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Study Protocol

Combined Transcriptome Analysis Reveals the Mechanism of ‘Shine Muscat’ Pollen Abortion Induced by CPPU and TDZ Treatment

by
Mengfan Ren
1,2,†,
Yixu Wang
1,2,†,
Siyi Yi
1,2,
Jingyi Chen
1,2,
Wen Zhang
3,
Haoran Li
1,2,
Ke Du
1,2,
Jianmin Tao
1,2,3,* and
Huan Zheng
1,2,*
1
Sanya Institute, Nanjing Agricultural University, Sanya 572024, China
2
College of Horticulture, Nanjing Agricultural University, Nanjing 210095, China
3
Institute of Horticultural Crops, Xinjiang Academy of Agricultural Science, Urumqi 830001, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2025, 11(5), 549; https://doi.org/10.3390/horticulturae11050549
Submission received: 25 March 2025 / Revised: 16 May 2025 / Accepted: 17 May 2025 / Published: 19 May 2025
(This article belongs to the Topic Grapevine and Kiwifruit Breeding Studies)
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Versions Notes

Abstract

:
N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU) and N-phenyl-1,2,3-thidiazole-5ylurea (TDZ) are plant growth regulators used for seedless treatment in grape. In this study, the flowers of ‘Shine Muscat’ (Vitis labruscana Bailey × V. vinifera L.) were treated with 3, 5, and 10 mg/L CPPU and TDZ one week before flowering. The results showed that both CPPU and TDZ treatments reduced the pollen germination rate and caused abnormal stamen and pollen grain phenotypes, resembling the male sterility observed in ‘Y_14’ (a novel grapevine germplasm derived from the self-progeny of ‘Shine Muscat’). Using RNA-seq technology, the stamens of flowers treated with 10 mg/L CPPU (CPPU_10), 10 mg/L TDZ (TDZ_10), and the control (CK) were analyzed. A total of 520 and 722 differentially expressed genes (DEGs) were identified in CPPU and TDZ treatments, respectively. GO and KEGG analyses revealed that the common pathways leading to pollen abortion in both treatments were primarily associated with hydrolase activity (acting on glycosyl bonds), phenylpropanoid biosynthesis, pentose and glucuronate interconversions, and ABC transporters. By comparing the DEGs across the three groups (Y_14 vs. SM, CPPU_10 vs. CK, TDZ_10 vs. CK), 16 DEGs exhibited similar expression patterns. Further tissue-specific expression analysis identified nine genes that were highly expressed in stamens and shared the same expression pattern in sterile lines. These findings provide a foundation for further studies on the impact of CPPU and TDZ treatments on grape stamen fertility.

