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Article

Transcriptome Analysis Reveals Pollination and Fertilization Mechanisms of Paeonia ostii ‘Fengdanbai’

by
Zhen Li
2,
Chi Xu
1,
Cancan Gu
1,
Shengxin Wang
1,
Wei Li
1,
Xiaolei Jiang
1,
Wanqiu Zhang
3,* and
Qing Hao
1,*
1
College of Landscape Architecture and Forestry, Qingdao Agricultural University, Qingdao 266109, China
2
College of Agriculture and Biology, Liaocheng University, Liaocheng 252000, China
3
Anhui Huatuo Academy of Traditional Chinese Medicine, Bozhou Vocational and Technical College, Bozhou 236800, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1082; https://doi.org/10.3390/horticulturae11091082
Submission received: 10 June 2025 / Revised: 28 July 2025 / Accepted: 1 August 2025 / Published: 8 September 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

Tree peony (Paeonia ostii) is widely cultivated in China as a traditional medicine and a new high-quality woody oil crop. Enhancing seed yield has become a primary breeding objective in the industrial development of oil tree peonies. Pollination and successful fertilization are essential for optimal seed yield. However, the molecular mechanisms underlying pollination and fertilization in P. ostii remain unclear. In this study, comparative transcriptomic and genetic analyses were conducted to investigate the pistils under different pollination periods of P. ostii ‘Fengdanbai’. Compared with pre-pollination, differentially expressed genes (DEGs) were screened from pistils 48 h after pollination, when most of the pollen tubes had reached the bottom of the style. Functional annotation indicated that these DEGs were involved in hormone signaling and carbohydrate metabolism pathways. Transcription factors and receptor-like kinases play a key role in pollen development, pollen tube growth, and carpel development. Key DEGs (PoUNE10 and PoLIM1) influenced pollination and fertilization and were characterized. Phylogenetic, promoter, and co-expression analyses suggest that they may affect plant pollination, fertilization, and seed yield through pathways such as hormone signaling and photosynthesis in P. ostii ‘Fengdanbai’. Our findings illustrate the molecular changes after pollination and fertilization in P. ostii ‘Fengdanbai’ and provide the molecular characterization of two key genes. These results provide insights into the molecular mechanisms underlying pollination and fertilization in tree peony and suggest potential candidate genes for molecular breeding aimed at improving seed yield in the species.

1. Introduction

The tree peony (Paeonia ostii) is a perennial shrub of the family Paeoniaceae, native to China. It is an important traditional medicinal plant and a high-quality woody oil crop due to the abundance of unsaturated fatty acids in its seed oil. These reach over 90% of the total fatty acid (FA), with α-linolenic acid (ALA) accounting for approximately 42% with a low ratio (<1) of n-6: n-3 [1]. Peony seed oil contains fat-soluble concomitants such as squalene, tocopherol, phytosterol, and plant polyphenol and is becoming a new food resource [2]. Owing to the strong development prospects of peony seed oil, the cultivation area of oil peony has exceeded 1.94 million acres in recent years. Increasing the seed yield of this woody oil plant is a key issue in its industrial development. Variations in the level of nutrients such as soluble sugars, starch, and crude fat during seed development may impact seed yield by affecting seed development and causing abortion in tree peony [3]. The number of carpels is also one of the factors affecting yield, and the regulatory mechanism of carpel number in P. rockii has been studied through comparative transcriptome analysis. Three MYBs have been characterized that regulate carpel variation [4]. Pollination and successful fertilization are crucial for seed numbers to affect seed yield. However, relatively few studies have been conducted on the pollination and fertilization of tree peonies, and the molecular mechanisms remain unclear.
The interaction between the pollen and the pistil is crucial for successful plant pollination and fertilization. To date, most studies on pollination and fertilization mechanisms in Arabidopsis have focused on pollen development, pollen tube growth, and signals for pollen tube guidance and rupture. This has substantially increased our knowledge of the genetic basis of pollination and fertilization. The integration of transcriptome with metabolomic data has shown that an increase in carbohydrate and lipid metabolism activated the in vitro germination of pollen tubes [5]. The number of specifically expressed genes increased significantly during pollen hydration and downward protrusion in Arabidopsis, including genes encoding calmodulin protein, receptor-like kinases (RLKs), and heat shock protein [6]. Compared with in vitro germination, pollen grain germination on the stigma requires more genes involved in signal transduction and pollen tube growth [7]. These signals affect pollen hydration, germination, pollen tube elongation, and sperm release. The mechanisms regulating pollen tube elongation and rupture are complex, involving two hub regulatory signaling cascades. One of them is crucial for pollen tube growth via RLKs’ interactions with cysteine-rich proteins (CRPs), anther-specific proteins (LAT52) [8], and the kinase partner protein [9]. Another important signaling factor is CRPs, which maintain pollen tube integrity during growth by interacting with the leucine-rich repeat extension protein (LRX) [10]. Therefore, RLKs and CRPs are likely to act as bridges that transduce intracellular and extracellular signals into the pollen cytoplasm by interacting with specific cytoplasmic components.
The transcription factor (TF) regulates the transition process of pollen tube guidance, which is divided into pre-ovular and ovular guidance. In Cucumis sativus, an ovary-expressed bHLH, CsALC (ALCATRAZ), functions in pollen tube emergence by promoting the expression of rapid alkalinization factor (CsRALF4/19) in the ovary during pollen tube guidance [11]. In Arabidopsis, ALC mediates fruit opening by driving the formation of a separation layer at the valve-replum border [12]. Another bHLH, unfertilized embryo sac 10 (UNE10), a homolog of ALC, is suggested to be involved in the fertilization process. The UNE10 mutant resulted in unfertilized ovules despite normal pollen tube attraction [13]. Studies have identified TFs that are expressed in different tissues and are involved in pollen development, pollen tube elongation, and seed development. The LIM transcription factor, which is highly expressed in pollen, has been identified in plants such as Petunia hybrida [14] and Triticum aestivum [15]. LIM2b is required for pollen tube elongation by interacting with a tethering protein involved in actomyosin transport (TAPE) [16]. Other LIMs involved in plant development influence the size of plant organs. AtWLIM2 is preferentially expressed in developing siliques, suggesting that it may be involved in embryonic development [17]. Another LIM, DA1, which acts as a ubiquitin receptor, is thought to restrict seed size [18]. SW16.1, which encodes an LIM from soybean, increases yield through metallothionein, a positive regulator of seed weight [19]. MYB, MADS-box, and WRKY regulate pollen tube guidance and synergistic cell differentiation [20].
Although the molecular mechanisms of pollination and fertilization have been partially clarified, the molecular mechanisms of tree peony remain unclear. In this study, we used the representative cultivar P. ostii ‘Fengdanbai’, which is the oil peony with the largest cultivation area. This was used as a research model to determine the molecular mechanism of pollination and fertilization of tree peonies, and we identified the TFs that affect seed yield. Comparative transcriptomes of the critical stages after pollination were obtained, and differentially expressed genes (DEGs) associated with pollination that may affect fertilization and seed yield were screened. The findings have provided new insights into the molecular mechanisms underlying pollination and fertilization of tree peony and the modulation affecting the seed yield of peony and potentially other plants.

