PF04043) from the Pfam database. To characterize the genomic distribution of the 93 MsPMEI genes in the alfalfa genome, a chromosomal localization map was constructed. The MsPMEI genes were distributed across eight chromosomes, with the fewest genes located on chr 5 (1 of 93) and the most on chr 8 (19 of 93). Based on their chromosomal positions, the two differentially abundant proteins identified in the proteomic analysis, MsG0180004784.01 and MsG0180004785.01, were designated MsPMEI14 and MsPMEI15, respectively (Fig. 4A). A phylogenetic tree was constructed based on of PMEI proteins between alfalfa and Arabidopsis, based on the 93 identified MsPMEI proteins and 114 AtPMEI members from Arabidopsis. The phylogenetic tree resolved the PMEI gene family into three Clades: Clade A, Clade B, and Clade C. Clade B contained the fewest members, while Clade A was the most populous (Fig. 4B). Both MsPMEI14 and MsPMEI15 were placed in Clade A, suggesting that they might share similar protein structures and biological functions.

Fig. 4.

Chromosomal localization of MsPMEI genes and phylogenetic analysis of PMEI proteins. A Locations of 93 MsPMEI genes on the chromosomes. B Phylogenetic relationships among PMEI proteins from M. sativa and Arabidopsis. Three clades are distinguished by different colors. Red asterisks indicate the two differentially abundant PMEI proteins identified in the proteomic analysis

Cis-regulatory element analysis of the promoter regions of the MsPMEI genes

To explore the regulatory functions of MsPMEI family members, the promoter regions of all 93 MsPMEI genes were analyzed using the PlantCARE online tool. A total of 13 major categories of cis-regulatory elements were identified within the MsPMEI promoter sequences. These elements encompassed several functional categories, including hormone-responsive elements (e.g., MeJA, ABA, auxin, GA, and SA responsiveness), light-responsive elements, developmental regulatory elements (e.g., endosperm development, anoxic-specific induction, cell differentiation, and seed development), and stress-responsive elements (e.g., low-temperature response, anaerobic induction, and defense and stress responsiveness). Notably, light-responsive elements were the most abundant, followed by ABA-, MeJA-, and GA-responsive elements (Fig. 5). The presence of these diverse cis-elements suggests that MsPMEI gene family members may be subject to complex transcriptional regulation in response to multiple abiotic stresses and hormonal signals.

Fig. 5.

Cis-regulatory elements in the promoter regions of the MsPMEI genes were analyzed, and the results were visualized using TBtools

Subcellular localization of MsPMEI14 and MsPMEI15 proteins

To investigate the subcellular localization of MsPMEI14 and MsPMEI15, the coding sequences (CDS) of each gene were individually fused to the pSuper::1300-GFP vector via homologous recombination and co-localized with organelle markers. Subcellular localization was subsequently examined by laser scanning confocal microscopy. The results showed that both MsPMEI14-GFP and MsPMEI15-GFP exhibited reticular and punctate localization patterns. When co-expressed with the ER marker RFP-HDEL, overlap of fluorescence signals was observed, indicating that the reticular localization represents ER localization. This result is consistent with the predictions from SignalP 6.0 (Fig. S4), which indicate that both proteins contain ER signal peptides. In addition, FM4-64 staining revealed that the punctate structures on the cell wall labeled by MsPMEI14-GFP and MsPMEI15-GFP co-localized with FM4-64-labeled punctate structures, suggesting that these proteins may localize to the endomembrane system and are likely associated with vesicular structures in the secretory pathway (Fig. 6). These results indicate that MsPMEI14 and MsPMEI15 are localized in the ER and vesicle-associated organelles and may be involved in intracellular transport and secretion processes.

Fig. 6.

Subcellular localization of MsPMEI14 and MsPMEI15. RFP-HDEL was used as an ER marker [25], and FM4-64 was used to label acidic vesicles in the cell. Scale bars = 10 μm

Discussion

Boron is an essential micronutrient for plant growth and development [26], and low boron significantly reduces seed yield and quality [27, 28]. Our previous studies have demonstrated that low-boron conditions inhibit pollen tube elongation; however, the specific regulatory mechanisms remain to be elucidated. In this study, immunofluorescence staining of pollen tubes with the monoclonal antibodies JIM5 and JIM7 under low-boron conditions revealed decreased levels of de-esterified pectin in the shank and subapical regions, accompanied by a reduced degree of methyl esterification at the pollen tube tip. Raman microscopic imaging further confirmed dynamic changes in methylesterified pectin under low-boron conditions. To identify potential regulators of pollen tube elongation under low-boron conditions, differentially expressed proteins in pollen tube samples subjected to varying boron concentrations were analyzed, revealing significant enrichment of PMEIs. Collectively, these findings indicate that pectin remodeling plays a pivotal role in the inhibition of pollen tube elongation under boron-deficient conditions.