1. Introduction

‘Shine Muscat’ (Vitis labruscana Bailey × V. vinifera L.) is a mid-late maturing table grape cultivar that is rich and juicy with a high nutritional value [1]. Under natural conditions, it produces seeded fruit, which impacts its texture and market appeal. In China, seedlessness is one of the favorite characteristics of table grapes for consumers [2,3]. The application of plant growth regulators offers a convenient and effective method for achieving seedlessness in grapes [4]. Recent studies have extensively explored the use of plant growth regulators for inducing seedlessness in grapes, providing a foundation for the high-quality development of the grape industry [5,6,7]. N-(2-chloro-4-pyridyl)-N′-phenylurea (CPPU), a plant growth regulator with cytokinin activity, promotes fruit setting and development when applied externally [8]. N-phenyl-1,2,3-thidiazole-5ylurea (TDZ), a plant growth regulator with both cytokinin-like and auxin properties [9], enhances cell enzyme activity, stimulates the synthesis of endogenous hormones, accelerates plant growth, improves fruit expansion, and can influence yield and quality [10,11]. Both CPPU and TDZ are commonly used in seedless treatment for grape cultivation [12]. He et al. [13] demonstrated that the combined use of GA3, SM, CPPU, and TDZ in ‘Shine Muscat’ grape effectively improved the seedless rate, achieving 100%. Zheng et al. [14] further investigated the application of plant growth regulators at different growth stages of ‘Shine Muscat’, finding that the highest seedless rate occurred when GA3, TDZ, and CPPU were used in full bloom.
Current research on CPPU and TDZ primarily focuses on fruit setting. CPPU treatment promotes grape berry setting by increasing energy status, as well as the Embden–Meyerhof pathway (EMP) and the tricarboxylic acid (TCA) cycle respiratory metabolic pathways, while decreasing the pentose phosphate pathway (PPP), as well as the Cytochrome C oxidase (CCO) and ascorbic acid oxidase (AAO) activity of respiratory terminal oxidase [15]. Exogenous CPPU treatment led to a significant reduction in endogenous cytokinin levels and the expression of the VlLOG11 gene, thereby promoting fruit setting in grapes [16]. TDZ has been reported to promote fruit setting in grapes [17,18], but the mechanism by which synthetic cytokinins like TDZ enhance fruit set remains unclear. It is hypothesized that TDZ enhances the retention of fruitlets by increasing the sink strength of fruits as they develop [19]. Limited research has been conducted on the seedless mechanism induced by CPPU and TDZ treatment. Seed formation is closely linked to the fertility of stamens and pistils, with sterility often resulting from abnormal morphology of pistils and stamens, difficulty in producing normal pollen, and low pollen germination rates. Plant growth regulators can influence reproductive growth by affecting pollen germination, thereby improving seedless rates and fruit setting [20]. Exogenous cytokinin application can inhibit pollen germination, with higher concentrations showing a stronger inhibitory effect [21,22]. Jiao et al. [23] and Wang et al. [24] both found that exogenous application of CPPU before flowering reduced grape pollen germination, potentially disrupting the balance of endogenous hormones.
Seedlessness in fruits is influenced by multiple factors. Among these, pollen fertility is one of the key factors affecting seed development. During the development of reproductive organs, plants require a significant accumulation of carbohydrates [25]. A lack of essential substances and energy needed for anther development is a critical factor resulting in male sterility [26]. Li et al. [27] conducted transcriptome sequencing using the male line NJCCMS1A and the maintainer NJCMS1A, identifying 365 differentially expressed genes (DEGs), which were significantly enriched in pathways related to carbohydrate and energy metabolism [27]. Sweet (sugars will eventually be exported transporter) proteins have recently been identified as a new type of sugar transporter. The expression of VvSWEET5a in grape flowers is significantly higher than that in other tissues, and it is preliminarily speculated that this gene may be related to grape reproductive development [28]. The thermo-sensitive genic male sterile line 05ms in eggplant regulates anther development through the expression of genes associated with pentose and gluconate conversion, carotenoid biosynthesis, and plant hormone signal transduction [29]. The phenylpropanoid biosynthesis pathway also impacts plant growth, water transport [30], and flower organ development [31,32]. Several genes in the phenylpropanoid pathway, such as PAL, CHI, and COMT, are specifically expressed in anthers and even the tapetum [33]. Glucanase, a member of the Glycoside Hydrolase family, plays a role in pollen development. In Arabidopsis thaliana, 50 glucanase-related genes have been identified. Among these, At3g23770 regulates microspore development through co-expression with tapetum-related male sterile protein MS2. Its early expression triggers callose degradation, leading to pollen abortion. At5g20390 and At5g64790 are specifically expressed in flowers or reproductive organs. During the middle and late stages of pollen development, the genes At5g20390 and At5g64790 are co-expressed with the tyrosine phosphatase gene AtPTEN1, which promotes pollen maturation [34,35]. β-glucanase enzymes, such as β-1,3-glucanase, are crucial for pollen development [36]. Studies have shown that low expression of the β-1,3-glucanase gene in sterile buds prevents timely callose degradation, impairing microspore release and causing pollen abortion [37,38].
In this study, ‘Shine Muscat’ grapes were used as the materials, and the flower morphological changes of the grapes treated with CPPU (3 mg/L, 5 mg/L, 10 mg/L) and TDZ (3 mg/L, 5 mg/L, 10 mg/L) were analyzed. Pollen viability was detected by the in vitro pollen germination method, and pollen morphology was observed by scanning electron microscope (SEM) and transmission electron microscope (TEM). The results showed that after applying high concentrations of CPPU and TDZ one week before flowering, pollen vitality decreased, pollen grains became deformed, and the formation of pollen exine was disrupted. This phenotype closely resembled that of ‘Y_14’, a male sterile line of ‘Shine Muscat’ [39,40]. To understand the specific regulatory mechanisms, DEGs from CPPU_10 vs. CK and TDZ_10 vs. CK were further analyzed by RNA-seq and enriched through GO and KEGG pathways. The results indicated that pollen abortion following 10 mg/L CPPU and 10 mg/L TDZ treatment was primarily linked to hydrolase activity acting on glycosidic bonds, pentose and glucuronate interconversions, phenylpropanoid biosynthesis, and energy metabolism. To identify key genes involved in pollen abortion, transcriptome data from CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM were integrated, revealing 20 DEGs, 16 of which exhibited consistent expression patterns across all three sterile lines. The expression patterns of these genes were analyzed across 54 grape tissues, leading to the identification of nine genes that were highly expressed in stamens and showed consistent patterns in the sterile lines. These results offer a foundation for researching the mechanisms by which CPPU and TDZ treatments lead to grape pollen abortion.

2. Materials and Methods

2.1. Plant Material

‘Shine Muscat’ and ‘Y_14’ were obtained from Bai Ma vineyard and Tang Shan Cui Gu vineyard, Nanjing Agricultural University, respectively. Bearing branches of ‘Shine Muscat’ with similar growth potential were selected, and only one inflorescence was retained per bearing branch for the experiment. Carry out normal flower and fruit management practices on fruit trees. The shoulder and middle parts of the inflorescence were removed before bloom, leaving a 3 cm section at the tip.
To investigate the effects of CPPU and TDZ on pollen fertility in ‘Shine Muscat’ grapevines, we conducted a comparative study involving seven experimental treatments. One week prior to flowering, inflorescences were dipped in treatment solutions containing either CPPU or TDZ at three concentrations (3, 5, and 10 mg/L), with an untreated control group (CK) serving as baseline. Each treatment was replicated 50 times to ensure statistical reliability.

2.2. Observation of Grape Flowers Phenotype and Determination of Pollen Viability

At full bloom, 30 flower buds from each treatment were randomly collected in the field. Flower phenotypes from different treatments and those from ‘Y_14’ were photographed for observation.
Before full bloom, five inflorescences were randomly selected from each treatment. Take off the anther, put it in a petri dish, and culture it at 25 °C for 24 h. After the anthers naturally split, pollen viability was determined using the in vitro germination method [41]. The method is as follows: Agar (1.00 g), sucrose (10.00 g), and borax (0.10 g) were weighed and placed in a beaker, followed by the addition of 100 mL of distilled water. The mixture was then heated to boiling in a microwave oven. A clean glass rod was used to dip the culture medium onto a glass slide, which was then allowed to cool. Pollen was applied to the surface of the medium using a clean brush. The medium was placed in a petri dish containing water-moistened gauze and incubated at 25 °C for 24 h. Observations were performed under an optical microscope, and the total number of pollen grains and the number of germinated pollen grains were counted in six fields of view for all treatments. The experimental data were sorted and analyzed by SPSS 20, and the significance of the data was tested by the Duncan method.