2. Materials and Methods

2.1. Plant Materials and Pollination

In this study, P. ostii ‘Fengdanbai’, the most widely cultivated oil peony cultivar, was used as the experimental material. The plants were cultivated in a tree peony germplasm resource nursery at the Institute of Botany, Chinese Academy of Sciences (Lat. 39°59′ N, 116°12′ E, Alt. 70 m). To avoid self-pollination and unwanted crosses with nearby plants, the flowers of the female parent were emasculated and covered with waxed paper bags one day (d) before they opened. Meanwhile, unopened flowers of the male parent were taken and dried at 25 °C to obtain dried pollen grains. These were applied to the stigma of the female parent two days later. Pistils in flower buds (S1) and pistils at 0 h (S2, unpollinated), 24 h (S3), and 48 h (S4) after pollination of open flowers were collected. Parts of them were immediately frozen in liquid nitrogen and then stored at −80 °C. The other ones were fixed with FAA (formalin: 70% ethanol: acetic acid = 1:18:1, v/v/v) solution for 24 h, preserved in 70% ethanol, and stored at 4 °C.

2.2. RNA Extraction, cDNA Library Construction, and Transcriptome Sequencing

The representative stages (S1, S3, and S4) were selected for transcriptome sequencing. Total RNA was isolated using an RNAprep Pure Plant Total RNA Extraction Kit (TIANGEN, Beijing, China) and quantified with the Agilent 2100 RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA). After purification with poly-T oligo-attached magnetic beads, the mRNA fraction was fragmented into small pieces. Then, the cleaved RNA fragments were reverse transcribed into cDNA libraries. Library sequencing was performed using the Illumina HiSeq X-Ten with sequencing strategy PE150 [21]. After removing low-quality reads, adapter-containing reads, and reads with unknown bases, clean reads were subjected to de novo assembly using Trinity.

2.3. DEG Annotation and Analysis

DEGs were determined using DESeq2 with parameters of Fold Change (FC) ≥ 2 and False Discovery Rate (FDR) < 0.01, based on the fragments per kilobase of transcript per million mapped fragments (FPKM) as expression data. The putative functions of the DEGs were determined using the Non-redundant Protein, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. GO and KEGG enrichment analyses were performed using the R-based GOSeq package and KOBAS 3.0, respectively [22]. Based on the FPKM file of transcriptome, DEGs with similar expression trends were clustered and classified using OmicShare (www.omicshare.com/tools/Home/Soft/trend, accessed on 20 March 2023.), and the number of classifications was chosen to be six trends. p < 0.05 was considered statistically significant.

2.4. Quantitative RT-PCR (qPCR) Analysis of DEGs

Total RNA was isolated from pistils at stages S1, S2, S3, and S4. First-strand cDNA was synthesized with 1 μg RNA using a PrimeScript™ RT Reagent Kit (TaKaRa, Tokyo, Japan). The qPCR reactions were performed with TB Green Premix Ex Taq II (Takara, Tokyo, Japan) in the Step One Plus system (ABI, Foster City, CA, USA) with three technical replicates, using PoTub as an endogenous control [23]. The primers are listed in Table S1A.

2.5. Gene Clone and Phylogenetic Tree Analysis

The coding sequence (CDS) of the unigenes c58692_g1 (PoUNE10) and c105707_g1 (PoLIM1) was isolated. The primers are listed in Table S1B. LIM and bHLH members of Arabidopsis and P. ostii ‘Fengdanbai’ were identified from the Carbohydrate-Active Enzyme (CAZY) database and P. ostii ‘Fengdanbai’ genome database on the CNGBdb platform with project number CNP0003098. Phylogenetic analysis was performed using MEGA X according to the neighbor-joining (NJ) and maximum likelihood (ML) statistical methods for the bHLH and LIM families, respectively. The Jones–Taylor–Thorton (JTT) model, with an estimated gamma-distributed (G) parameter, was selected as the best-fitting amino acid substitution model. A bootstrap analysis with 1000 replicates was performed in each case. To deal with short insertions/deletions, the “pairwise deletion” setting was used in NJ analysis, and “partial deletion” with a 50% site coverage cutoff was used in the ML analysis.