Pollen tubes elongate rapidly through continuous secretion and assembly of new plasma membrane and cell wall materials at the tube tip. Accordingly, the pollen tube tip must maintain sufficient plasticity to withstand the turgor pressure required for elongation and to prevent tube rupture. The pollen tube tip is composed almost entirely of a pectin network [11]. Pectins are synthesized in the Golgi apparatus in a highly methylesterified form and are transported to the pollen tube tip via secretory vesicles. During pollen tube elongation, highly methylesterified pectins are de-esterified by pectin methyl esterases [29]. The abundance and relative proportions of methylesterified and de-esterified pectins are major determinants of the shape and structural integrity of growing pollen tubes [30, 31]. Disruption of the balance between methylesterified and de-esterified pectins at the pollen tube tip affects polar elongation and ultimately leads to swelling of the tube apex [15]. The monoclonal antibodies JIM5 and JIM7 specifically recognize de-esterified and methylesterified pectins, respectively [32]. In our study, JIM5-labeled de-esterified pectin was mainly localized at the shank under B2 conditions but aberrantly accumulated at the tip under B1 conditions. Conversely, JIM7-labeled methylesterified pectin shifted from an apical distribution under B2 conditions to a shank localization under B1 conditions. These observations demonstrate that low-boron conditions disrupt the normal distribution of methylesterified and de-esterified pectins along the pollen tube, thereby impairing polar elongation. These findings are consistent with previous reports in M. domestica, in which treatment of pollen tubes with the boron inhibitor phenylboronic acid (PBA), followed by JIM5 and JIM7 immunofluorescence staining, revealed increased accumulation of de-esterified pectin at the pollen tube tip, ultimately inhibiting elongation [26].

The degree of pectin methylesterification is regulated by the PME family and influences plant growth and development by altering the biomechanical properties of the cell wall, including seed germination, pollen tube development, and stomatal opening. PME activity is essential for pollen tube elongation and integrity [33]. Overexpression of AtPME1 in Arabidopsis affects pollen tube morphology and growth [34]. In addition to transcriptional levels, protein modifications, and intracellular pH, PME activity is further controlled by post-translational mechanisms mediated by PMEIs [35]. PMEIs were first identified in Actinidia chinensis, where they specifically bind to PME to form a reversible, high-affinity 1:1 complex in a pH manner [36]. Numerous studies have demonstrated that PMEIs play an important role in maintaining cell wall pectin methylesterification and are involved in plant growth and responses to various biotic and abiotic stresses. In Pisum sativum, cold-acclimated seedlings enhance frost tolerance by increasing the level of esterified pectin [37]. In Camellia sinensis, pectin activity, pectin content, and the degree of methylesterification change dynamically with temperature [23]. In Oryza sativa, overexpression of OsPMEI28 results in plant dwarfism [24]. Overexpression of the PMEI from Actinidia chinensis in Triticum aestivum decreases endogenous PME activity and significantly alleviates disease symptoms caused by fungal pathogens or Fusarium [38]. Genome-wide analysis is a key approach for elucidating the biological roles of PMEI genes. Large PMEI gene families have been identified across multiple plant species, including Arabidopsis (114) [22], Glycine max (170) [39], Camellia sinensis (51) [23], Linum usitatissimum (49) [40], and Zea mays (49) [41]. However, PMEI gene family members in alfalfa have not yet been systematically characterized prior to this study. Here, we identified 93 MsPMEI genes from the Zhongmu No. 1 alfalfa genome, a number exceeding that reported in gramineous species, possibly attributable to whole-genome duplication (WGD) events. Transcription factors interact with cis-regulatory elements in the promoters of target genes to modulate downstream gene expression and influence a broad range of biological processes [42]. In this study, we found that the promoter regions of MsPMEIs harbor numerous cis-elements related to phytohormone responsiveness, abiotic and biotic stress responsiveness, and light responsiveness. These findings indicated that PMEIs played an important role in regulating plant defense signaling and stress responses. This is consistent with reports that members of the BaPMEI gene family in rapeseed also contain numerous cis-regulatory elements [19]. Furthermore, phylogenetic analysis revealed that MsPMEIs family members were evolutionarily conserved and closely related to AtPMEIs family members from Arabidopsis, indicating functional conservation between the two species.