2.3. Cytological Observation of Mature Pollen Grains

Before full bloom, the flowers were made into paraffin sections and observed. Flowers from the treatments were collected and fixed in an FAA solution (5 parts formalin, 5 parts acetic acid, and 90 parts ethanol by volume), then stored at 4 °C. The fixed materials were processed into paraffin sections, which were observed and photographed under a microscope [42].

2.4. Scanning Electron Microscope (SEM) and Transmission Electron Microscopy (TEM) Analysis of Pollen Grains Morphology

Collect the flower buds at the beginning of flowering, take them back to the laboratory, peel off the anthers, and dry them at 25 °C. The naturally dehisced pollen, which was treated with CK, 10 mg/L CPPU, and 10 mg/L TDZ, along with the pollen of ‘Y_14’, were collected and analyzed by SEM and TEM following the method described by Crawford [43]. We measured the size of the pollen grains with ImageJ 1.8.0.

2.5. Transcriptome Sequencing Sample Preparation and Sample RNA Extraction and Quality Control

Flowers treated with 10 mg/L CPPU, 10 mg/L TDZ, and CK were collected after four days. Anthers (1.0 g) were kept on ice, frozen in liquid nitrogen immediately, and stored at −80 °C for later use. Three biological replicates were taken for each treatment. RNA was extracted from the above 9 samples. The concentration and integrity of the RNA were evaluated using spectrophotometry and 1% agarose gel electrophoresis.

2.6. cDNA Library Construction and High-Throughput Sequencing Analysis

After RNA quality was confirmed, the samples were sent to Beijing Novozymes Biologicals for cDNA library construction using the library preparation kit (NEB, Ipswich, MA, USA). Three replicates were prepared for each treatment, resulting in a total of nine gene libraries. Libraries were tested and sequenced using Illumina technology.
To ensure data quality and reliability, reads containing adapters, N bases, or low-quality sequences were removed from the raw reads to obtain clean reads. The clean data were then analyzed for Q20, Q30, and GC content. HISAT2 v2.0.5 was used to align the clean reads with the grape reference genome (https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/003/745/GCF_000003745.3_12X/, accessed on 20 May 2024) to map their locations. Gene expression levels were calculated using Feature Counts and quantified as FPKM.

2.7. Screening and Enrichment Analysis of Differentially Expressed Genes (DEGs)

Differential expression analysis was performed using DESeq2 software (1.20.0) for comparisons between treatments. Genes with |log2(fold change)| ≥ 1 and padj ≤ 0.05 were identified as DEGs. Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the DEGs were performed using ClusterProfiler (3.8.1), with padj ≤ 0.05 set as the significance threshold. Enrichment results were visualized using Cytoscape 10.2. The key pathways are those that are significantly enriched both in CPPU_10 vs. CK and TDZ_10 vs. CK.

2.8. Expression Analysis of DEGs in Grapevine Tissues

The expression of DEGs from the comparisons of CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM was further analyzed in the transcriptome of 54 different grape tissues (NCBI accession number GSE36128) [44]. A heatmap of the DEGs was constructed using GraphPad Prism 9.5 software, and gene expression levels were normalized using [Log2(FPKM+1)] [45].

2.9. qRT-PCR Analysis

Nine genes were randomly selected for qRT-PCR. Snap Gene 5.2.4 was used to design primers, with the specific sequences listed in Table S1. RNA was extracted from each sample following the method described previously, and cDNA was synthesized using the Takara Reverse Transcription Kit. Gene expression levels were measured using the Quant Studio5 real-time PCR system. The RT-qPCR reaction conditions were 95 °C for 4 min, 95 °C for 20 s, 60 °C for 20 s, 72 °C for 40 s, for a total of 40 cycles, followed by a melting curve detection from 65 to 95 °C. The experiments were conducted in triplicate using VvActin as the internal reference gene. The relative expression levels of each gene in different treatments were calculated using the 2−△△CT [46]. The qRT-PCR data were visualized using GraphPad Prism 9.5 software. All qPCR assays were run with three technical replicates. The experimental data were sorted and analyzed by SPSS 20, and the significance of the data was tested by the Duncan method.