2.6. Promoter Cis-Acting Elements and Gene Co-Expression Analysis

The 2000 bp upstream sequence of the PobHLHs and PoLIMs was selected as promoter sequences. Cis-elements were identified from PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/, accessed on 16 February 2025.), and TF binding elements were predicted from PlantRegMap (https://plantregmap.gao-lab.org, accessed on 18 February 2025.). A heatmap of promoter elements was visualized by TBtools v1.098. Co-expression networks were predicted via the string database (https://www.string-db.org, accessed on 18 February 2025.) and visualized via Cytoscape v3.8.

2.7. Statistical Analysis

Statistical significance was determined using SPSS v19.0 and Duncan’s test. In all figures, data are represented as the mean ± standard error (SE).

3. Results

3.1. Critical Periods for the Pollen Tube Growth

The pollen grains germinated at 0.5 h, and the lengths of the pollen tubes were about 2~3 times the diameter of the pollen grains (Figure 1A). An abundance of pollen grains germinated and penetrated the stigma at 12 h after pollination (Figure 1B). Meanwhile, the pollen tubes grew and elongated in bundles along the style at 24 h after pollination (Figure 1C). Finally, the pollen tubes reached the bottom of the style at 48 h after pollination (Figure 1D). These results are consistent with Chen’s findings on pollen tube growth in peony [24] and suggest that 24 h and 48 h after pollination are critical periods for the pollen tubes to elongate polarly and enter the ovule with guidance. Considering that the stigma began to secrete in the S2 stage, while it had not yet secreted in the S1, with no interference from stigmatic secretion, it conforms more to the pre-pollination stage. Therefore, the representative stages (S1, S3, and S4) were selected for transcriptome sequencing.

3.2. Pistil Transcriptome Analysis

To elucidate the molecular mechanism underlying pollination, transcriptome analysis was performed on pistils at three critical developmental stages after pollination, S1, S3, and S4. A total of 46,000,911 clean reads were generated on average, with a Q30 value of 96.74% (Table S2A). The lengths of the transcripts varied from 201 bp to 16 bp and 485 bp, with an N50 value of 1307 bp, after de novo assembly of the clean reads (Table S2B). Transcripts were further assembled into 113,603 unigenes, with 770 unigenes encoding TFs belonging to 36 families, of which MYB (178), bHLH (78), CCCH (67), Homeobox (60), and C2H2 (57) were the most overrepresented TF families (Table S2C). GO terms were assigned to 25,778 unigenes, including biological process, molecular function, and cellular component categories (Figure 2). The cellular processes (GO:0009987), metabolic processes (GO:0008152), binding (GO:0005488), and catalytic activity (GO:0003824) were the most significantly enriched. KEGG analysis suggested that carbohydrate metabolism was the most enriched pathway, including glycolysis, citrate cycle, and pentose phosphate pathways. Some unigenes were enriched in lipid metabolism, including FA biosynthesis, elongation, and degradation (Figure S1).

3.3. DEGs Relating to the Pollination Process of Pistils

The expression of unigenes in the pistils at different pollination stages was analyzed. The results show that 91,098 genes were co-expressed at the three different stages, and 978, 2041, and 1465 genes were specifically expressed at stages S1, S3, and S4, respectively (Figure S2A). A total of 29,258 DEGs were identified by comparing all stages (Figure S2B). A total of 18,075 and 16,653 genes were significantly differentially expressed in the pistil at S4 and S3, respectively, compared to S1. In contrast, 7582 genes were differentially expressed between pistils at S3 and S4 (Figure S2B). This indicates that significant changes in gene expression occurred before and after pollination; however, the changes were attenuated when comparing 24 h and 48 h after pollination.
The gene expression pattern may affect its function. Therefore, a series of tests of cluster analysis of DEGs were performed to obtain dominant gene clusters with similar expression patterns. Six clusters were grouped according to similarity in expression profiles, and most of the DEGs belonged to two clusters, that is, 4400 and 1218 DEGs in Cluster I and Cluster II, respectively (Figure 3A,B). The DEGs in Cluster I were upregulated. GO analysis showed that the DEGs were enriched in photosynthesis (GO:0015979), polysaccharide catabolism (GO:0000272), and flavonoid biosynthetic processes (GO:0009813). KEGG analyses showed that these genes were enriched in the biosynthesis of various plant secondary metabolites (ko00999), starch, and sucrose (ko00500) (Figure 3C,D). In contrast to Cluster I, DEGs in Cluster II were downregulated at S3 and remained stable. The results of the GO and KEGG analyses show that DEGs were enriched in response to endogenous stimulus, plant-type secondary cell wall biogenesis, and cutin, suberine, and wax biosynthesis (Figure S3). Considering these functional terms, we propose that multiple biological processes affect pollination and fertilization in plants, in which polysaccharide metabolism, cell wall organization, and phytohormones may play important roles.