Most secretory proteins in plants are transported via the conventional secretory pathway, passing through the ER, Golgi apparatus, and trans-Golgi network (TGN) before reaching the plasma membrane (PM) [43]. PMEI is a known regulator of pectin methylesterification in the cell wall, and its function depends on precise spatial delivery. The subcellular localization of MsPMEI14 and MsPMEI15 in the ER and vesicle-like structures suggested that both proteins were secreted to the pollen tube cell wall via the CPS pathway. Previous studies have shown that under low boron conditions, the cross-linking structure of rhamnogalacturonan II (RG-II) formed via borate ester bonds is disrupted, thereby affecting the integrity of the pollen tube tip cell wall [26]. Consistent with our findings, under low-boron conditions, the secretion and Golgi-directed transport of MsPMEI14- and MsPMEI15-related pectin proteins are impaired, leading to severe disruption in the content and distribution of methylesterified pectin at the pollen tube tip, ultimately inhibiting pollen tube elongation. This finding is consistent with the established model of PMEI protein trafficking in pollen tubes and provides an important mechanistic basis for understanding how methylesterified pectin regulates pollen tube growth under low boron conditions in alfalfa.

Rapid polar growth of the pollen tube depends on the coordinated action of multiple cellular processes and is regulated by the interaction of complex signaling pathways involving Ca²⁺, Rop GTPases, protons (H⁺), pH, the cytoskeleton, and reactive oxygen species (ROS) [44]. The inhibition of pollen tube elongation under low-boron conditions is similarly regulated by multiple signaling pathways, and our previous studies showed that ROS abnormally accumulate in pollen tubes under low-boron conditions; meanwhile, we identified a key transcription factor, MsWRKY49, which regulated pollen tube elongation by mediating ROS homeostasis under low-boron conditions [6]. In this study, we found that pectin deposition in the pollen tube cell wall may be critical for boron-promoted pollen tube elongation, and identified MsPMEI14 and MsPMEI15 as potential regulatory factors involved in this process. This providing a candidate target for future investigations into the mechanisms by which boron regulates pollen tube elongation. We acknowledge that this study has several limitations. The conclusions are primarily based on proteomic analysis and lack direct functional validation; future studies should therefore employ genetic and biochemical approaches to validate the regulatory mechanisms proposed in this study.

Conclusion

In this study, morphological observations revealed that the spatial distribution of pectin in pollen tubes was closely associated with boron concentrations. Proteomic analysis demonstrated that this effect was linked to significant enrichment of pectin methylesterase activity and carbohydrate metabolic processes. Volcano plot analysis of differentially expressed proteins shared across three treatment groups identified two pectin methylesterase inhibitors, MsPMEI14 and MsPMEI15. Phylogenetic analysis and cis-regulatory element analysis of the MsPMEI gene family in alfalfa further indicated that PMEIs were evolutionarily conserved and might play potential roles in abiotic stress resistance. Subcellular localization experiments demonstrated that both MsPMEI14 and MsPMEI15 localized to the ER and acidic vesicular compartments, confirming that these two proteins deliver regulatory cargo to the cell wall via the CPS. Collectively, these findings provide new insights into the relationship between pollen tube growth and the spatial distribution of pectin under low boron conditions, and lay a foundation for further elucidating the molecular mechanisms by which boron regulates cell wall dynamics during pollen tube elongation.

Authors’ contributions

**Juan Wang** : Writing – original draft, Visualization, Validation, Supervision, Software, Resources, Methodology, Formal analysis, Data curation, Conceptualization. **Liru Dou** and **Peisheng Mao** : Writing – review & editing, Supervision, Project administration, Investigation, Funding acquisition.

Funding

This research was funded by the earmarked fund for CARS (CARS-34) and the 2115 Talent Development Program of China Agricultural University.

Data availability

The dataset supporting the conclusions of this article is available in the iProX partner repository (PXD075180) repository, the data link is https://www.iprox.cn//page/project.html? id=IPX0015949000.

Declarations

Ethics approval and consent to participate

No specific permits were required, and sample collection was conducted in accordance with local conservation regulations.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.