3. Results

3.1. CPPU and TDZ Treatments Changed the Flower Phenotype and Led to Pollen Abortion

Compared to the control, the filaments of ‘Shine Muscat’ were shortened and wrapped more tightly around the pistil after treatment with CPPU or TDZ, and the ovaries were enlarged. With increasing concentrations of CPPU, the ‘Shine Muscat’ filaments wrapped the pistil more tightly. In TDZ treatments, the filaments were tightly wrapped around the pistil, and the anthers showed difficulty in dehiscing (Figure 1a). The pollen germination rate of ‘Shine Muscat’ treated with CPPU and TDZ was significantly lower compared to the control (Table 1), with no germination observed in TDZ-treated pollen. In CPPU treatments, the pollen germination rate decreased as the concentration of CPPU increased, with 0% germination observed at 10 mg/L CPPU (Figure 1b). Paraffin sections of flowers treated with CPPU and TDZ were also examined (Figure 1c). Pollen from the control group appeared oval and mostly stained blue. In CPPU and TDZ treatments, a higher proportion of empty and abnormal pollen grains were observed.
SEM analysis of the pollen grains from ‘Shine Muscat’ treated with CK, 10 mg/L CPPU, and 10 mg/L TDZ revealed clear differences between the experimental groups and the control (Figure 2a). The pollen grains of the control group were oblong and featured three germinal furrows. After treatment with 10 mg/L CPPU or TDZ, most of the pollen grains became wrinkled and irregular, with triangular or deformed shapes and unclear germination grooves (Figure 2a). Quantitative analysis of pollen grain dimensions and deformation rates revealed significant treatment effects (Table 2). Pollen grains treated with 10 mg/L CPPU and TDZ exhibited 41.97% and 37.25% shorter polar axes, respectively, compared to the control (CK). While the equatorial axis in TDZ_10 showed no statistical difference from CK, CPPU_10 treatment resulted in a 28.84% reduction in equatorial length. The P/E ratio demonstrated marked morphological alterations: CK maintained typical prolate shapes (1.95 ± 0.20), whereas both TDZ_10 (1.15 ± 0.17) and CPPU_10 (1.17 ± 0.05) approached subprolate configurations. Strikingly, deformation rates reached 100% in both treatment groups, contrasting with 23.8% in CK. These findings indicate that 10 mg/L concentrations of CPPU and TDZ induce profound morphological disruptions, with TDZ causing more severe equatorial axis compression while CPPU affected both axes proportionally. TEM analysis of the internal structure of the pollen grains further revealed that, in the control group, the pollen exhibited a continuous layer of bacula and tectum, with abundant cytoplasm, organelles, starch, and other nutrients (Figure 2b). In contrast, after treatment with 10 mg/L CPPU or TDZ, the pollen nuclei and organelles were completely disintegrated, leaving behind a contracted and deformed cell structure. The pollen wall was abnormally developed, with the bacula absent and replaced by a continuous dot-like structure, and the intine was degraded (Figure 2b).

3.2. Transcriptome Sequencing Analysis to Find the Shared Pathways Responsible for Pollen Abortion

Both high concentrations of CPPU and TDZ treatments resulted in pollen abortion phenotypes. To determine whether they shared a mechanism of abortion, we constructed the anther transcriptome database for ‘Shine Muscat’ grapes under three treatments (CK, 10 mg/L CPPU, and 10 mg/L TDZ), with a total of nine samples analyzed. As shown in Table S2, a total of 436,999,874 clean reads were obtained. The average alignment percentage of reads to the genome across all samples was 68.74%, with an average single-read alignment rate of 66.96%. After filtering the raw data and assessing the sequencing error rate and GC percentage, the clean reads yielded an average Q30 value of 96.60% and an average GC percentage of 45.93%. The sequencing error rate across all samples was 0.01%, and the correlation coefficients among replicates were consistently above 0.98, indicating high reproducibility among the samples (Figure S1). These data confirm that the quality of the sequencing data meets the analysis requirements.
The differentially expressed genes of CPPU_10 vs. CK and TDZ_10 vs. CK were analyzed. In CPPU_10 vs. CK, 520 DEGs were identified, with 277 up-regulated and 243 down-regulated. In the TDZ_10 vs. CK comparison, 755 DEGs were identified, with 448 up-regulated and 307 down-regulated (Figure 3a). Among these, 185 DEGs were up-regulated, and 115 were down-regulated in both treatments (Figure 3b,c).
GO enrichment and KEGG pathway analysis of the DEGs were performed using ClusterProfiler (3.8.1), with padj ≤ 0.05 set as the significance threshold. GO annotation and enrichment analyses were conducted on the DEGs from both CPPU_10 vs. CK and TDZ_10 vs. CK treatments. The top 30 GO terms are shown in Figure 4. Thirteen GO terms were enriched in both treatments, including carbohydrate metabolic process (GO:0005975), external encapsulating structure organization (GO:0045229), cell wall organization or biogenesis (GO:0071554), extracellular region (GO:0005576), and hydrolase activity, hydrolyzing O-glycosyl compounds (GO:0004553), among others. Notably, hydrolase activity, hydrolyzing O-glycosyl compounds (GO:0004553) and hydrolase activity, acting on glycosyl bonds (GO:0016798), were significantly enriched in both CPPU_10 vs. CK and TDZ_10 vs. CK.
KEGG enrichment analysis was performed on the DEGs to identify the relevant metabolic and signaling pathways. Among the top 20 KEGG pathways enriched in both CPPU_10 vs. CK and TDZ_10 vs. CK (Figure 5), 13 pathways were significantly enriched, including pentose and glucuronate interconversions (vvi00040), steroid biosynthesis (vvi00100), ubiquinone and other terpenoid-quinone biosynthesis (vvi00130), starch and sucrose metabolism (vvi00500), and plant hormone signal transduction (vvi04075). DEGs in both treatments were significantly enriched in pentose and glucuronate interconversions (vvi00040), phenylpropanoid biosynthesis (vvi00940), and ABC transporters (vvi02010).