3.4. Expression Patterns of Pollination and Fertilization Related DEGs in P. ostii ‘Fengdanbai’ Pistil

To identify candidate genes related to pollination, DEGs associated with pollination and fertilization were screened based on GO annotation. The results show that 17, 10, 3, 3, and 3 DEGs were involved in pollen tube growth and guidance, embryo sac development, pistil development, double fertilization, and pollen germination, respectively (Table S3A). A total of 126 DEGs encoding RLK and the functional proteins interacting with them were screened, of which 46 DEGs encoded the leucine-rich repeat receptor protein kinase (LRR), 25 DEGs encoded the G-type S-locus receptor protein kinase (G-SRK), and 22 DEGs encoded the cysteine-rich receptor protein kinase (CRK) (Table S3B). Among them, 24 RLKs involved in pollen development, pollen recognition, pollen tube reception, and embryo sac development were annotated using the GO database. Other RLKs, including FERONIA (FER) and rapid alkalinization factor (RALF), have also been screened and suggested to be involved in pollen tube elongation in previous studies.
Annotation analysis showed that 180 DEGs encoded TFs belonging to 16 families (Table S3C), of which MYB (42), ZF (zip finger, 40), bHLH (23), NAC (22), homeobox (15), MADS-box (8), and LIM (7) were the most overrepresented. Some members were involved in biological processes, including double fertilization (c58692_g1), embryo development (c102454_g1), and pollen-specific expression (c105707_g1).
To investigate the potential functions of these DEGs, their expression patterns in the pistils after pollination were further analyzed using the expression profiles of transcriptome data (Figure 4). The expression trend was not consistent among the DEGs encoding RLK, of which DEGs encoding LRR, G-type lectin S-receptor, RALF, and FER were upregulated. Meanwhile, the other half was downregulated after pollination. However, almost all DEGs encoding CRP were significantly upregulated at 24 h or 48 h after pollination, except for two (c8809_g1 and c63450_g1). The expression trends of most DEGs encoding TFs were consistent, with DEGs encoding bHLH, NAC, MADS-box, and LIM being upregulated after pollination. In contrast, a substantial proportion of DEGs encoding MYB were downregulated at S3.

3.5. Validation of DEGs Expression Profiles

The expression profiles were confirmed using qPCR in P. ostii ‘Fengdanbai’ pistils at the S1, S2, S3, and S4 stages. Based on the functional annotation, 15 DEGs were selected for qPCR analysis, and the expression pattern was consistent with the transcriptome data (Figure S4). The expression of c57273_g1 was upregulated first and then significantly downregulated, peaking at S3. This encodes a calcium-dependent protein kinase, suggesting the regulation of pollen tube growth through calcium transport and release. c44070_g1 and c58692_g1 of the bHLH family were significantly upregulated, with the highest expression observed at S4. The expression of other representative TFs, such as MYB c70223_g4 and c69319_g1, associated with circadian rhythm-mediated reproductive growth, was also upregulated. c105707_g1 showed no significant difference in expression between S1 and S2 but was significantly upregulated at S3 and S4, suggesting an important role in the late stages after pollination. The integration of gene functional annotation and expression results shows that c58692_g1 and c105707_g1 may act as key genes for pollination and fertilization. Among them, c58692_g1, a homolog of UNE10 that plays a key role in the double fertilization process, was upregulated 91 times at S4 compared to S2. c105707_g1 encode a LIM with two SF3 domains associated with pollen-specific expression and embryo development.

3.6. PobUNE10 and PoLIM1 Are Candidate Regulators of Pollination and Fertilization

To explore the molecular characteristics and potential functions of c58692_g1 and c105707_g1, their CDSs were isolated and matched with the genome of P. ostii. The CDS of c58692_g1 was 1338 bp in length and encoded a bHLH with 445 amino acids, namely, PoUNE10 (Figure S5A). The CDS of c105707_g1 is 576 bp in length and encodes a 191 amino acid pollen-specific protein PoLIM1, containing two conserved zinc finger domains, that is, cd09440 and cd09441, the LIM domain of pollen-specific protein SF3 with a Zn binding site (Figure S5B).
To confirm the functional differentiation of bHLH and LIM in P. ostii ‘Fengdanbai’, the bHLH and LIM family members were identified from the tree peony genome. A total of 169 PobHLH members were divided into 25 subfamilies. Phylogenetic analysis showed that PoUNE10 corresponds to Pos.gene61265 in the tree peony genome and is a direct homolog of UNE10 (At4g00050) in A. thaliana, which belongs to subfamily VIIa + b (Figure 5). Based on the results of the homology search, the tree peony contained 11 genes belonging to the VIIa + b subfamily. The members of this subfamily were mainly involved in the regulation of carpel development, fruit dehiscence, and double fertilization.
To explore the regulatory relationships and protein interactions of the PobHLHs in the VIIa + b subfamily, the cis-elements of their promoters were analyzed, and the interacting proteins were predicted (Figure 6A). Different types of cis-elements were distributed on the promoters of VIIa + b subfamily PobHLHs. Light-responsive elements, stress-responsive elements, and anaerobic induction elements were the most abundant abiotic factor-responsive elements. Among the hormone-responsive elements, abscisic acid, MeJA, and gibberellin-responsive elements were the most numerous. Endosperm expression elements were also distributed on their promoter, such as PoUNE10 and Pos.gene52310. In addition, a large number of TF binding elements, including the MYB, ERE, and MYC binding elements, have been identified. Other TF binding elements on the promoters were checked (Figure 6B). The results show that the Pos.gene47735 promoter contains the largest amount of TF binding elements. The ERE and Dof binding elements are more abundant on the promoters of genes such as PoUNE10 and Pos.gene8505.
To explore the functional relationship between PoUNE10 and other genes, co-expression networks were constructed. A total of 78 genes were co-expressed with 10 PobHLHs of VIIa + b subfamily (Figure 6D). PoUNE10, Pos.gene8505, and Pos.gene54923 shared more co-expressed genes. Genes with better connectivity to VIIa + b subfamily genes include PHYA and PHYB encoding phytochrome, RGA, RGL2, and GAI encoding DELLA proteins, ELF3 encoding an EARLY FLOWERING Protein, and two genes encoding B3 domain transcription factor ABI3 and the bZIP transcription factor HY5. KEGG enrichment analysis of the proteins in the interactions network showed that these genes are mainly involved in circadian rhythm and plant hormone signal transduction pathways (Figure 6C). In addition, PoUNE10 was co-expressed with UPB1, which modulates the balance between cellular proliferation and differentiation.
Sixteen LIM members were identified from the P. ostii ‘Fengdanbai’ genome. Phylogenetic analysis of the LIM members showed that the LIM family was divided into five subfamilies. PoLIM1 corresponds to Pos.gene65789 in the tree peony genome and was clustered into the WLIM1 subfamily with Pos.gene72368 and Pos.gene25784 (Figure 7). Pos.gene43700 belonged to the WLIM2 subfamily. Pos.gene63261 and Pos.gene54563 belonged to the PLIM2 subfamily. The WLIM and PLIM subfamilies are mainly involved in plant growth and pollen development. A small number of peony LIMs were classified into the DA1 and DAR subfamilies, which are likely involved in the response to stress and regulation of seed size.
The cis-elements of promoters of the PoLIM gene were analyzed (Figure 8A). Light-responsive elements, stress-responsive elements, and wound-responsive elements were the most numerous abiotic factor-responsive elements. MeJA- and ABA-responsive elements are the most abundant plant hormone-responsive elements, with MeJA-responsive elements predominantly distributed on the promoters of the WLIM1 subfamily gene, such as PoLIM1. A large number of MYB and MYC binding sites also exist on the PoLIMs promoter. Prediction of TF binding sites on the promoter of PoLIMs showed a high number of MYB and Dof binding sites (Figure 8B). However, fewer TF binding sites existed on the PoLIM1 promoter, with MYB, AP2/ERF, and BBR-BPC binding sites dominating.
Ten genes co-expressed with fourteen PoLIMs, including genes encoding a GPI-anchored protein, a ubiquitin receptor, an expansin protein, a zinc finger protein, and TFs (Figure 8D). The annotation of these genes to the KEGG pathway showed that the TCP transcription factor was associated with the circadian rhythm pathway, and ten PoLIM genes, including PoLIM1, were associated with the cytoskeleton in muscle cells pathway, which is related to the process of pollen tube elongation in plants (Figure 8C).

4. Discussion

4.1. Hormone Signaling, Carbohydrate Metabolism, and Calcium-Dependent Kinases Are Essential for Pollination and Fertilization

Pollination and fertilization are key factors that affect plant yield. As a representative germplasm of oil peony, the yield of P. ostii ‘Fengdanbai’ limits the development of the oil peony industry. As information on the mechanisms of pollination and fertilization in tree peonies remains limited, we examined the molecular mechanisms before and after pollination using P. ostii ‘Fengdanbai’ as a model. A large number of DEGs related to hormonal signaling and carbohydrate metabolism were identified 48 h after pollination, the period during which pollen tubes were thought to grow into the ovule. Two key TFs, namely, PoUNE10 and PoLIM1, were characterized, and their gene families were identified and analyzed by bioinformatics. The findings have provided insight into the molecular mechanisms of tree peony pollination and fertilization processes and offer candidate genes to enhance yields.
Pollen germination and pollen tube elongation after pollination involve multiple signaling pathways. Indole acetic acid, gibberellin, abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA) are associated with reproductive processes [25]. The exogenous MeJA and SA promoted pollen germination and pollen tube elongation [26]. CoCOI1, CoJAZ1, and CoMYC, which are the core genes involved in jasmonate signal transduction, are associated with self-crossing affinity in oil tea [27]. In the present study, 53 downregulated and 15 upregulated DEGs were enriched in the plant hormone signal transduction pathway, with the most significant response to cytokinin, auxin stimulus, ABA, and MeJA. Comparative transcriptome analysis of crossbred Solanum melongena also showed that DEGs were most enriched in plant hormone transduction pathways, which is similar to the results of our study [20].
Functional enrichment analysis indicated that the expression of genes related to carbohydrate metabolism and photosynthetic pathways was significantly upregulated after pollination. Disruption of the carbohydrate balance in the pistil is the main cause of reduced pollen tube elongation and reproductive failure [28]. In the rice-abortive mutant (feb, fertilization barrier), the genes related to carbohydrate metabolism and signal transduction pathways were enriched in the guided growth of pollen tubes [29]. Rapid pollen tube growth requires the uptake of sucrose or its hydrolytic products from the apoplast of the surrounding tissues. Sugar transporter proteins in apple and cucumber both positively regulate pollen tube growth, with the absence of CsHT1 in cucumber leading to a decrease in seed number per fruit and seed size [30,31]. Meanwhile, reproductive growth is a highly energy-consuming process and is mainly supported by the photoassimilation supply. Many studies have shown that reproductive tissues not only serve as resource sinks but are also known to be photosynthetically active [32]. Carpels of plants such as tree peony are essentially a modified leaf that has folded into a tubular structure enclosing the ovules, and photosynthesis-related genes are actively expressed in the carpel at the post-pollination stage. In spinach, the central carbon metabolic pathway was significantly activated after pollination and governed by carbohydrate metabolism and photosynthesis pathways [33]. In the pollination-induced fruit set of tomato, photosynthesis-related genes were upregulated during the flower-to-fruit transition [34]. These findings corroborate the results of this study in which upregulated expressed genes were enriched in the carbohydrate metabolism and photosynthesis pathways, and both suggest that the activation of carbohydrate metabolism and photosynthesis-related genes is an integral regulatory component of the reproductive process.
Many RLKs were found to be involved in pollination and fertilization processes, according to functional annotation. Among the RLKs, LRR accounted for the largest proportion, followed by G-SRK and CRK. These results are consistent with studies on the transcriptomes of a variety of plant reproductive tissues and suggest an important role for RLKs in reproductive biology [35]. These kinases affect pollination and fertilization processes through hormone signaling, carbohydrate metabolism pathways [36,37], and calcium-modulated signaling that affects pollen development and pollen tube integrity. Pollen tube rupture and sperm release are dependent on the calcium signaling-mediated FER–LRE–NTA pathway [38]. FER has been previously described as an RLK involved in pollen tube elongation, where it interacts with the glycosyl-phosphatidyl inositol-anchored protein LRE to regulate pollination and fertilization via the recruitment of the calmodulin-gated channel NTA. PoSERK, a critical receptor-like kinase, was involved in tree peony zygotic embryo development by interacting with PorbcL [39]. In the present study, more than half of the DESs encoding G-SRK were associated with pollen recognition and calcium ion modulation. This suggests that numerous calcium-dependent kinases play a key role in regulating pollen tube growth.