3.3. Conjoint Transcriptome Analysis to Screen Key Genes

The phenotype of grape flowers treated with 10mg/L CPPU and 10mg/L TDZ was similar to that of the male sterile line ‘Y_14’ (Figure 6a). The pollen grains were distorted and shrunk, with nuclei, organelles, and starch inclusions visibly absent. Additionally, the bacula on the pollen exine was not clearly defined, and the tectum layers of the pollen grains were incomplete (Figure 6b,c). These characteristics closely mirrored the pollen grain morphology observed in the CPPU and TDZ treatments.
To explore whether the pollen abortion caused by CPPU and TDZ treatments shared differentiated genes with natural male sterile grape pollen, we performed a conjoint analysis using the Y_14 transcriptome data at the mononuclear stage, along with the CPPU and TDZ treatment groups. We compared DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM and identified 20 genes that were differentially expressed across the three groups (Figure 7a, Table S3). Sixteen of these DEGs exhibited consistent expression patterns in the sterile lines (Figure 7b). Among them, 10 genes, including CESA4, EXO70H1, NPF2.11, CER2, UGT83A1, MBF1C, XTH23, MYB101, HSP23.6, and NMT3, were all down-regulated in the sterile lines, and CESA4 showed a particularly significant down-regulation in the three sterile lines. CLSC5, NLTP6, PDR2, CAJ1, ERF109, and novel.123 were up-regulated in male sterile lines.
To elucidate the tissue-specific expression patterns of these candidate genes, we conducted comprehensive expression profiling leveraging publicly available transcriptomic datasets from the NCBI SRA database (Accession: GSE36128). The normalized RNA-seq data encompassing 54 organ-specific samples (including leaves, roots, fruits, and reproductive tissues at various developmental stages) revealed expression enrichment of these genes. Some newly identified genes were not found in this dataset. Upon examining the expression levels, 12 genes were specifically expressed in different tissues (Figure 8). Our primary focus was on genes expressed in stamens. Except for EXO70H1 and NMT3, all other genes were highly expressed in stamens, with NLTP6 and CER2 showing particularly high expression. However, most of these genes were almost undetectable in pollen, except for MYB101 and NLTP 6. Combining these findings with the transcriptome joint analysis, we identified nine genes—ERF109, NPF2.11, CAJ1, MBF1C, MYB101, HSP23.6, CESA4, CER2, and NLTP6—that not only exhibited consistent expression patterns but also showed higher expression in stamens. Functional annotation of the hub genes listed in Table 3 revealed that their homologous genes play roles in reproductive development, with strong associations with the biological processes of anther wall tapetum, pollen tube growth, pollen exine, and cell wall formation. ERF109 [47] and NPF2.11 [48] were important genes involved in flavonoid synthesis and the transport of flavonoids from tapetum cells to pollen walls. CESA4 [49], CER2 [50], and NLTP6 [51,52] were functionally characterized as key components of pollen wall biogenesis. CAJ1 [53], MBF1C [54,55], and HSP23.6 [56] are heat shock proteins involved in pollen tube growth. MYB101 [57] is involved in the regulation of pollen tube growth. According to evolutionary theory, orthologs in the grapevine are likely to exhibit analogous biological roles.
Expression of DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM in 54 tissues of grape (note: for a detailed explanation of the tissue names, refer to the website: https://www.ncbi.nlm.nih.gov/geo/geo2r/?acc=GSE36128, accessed on 19 December 2024).

3.4. The qRT-PCR Validation of Related Differential Genes

The qRT-PCR validation results indicated that the expression trends of the selected genes were largely consistent with those observed in RNA-seq, confirming the reliability of the transcriptome data (Figure 9).
The y-axis on the left represents the relative expression levels from qRT-PCR, while the y-axis on the right represents the FPKM values of the genes. The error bars denote the standard deviation (SD) of three independent biological replicates, with each replicate consisting of three technical replicates.

4. Discussion

4.1. The Treatment of CPPU and TDZ Resulted in Pollen Abortion of ‘Shine Muscat’

In this study, the pollen germination rate of the ‘Shine Muscat’ grape decreased as the concentration of CPPU increased, suggesting that CPPU effectively inhibits pollen germination. In contrast, the germination rate was completely inhibited at all concentrations of TDZ, indicating that TDZ had a full inhibitory effect on pollen germination in the ‘Shine Muscat’ grape. Grapes treated with CPPU and TDZ exhibited abnormal flower phenotypes, where the filaments not only shortened and curved but also wrapped around the pistil. Paraffin sectioning revealed that mature pollen grains were concave, shriveled, and lightly stained (Figure 1).
SEM and TEM analysis of the pollen grains showed that both CPPU and TDZ treatments caused the grains to become concave, with missing inclusions. The bacula layer structure was unclear, and the intine layer was degraded under both 10 mg/L CPPU and 10 mg/L TDZ treatments (Figure 2). These morphological changes resembled the characteristics of male sterility [39,45,58,59,60,61]. Excessive cytokinin will affect stamen development [62,63]. Cytokinin signal transduction members AHK2, AHK3, and AHK4/CRE1 participate in the regulation of tapetum degeneration and ultimately affect pollen viability [64]. Based on these observations, we suggest that the application of high concentrations of CPPU and TDZ one week before flowering in ‘Shine Muscat’ leads to pollen abortion, and TDZ will make the pollen abortion more effective.