4.2. PoUNE10 and PoLIM1 Are Involved in the Process of Pollination and Fertilization

c58692_g1 encoded a bHLH transcription factor, namely, PoUNE10. The homolog of PoUNE10 in Arabidopsis is the unfertilized embryo sac 10 (UNE10), of which the mutation affects embryo sac functions with unfertilized ovules but normal pollen tube attraction [13]. Phylogenetic tree analysis showed that PoUNE10 is also a homolog of SPATULA (SPT) and ALC, which belong to the subfamily VII (a + b) of bHLH. SPT and ALC are involved in dehiscence zone formation in Arabidopsis [40]. They also play a key role in regulating fruit development in cucumber and tomato plants. Mutation of CsSPT results in a 60% reduction in female fertility, with seeds produced only in the upper portion of the fruit. Stigmas in the spt and alc double mutants turned outward with a defective papillae identity, which resulted in no path for pollen tube extension and no fertilized ovules [41]. SPT and ALC are closely related TFs with unequal redundant functions. SPT is known to support septum, style, and stigma development in flowers. Meanwhile, ALC is involved in dehiscence zone development in fruits. However, their roles are sometimes interchanged [40]. PoUNE10 was highly expressed at 48 h after pollination, a period that coincided with the period of fertilization after pollination of the tree peony, suggesting that PoUNE10 may affect the pollination and fertilization process.
bHLHs in the VII (a + b) subfamily are also recognized as phytochrome-interacting factors (PIFs), among which UNE10 is also known as PIF8. In Arabidopsis, PIF8, ALC, and SPT were highly expressed during seed development. Although the high levels of ALC and SPT expression are consistent with their roles in pistil development, the function of PIF8 in the reproductive process is unclear [42]. PIFs are associated with photosynthesis and photomorphogenesis [43]. In this study, PobHLHs in the VII (a + b) subfamily not only possess a large number of light-responsive elements on their promoter but are also co-expressed with genes encoding PHYA, PHYB, and HY5, which is consistent with their known function in photosynthesis and photomorphogenesis. In addition, PoUNE10 was co-expressed with gene encoding the UPB1 transcription factor involved in cellular value addition. A similar phenotype was observed in tomatoes. SlyALC/SPT, another homolog of UNE10, positively regulates cell proliferation in floral organs. The suppression of ALC/SPT expression in Capsicum annuum and Solanum lycopersicum resulted in decreased leaf size and pigmentation [44]. Therefore, PoUNE10 may play multiple roles in pollination and seed development, thus improving the seed yield.
c58692_g1 (PoLIM1) was identified encoding a LIM transcription factor belonging to the WLIM1 subfamily. The LIM family members of P. ostii were categorized into five subfamilies, in which the WLIM1, WLIM2, PLIM2, and PLIM2-like subfamilies were significantly separated from the DA1 and DAR subfamilies into two clusters. This is similar to the phylogeny of the LIM family in Brassica rapa and Populus tremula × P. alba [45]. These results also suggested that the PLIM2-like subfamily is found only in some species, such as P. tremula × P. alba and P. ostii ‘Fengdanbai’. The WLIM1, WLIM2, PLIM2, and PLIM2-like subfamilies have two LIM domains that are associated with growth and development, actin bundling, lignin biosynthesis, and pollen tube development and elongation. Meanwhile, the DA1&DAR subfamily with a single LIM domain is involved in the response to stress and seed size restriction [46]. Some studies have further subdivided the WLIM1 subfamily into two parts, namely, βLIM1 and PLIM1 [47]. However, no significant evidence supported the categorization of LIM members in P. ostii ‘Fengdanbai’.
Abundant MeJA-responsive elements exist on the PoLIM1 promoter, suggesting that PoLIM1 may be regulated by MeJA signaling. The content of MeJA is higher in flowers and seeds than in stems and leaves, with roles in regulating stamen development and plant fertilization, and MeJA signaling forms a cascade regulatory network with MYB and MYC transcription factors [48]. In this study, many MYB and MYC transcription factor binding motifs were present on the PoLIM promoter; however, whether it is undergoing cascade regulation by MeJA signaling still needs to be further investigated. In this study, genes in the PoLIMs co-expression network were enriched in the cytoskeleton in the muscle cell pathway. We also identified the genes encoding the GPI-anchored protein, the ubiquitin receptor, and expansion as the top co-expressed genes with PoLIMs. Among them, four genes encoding GPI-anchored proteins were co-expressed with PoLIM1, which was highly expressed in the pollinated pistil at the S3 and S4 stages. GPI-anchored proteins are known to be involved in actin filament binding and regulation of pollen tube integrity [49]. LIMs have also been recognized as regulators of the actin cytoskeleton [50], as well as pollen development and growth [15], which suggests a similar relationship centered on LIMs. This highlights their potential role in both the development process and formation of the pollen tube and provides an initial focus for the necessary functional validation of key genes.