4.2. Major Pathways Regulating Pollen Abortion in Grape

In this study, transcriptome analysis of anthers treated with 10 mg/L CPPU and 10 mg/L TDZ was conducted using RNA-seq. The experimental data revealed 520 DEGs in CPPU-treated anthers and 755 DEGs in TDZ-treated anthers, compared to the control group (Figure 3).
The DEGs were analyzed through GO and KEGG enrichment to explore the specific pathways and mechanisms underlying the effects of CPPU and TDZ on the stamens. Among the GO terms enriched in both CPPU_10 vs. CK and TDZ_10 vs. CK, six were related to the cell wall. These included “external encapsulating structure organization” (GO:0045229), “cell wall organization or biogenesis” (GO:0071554), “cell wall” (GO:0005618), “cell wall organization” (GO:0071555), “external encapsulating structure” (GO:0030312), and the “anchored component of membrane” (GO:0031225). Of these, “external encapsulating structure organization” (GO:0045229) and “external encapsulating structure” (GO:0030312) are directly associated with the development of the pollen wall. Hydrolase activity, specifically “hydrolase activity, hydrolyzing O-glycosyl compounds” (GO:0004553) and “hydrolase activity, acting on glycosyl bonds” (GO:0016798), was significantly enriched in both CPPU_10 vs. CK and TDZ_10 vs. CK (Figure 4). Glycosidic hydrolases are enzymes that cleave glycosidic bonds in carbohydrates, playing key roles in regulating plant developmental processes and responses to both biotic and abiotic stresses [65]. During pollen formation, the tapetum secretes various substances, including hydrolases that control polysaccharide metabolism and provide essential nutrients for microspore development [66]. For example, β-1,3-glucanase [67], pectin methyl esterase [68], and galacturonidase [69] all show hydrolase activity and are related to pollen abortion. Zhang et al. [70] demonstrated that LbGlu1 (β-1,3-glucanase), a hydrolase, significantly affects male sterility in L. barbarum.
Both CPPU_10 vs. CK and TDZ_10 vs. CK significantly enriched three KEGG pathways: Pentose and glucuronate interconversions (vvi00040), ABC transporters (vvi02010), and phenylpropanoid biosynthesis (vvi00940) (Figure 5). Disruptions in sugar metabolism and transport within the anthers can severely impair pollen development and result in male sterility [71]. The pathway of pentose and glucuronate interconversion is crucial in maintaining pollen growth and development. Compared with the maintainer gene line, many key genes identified in the upland cotton GMS line are involved in pentose and glucuronate interconversions, starch and sucrose metabolism, and galactose metabolism [72]. Transcriptome analysis of cytoplasmic male sterile lines and related maintainer lines in soybean revealed 30 DEGs in pollen, which participated in the pentose and glucuronate interconversions [73]. DEGs enriched in this pathway in both CPPU_10 vs. CK and TDZ_10 vs. CK are mainly involved in cell wall metabolism, including polygalacturonase (PGLRs) and pectin methyl esterase (PMEs), highlighting the importance of pentose and glucuronic acid interconversion in cell wall development and affecting fertility. ABC transporters are a large, diverse superfamily [74] involved in the transport of various biomolecules, such as sugars, lipids, polysaccharides, alkaloids, and steroids [75]. ABCG26, an Arabidopsis ATP-binding cassette transporter, is essential for sporopollenin accumulation, with mutations in this gene causing significantly reduced fertility and preventing the release of pollen from mature anthers [76,77]. Phenylpropane and its derivatives are significant for the synthesis of sporopollenin and pollen wall development in plants [78,79]. Sporopollenin is a key component of the pollen exine, protecting male gametes from environmental stresses [80]. Chavan [81] found that pollen sterility in the grape seedless mutant is associated with the down-regulation of the genes involved in cell wall development and pollen tube growth. Five of these VvKCS (VvKCS6/15/19/20/24) genes were involved in the fatty acid elongation pathway, which may ultimately affect the structural integrity of the pollen wall in grape [40]. The over-expression of the grape stilbene synthase gene affected flavonoid metabolism in tomato, resulting in complete male sterility, and significant depletion of the precursors of lignin and sporopollenin biosynthesis resulted in pollen ablation [82].
The results showed that the treatment of CPPU and TDZ led to abnormal transcription regulation in some important pathways, especially those related to hydrolase activity, pentose and glucuronate interconversions, phenylpropanoid biosynthesis, and energy metabolism. These pathways are closely related to the synthesis of the pollen wall. Our previous experiments observed abnormal pollen wall formation following 10 mg/L CPPU or TDZ treatments. Combined with GO and KEGG enrichment analysis, we hypothesize that the primary cause of pollen abortion may be linked to the disrupted development of the pollen wall.

4.3. Major Genes Regulate Pollen Abortion in Grape

The flower phenotype and pollen grain morphology of grapes treated with 10 mg/L CPPU or TDZ closely resembled those of the male sterile line Y_14 (Figure 6). To explore the underlying mechanisms, transcriptome analysis was performed for the CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM groups. A total of 20 genes were identified, 16 of which exhibited a similar expression profile across these three groups (Figure 7). Further analysis of the expression of these 16 genes in 54 grape tissues revealed that 9 of them were highly expressed in the stamens and showed the same expression pattern in male sterile lines (Figure 8). These genes (CESA4, CER2, NPF2.11, MBF1C, MYB101, CAJ1, HSP23.6, NLTP6, and ERF109) are likely involved in pollen abortion.
The homologous genes of CESA4, CER2, NPF2.11, MBF1C, MYB101, and CAJ1 in Arabidopsis thaliana are shown in Table 3. These genes are known to be related to fertility. Cellulose synthase (CesA) is a key enzyme responsible for cellulose synthesis in the plant plasma membrane, with cellulose playing a critical role in the pollen wall. Different cellulose synthase proteins, such as CESA4, CESA7, and CESA8, are involved in cellulose biosynthesis in different tissues [83]. Very-long-chain fatty acid (VLCFA) lipids in the pollen coat are crucial for pollen hydration [84]. BAHD acyltransferase CER2/CER2-like proteins are essential for VLCFA synthesis [85]. In low-humidity environments, AtCER2 acts with AtCER2L2 to affect sterility [86]. The cer2 cer2l2 Arabidopsis double mutant leads to hydration defects [50]. AtNPF2.8 and AtNPF2.11 belong to the proton-dependent oligopeptide transporter (POT/PTR) family. AtNPF2.8/fst1 (flavanol-sophoroside-transporter 1) is specifically expressed in the tapetum and is essential for the accumulation of flavonol glycosides on pollen surfaces [48]. AtMBF1c overexpression increased seed yield in Arabidopsis and Glycine max [54,55], indicating its regulatory effect on plant fertility. MYB97, MYB101, and MYB120 are specifically expressed in mature pollen and pollen tubes [87]. Further study demonstrated that the pollen germination rate in the Arabidopsis triple mutant myb97 myb101 myb120 decreased, leading to fertilization defects and partial male sterility [57]. CAJ1 is a molecular chaperone protein in the DnaJ family. The J-domain protein J3 in Arabidopsis is involved in integrating flowering signals [53].
The homologous gene of HSP23.6 in wheat is TaHsp23.5. In BNS male-sterile wheat, this small heat shock protein gene (such as hsp23.5) shows significant down-regulation, potentially contributing to sterility [56]. The homologous genes of NLTP6 in wheat and tobacco are TansLTP3 and NtnsLTP, respectively. Three nsLTPs exhibit coordinated down-regulation (mRNA/protein) in the tobacco CMS line MSYY87 [51]. Zaidi et al. [52] identified a Triticeae anther-expressed type III nsLTP that potentially plays a role in pollen cell wall formation. In our study, NLTP6 was highly expressed in sterile lines, suggesting that it may influence lipid synthesis. The homologous gene of ERF109 in Chinese cabbage is BrERF109. Silencing BrERF109 can promote flavonoid biosynthesis [47]. Flavonoid synthesis is crucial for the normal development of pollen, and disruption of this pathway can affect extine formation [88]. The above studies suggest that important roles of these genes in the regulation of male sterility in plants, but knowledge on the relationship between genes and the fertility of pollen cells is limited, especially in grape.
In summary, pollen abortion of ‘Shine Muscat’ grape induced by 10 mg/L CPPU and TDZ treatments results from multiple factors and genes. A flowchart illustrating the experimental results of this study is presented (Figure 10). Our findings advance understanding of plant growth regulator-induced male sterility mechanisms, offering a theoretical basis for the breeding of the ‘Shine Muscat’.