5. Conclusions

In this study, we performed comparative transcriptomic and genetic analyses on pistils during different pollination stages of P. ostii ‘Fengdanbai’. The pollen tubes grew rapidly and polarly at 24 h after pollination, and most of them reached the bottom of the style at 48 h after pollination. Compared to the pre-pollination stage, numerous DEGs were identified in pistils 48 h post-pollination, coinciding with the time when most pollen tubes had reached the base of the style. These DEGs were associated with hormone signaling and carbohydrate metabolism pathways. Transcription factors and receptor-like kinases play significant roles in pollen development, pollen tube growth, and carpel development. Two key DEGs, namely, PoUNE10 and PoLIM1, were isolated and further studied through bioinformatics. Phylogenetic tree, promoter element, and co-expression network analyses suggest that these two genes may affect the pollination and fertilization of P. ostii ‘Fengdanbai’ through plant hormone signaling and photosynthesis pathways. These findings provide insights into the molecular mechanisms underlying pollination and fertilization in tree peony and suggest potential candidate genes for molecular breeding aimed at improving seed yield.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11091082/s1, Figure S1: KEGG analysis of all assembled unigenes in pistil of P. ostii ‘Fengdanbai’. Figure S2: Uningene (A) and DEG number (B) in pistils of P. ostii ‘Fengdanbai’. S1, S3, and S4 represent pistils in flower buds, pistils at 24 h and 48 h after pollination, respectively. Figure S3: GO and KEGG analyses of DEGs in Cluster II. Rich Factor represents the value of enrichment factor, which is the quotient of foreground value (the number of DEGs). The larger the value, the more significant the enrichment. Coloring indicates p-values, with higher values in red and lower values in blue. The lower the p-value, the more significantly enriched. Point size indicates the number of DEGs. Figure S4: qPCR analysis of DEGs. Lowercase letters a and b indicate significant differences (p < 0.05). S1, S2, S3, and S4 represent pistils in flower buds, unpollinated pistils, and pollinated pistils at 24 h and 48 h after pollination, respectively. Figure S5: Nucleotide and deduced amino acid sequences of PoUNE10 (A) and PoLIM1 (B). 3′UTR and 5′UTR sequences of PoUNE10 are underlined with dotted lines. LIM conserved domains are underlined. The DNA and protein binding sites of PoUNE10 are shaded in yellow and green, and the zinc binding sites of the zinc finger domain of PoLIM1 are shaded in blue. Table S1A: Primer list of primers used for qPCR. Table S1B: Primer list used for gene isolation. Table S2A: Sequencing data statistics. Table S2B: Assembly data statistics. Table S2C: TF families. Table S3A: DEGs associated with pollination and fertilization based on GO annotation. Table S3B: DEGs encoding RLK. Table S3C: DEGs encoding transcription factors.

Author Contributions

Conceptualization and methodology, Q.H. and Z.L.; software and formal analysis, C.X.; validation, C.G. and S.W.; investigation, W.L.; data curation, X.J.; writing—original draft preparation, Z.L.; writing—review and editing and supervision, Q.H. and W.Z.; funding acquisition, Q.H. and Z.L. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Shandong Provincial Natural Science Foundation (ZR2022MC163), the Major research project supported by Anhui Huatuo Academy of Traditional Chinese Medicine (BZKZ2417), the Research Foundation for Advanced Talents of Qingdao Agricultural University (663/1121036), and the Open Project of Liaocheng University Landscape Architecture Discipline (319462212).