5. Conclusions

In this study, different concentrations of CPPU and TDZ were applied to the flowers of ‘Shine Muscat’ one week before flowering. The treatment with 10 mg/L CPPU and TDZ induced morphological changes in the flowers, and the pollen germination rate decreased. Using RNA-seq technology, we compared the GO and KEGG pathways enriched by CPPU_10 vs. CK and TDZ_10 vs. CK. This revealed that the DEGs primarily affected molecular functions, particularly hydrolase activity, and were enriched in pathways related to pentose and glucuronate interconversions, phenylpropanoid biosynthesis, and energy metabolism. Combined with the transcriptome data of SM vs. Y_14, nine hub genes were identified, which may be involved in the regulation of pollen abortion. In future studies, functional verification experiments can be conducted to elucidate the specific functions of these hub genes in pollen development and their regulatory networks. Additionally, this study provides a novel approach to inducing male sterility phenotypes using exogenous hormones, which could facilitate breeding and benefit subsequent breeding efforts. The experimental results provide valuable insights for further investigating the effects of CPPU and TDZ on grape male sterility.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11050549/s1, Figure S1: Pearson correlation between samples; Table S1: Primers of qRT-PCR; Table S2: The quality transcriptome data; Table S3: The description of 20 genes.

Author Contributions

M.R. and Y.W.: Writing—original draft, Methodology, Formal analysis, Data curation, and Conceptualization. S.Y. and J.C.: Writing—review and editing, Visualization, and Validation. W.Z.: Writing—review and editing. H.L. and K.D.: Writing—review and editing, and Investigation. J.T. and H.Z.: Writing—review and editing, Methodology, and Funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the Achievement Transformation Fund project of Key R&D Technology Commissioner Projects in Hainan Province (ZDYF2024KJTPY008), the China Agriculture Research System of MOF and MARA (CARS-29), Tianshan Innovation of Team Xinjiang China (2022D14014), the Special Project of Science and Technology Special Commissioner Groups Serving Key Industrial Chains (2024N0068), and the National Natural Science Foundation of China (31901975), Fuan Grape Science and Technology Backyard, Jiangsu Province (Pukou) grape science and technology Backyard.

Data Availability Statement

The original data we used were transcriptome data with submission number CRA023343 in GSA (Genome Sequence Archive—CNCB-NGDC).

Conflicts of Interest

The authors declare that they have no competing interests.