Data Availability Statement

The original contributions presented in the study are included in the article; further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Pollen grain germination on the stigma and pollen tube growth in the style of P. ostii ‘Fengdanbai’. (A) Pollen grains germinated at 0.5 h after pollination, and pollen tubes grew 2 times longer than pollen grains. (B) Pollen grains germinated at 12 h after pollination. (C) Pollen tubes grew in clusters at 24 h after pollination. (D) Pollen tubes grew into the style bottom at 48 h after pollination. Pg: Pollen grain; Pt: Pollen tube. Scale bars = 20 µm.
Figure 1. Pollen grain germination on the stigma and pollen tube growth in the style of P. ostii ‘Fengdanbai’. (A) Pollen grains germinated at 0.5 h after pollination, and pollen tubes grew 2 times longer than pollen grains. (B) Pollen grains germinated at 12 h after pollination. (C) Pollen tubes grew in clusters at 24 h after pollination. (D) Pollen tubes grew into the style bottom at 48 h after pollination. Pg: Pollen grain; Pt: Pollen tube. Scale bars = 20 µm.
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Figure 2. Gene Ontology (GO) analysis of all assembled unigenes in pistil of P. ostii ‘Fengdanbai’.
Figure 2. Gene Ontology (GO) analysis of all assembled unigenes in pistil of P. ostii ‘Fengdanbai’.
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Figure 3. Function enrichment analysis of differentially expressed genes (DEGs). (A) DEG number in different clusters. (B) Different clusters based on p-values. Clusters with p-values less than 0.05 are highlighted in colors. (C,D) GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs in Cluster I. Rich factor represents the value of enrichment factor, which is the quotient of foreground value (the number of DEGs). The larger the value, the more significant the enrichment. High p-values are shown in red and lower p-values are in blue. The lower the p-value, the more significantly it is enriched. Point size indicates the number of DEGs.
Figure 3. Function enrichment analysis of differentially expressed genes (DEGs). (A) DEG number in different clusters. (B) Different clusters based on p-values. Clusters with p-values less than 0.05 are highlighted in colors. (C,D) GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs in Cluster I. Rich factor represents the value of enrichment factor, which is the quotient of foreground value (the number of DEGs). The larger the value, the more significant the enrichment. High p-values are shown in red and lower p-values are in blue. The lower the p-value, the more significantly it is enriched. Point size indicates the number of DEGs.
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Figure 4. Expression patterns of genes encoding receptor-like kinases and transcription factors involved in pollination and fertilization. The color bar indicates log2-based FPKM with higher values in red and lower values in blue. S1, S3, and S4 represent unpollinated pistils, pistils at 24 h, and pistils at 48 h after pollination, respectively.
Figure 4. Expression patterns of genes encoding receptor-like kinases and transcription factors involved in pollination and fertilization. The color bar indicates log2-based FPKM with higher values in red and lower values in blue. S1, S3, and S4 represent unpollinated pistils, pistils at 24 h, and pistils at 48 h after pollination, respectively.
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Figure 5. Phylogenetic tree of bHLH family from P. ostii ‘Fengdanbai’ and A. thaliana. Subfamilies are labeled in different colors, and bHLH from P. ostii ‘Fengdanbai’ are shaded in pink. PoUNE10 is marked with a star.
Figure 5. Phylogenetic tree of bHLH family from P. ostii ‘Fengdanbai’ and A. thaliana. Subfamilies are labeled in different colors, and bHLH from P. ostii ‘Fengdanbai’ are shaded in pink. PoUNE10 is marked with a star.
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Figure 6. Promoter elements and co-expression networks of PobHLHs in VIIa + b subfamily. (A) Cis-elements acquired from PlantCARE. (B) TF binding elements predicted from PlantRegMap. (C) KEGG pathway enriched for genes in co-expression networks. (D) Genes co-expressed with PobHLHs in VIIa + b subfamily. The color bar indicates statistical data with higher values in red and lower values in blue. Point size indicates the number of genes.
Figure 6. Promoter elements and co-expression networks of PobHLHs in VIIa + b subfamily. (A) Cis-elements acquired from PlantCARE. (B) TF binding elements predicted from PlantRegMap. (C) KEGG pathway enriched for genes in co-expression networks. (D) Genes co-expressed with PobHLHs in VIIa + b subfamily. The color bar indicates statistical data with higher values in red and lower values in blue. Point size indicates the number of genes.
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Figure 7. Phylogenetic tree of LIM family from P. ostii ‘Fengdanbai’ and A. thaliana. Subfamilies are labeled in different colors. PoLIM1 is marked with a triangle.
Figure 7. Phylogenetic tree of LIM family from P. ostii ‘Fengdanbai’ and A. thaliana. Subfamilies are labeled in different colors. PoLIM1 is marked with a triangle.
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Figure 8. Promoter elements and co-expression networks of PoLIMs. (A) Cis-elements acquired from PlantCARE. (B) TF binding elements predicted from PlantRegMap. (C) Genes co-expressed with PoLIMs. (D) Pathways enriched by co-expression networks and genes in these pathways. The color bar indicates statistical data with higher values in red and lower values in blue. Point size indicates the number of genes.
Figure 8. Promoter elements and co-expression networks of PoLIMs. (A) Cis-elements acquired from PlantCARE. (B) TF binding elements predicted from PlantRegMap. (C) Genes co-expressed with PoLIMs. (D) Pathways enriched by co-expression networks and genes in these pathways. The color bar indicates statistical data with higher values in red and lower values in blue. Point size indicates the number of genes.
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Li, Z.; Xu, C.; Gu, C.; Wang, S.; Li, W.; Jiang, X.; Zhang, W.; Hao, Q. Transcriptome Analysis Reveals Pollination and Fertilization Mechanisms of Paeonia ostii ‘Fengdanbai’. Horticulturae 2025, 11, 1082. https://doi.org/10.3390/horticulturae11091082

AMA Style

Li Z, Xu C, Gu C, Wang S, Li W, Jiang X, Zhang W, Hao Q. Transcriptome Analysis Reveals Pollination and Fertilization Mechanisms of Paeonia ostii ‘Fengdanbai’. Horticulturae. 2025; 11(9):1082. https://doi.org/10.3390/horticulturae11091082

Chicago/Turabian Style

Li, Zhen, Chi Xu, Cancan Gu, Shengxin Wang, Wei Li, Xiaolei Jiang, Wanqiu Zhang, and Qing Hao. 2025. "Transcriptome Analysis Reveals Pollination and Fertilization Mechanisms of Paeonia ostii ‘Fengdanbai’" Horticulturae 11, no. 9: 1082. https://doi.org/10.3390/horticulturae11091082

APA Style

Li, Z., Xu, C., Gu, C., Wang, S., Li, W., Jiang, X., Zhang, W., & Hao, Q. (2025). Transcriptome Analysis Reveals Pollination and Fertilization Mechanisms of Paeonia ostii ‘Fengdanbai’. Horticulturae, 11(9), 1082. https://doi.org/10.3390/horticulturae11091082

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