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Figure 1. Flower phenotype, pollen germination in vitro, and paraffin section observation of ‘Shine Muscat’ after CPPU and TDZ treatments. (a) Flower phenotype of ‘Shine Muscat’ treated with CK, CPPU, and TDZ; (b) pollen germination of ‘Shine Muscat’ treated with CK, CPPU, and TDZ in vitro; (c) paraffin sections of anthers treated with CPPU and TDZ (note: CK: water; CPPU_3: 3 mg/LCPPU; CPPU_5: 5 mg/LCPPU; CPPU_10: 10 mg/LCPPU; TDZ_3: 3 mg/LTDZ; TDZ_5: 5 mg/LTDZ; TDZ_10: 10 mg/LTDZ).
Figure 1. Flower phenotype, pollen germination in vitro, and paraffin section observation of ‘Shine Muscat’ after CPPU and TDZ treatments. (a) Flower phenotype of ‘Shine Muscat’ treated with CK, CPPU, and TDZ; (b) pollen germination of ‘Shine Muscat’ treated with CK, CPPU, and TDZ in vitro; (c) paraffin sections of anthers treated with CPPU and TDZ (note: CK: water; CPPU_3: 3 mg/LCPPU; CPPU_5: 5 mg/LCPPU; CPPU_10: 10 mg/LCPPU; TDZ_3: 3 mg/LTDZ; TDZ_5: 5 mg/LTDZ; TDZ_10: 10 mg/LTDZ).
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Figure 2. Morphology observation of the pollen grains of ‘Shine Muscat’ after CPPU and TDZ treatments. (a) SEM observation of ‘Shine Muscat’ pollen grains treated with 10 mg/L CPPU and 10 mg/L TDZ; (b) TEM observation of ‘Shine Muscat’ pollen grains treated with 10 mg/L CPPU and 10 mg/L TDZ.
Figure 2. Morphology observation of the pollen grains of ‘Shine Muscat’ after CPPU and TDZ treatments. (a) SEM observation of ‘Shine Muscat’ pollen grains treated with 10 mg/L CPPU and 10 mg/L TDZ; (b) TEM observation of ‘Shine Muscat’ pollen grains treated with 10 mg/L CPPU and 10 mg/L TDZ.
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Figure 3. Statistical analysis of differentially expressed genes in CPPU_10 vs. CK and TDZ_10 vs. CK. (a) Statistics of the number of DEGs; (b) Venn plots of up-regulated DEGs in CPPU_10 vs. CK and TDZ_10 vs. CK; (c) Venn plots of down-regulated DEGs in CPPU_10 vs. CK and TDZ_10 vs. CK.
Figure 3. Statistical analysis of differentially expressed genes in CPPU_10 vs. CK and TDZ_10 vs. CK. (a) Statistics of the number of DEGs; (b) Venn plots of up-regulated DEGs in CPPU_10 vs. CK and TDZ_10 vs. CK; (c) Venn plots of down-regulated DEGs in CPPU_10 vs. CK and TDZ_10 vs. CK.
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Figure 4. GO annotation and enrichment in CPPU_10 vs. CK and TDZ_10 vs. CK. Note: The colors of the connecting lines represent the significance of GO and KEGG in each combination.
Figure 4. GO annotation and enrichment in CPPU_10 vs. CK and TDZ_10 vs. CK. Note: The colors of the connecting lines represent the significance of GO and KEGG in each combination.
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Figure 5. KEGG enrichment in CPPU_10 vs. CK and TDZ_10 vs. CK. Note: The colors of the connecting lines represent the significance of GO and KEGG in each combination.
Figure 5. KEGG enrichment in CPPU_10 vs. CK and TDZ_10 vs. CK. Note: The colors of the connecting lines represent the significance of GO and KEGG in each combination.
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Figure 6. Phenotype observation of flower and pollen grains of ‘Y_14’. (a) The phenotype of ‘Y_14’ flowers; (b) TEM observation of ‘Y_14’ pollen grains; (c) SEM observation of ‘Y_14’ pollen grains.
Figure 6. Phenotype observation of flower and pollen grains of ‘Y_14’. (a) The phenotype of ‘Y_14’ flowers; (b) TEM observation of ‘Y_14’ pollen grains; (c) SEM observation of ‘Y_14’ pollen grains.
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Figure 7. The DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM. (a) Venn plots of DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM; (b) the expression of DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM.
Figure 7. The DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM. (a) Venn plots of DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM; (b) the expression of DEGs in CPPU_10 vs. CK, TDZ_10 vs. CK, and Y_14 vs. SM.
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Figure 8. Heatmap of key gene expression in 54 grape tissues.
Figure 8. Heatmap of key gene expression in 54 grape tissues.
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Figure 9. Verification of the relative expression levels of differentially expressed genes in the transcriptome by qRT-PCR. Note: The left y-axis shows the relative gene expression levels determined by RT-qPCR, while the right y-axis shows the FPKM levels determined by RNA-seq. The different letters indicate significant differences (p ≤ 0.05).
Figure 9. Verification of the relative expression levels of differentially expressed genes in the transcriptome by qRT-PCR. Note: The left y-axis shows the relative gene expression levels determined by RT-qPCR, while the right y-axis shows the FPKM levels determined by RNA-seq. The different letters indicate significant differences (p ≤ 0.05).
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Figure 10. The pathways and possible key regulatory genes related to pollen abortion.
Figure 10. The pathways and possible key regulatory genes related to pollen abortion.
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Table 1. Effect of CPPU and TDZ on pollen viability of ‘Shine Muscat’.
Table 1. Effect of CPPU and TDZ on pollen viability of ‘Shine Muscat’.
TreatmentsTotal Number of
Pollen Grain		Number of Germinated
Pollen Grains		Germination Rate (%)
CK35120357.83 ± 6.43 a
CPPU_333311534.39 ± 4.30 b
CPPU_52986923.28 ± 3.95 c
CPPU_1023400 d
TDZ_321600 d
TDZ_526100 d
TDZ_1038000 d
Note: The different letters in the same column indicate significant differences (p ≤ 0.05).
Table 2. Pollen morphological size and deformation rate of ‘Shine Muscat’ after CPPU and TDZ treatments.
Table 2. Pollen morphological size and deformation rate of ‘Shine Muscat’ after CPPU and TDZ treatments.
TreatmentsPolar Axis (μm)Equatorial Axis (μm)P/E RatioPollen ShapeGerminalDeformation Rate
CK30.90 ± 0.91 a15.95 ± 1.69 a1.95 ± 0.20 aProlateThree23.80% b
TDZ_1017.93 ± 1.96 b15.88 ± 3.33 a1.15 ± 0.17 bSub prolateNone100.00% a
CPPU_1019.39 ± 1.05 b11.35 ± 0.68 b1.71 ± 0.05 aProlateNone100.00% a
Note: The different letters in the same column indicate significant differences (p ≤ 0.05).
Table 3. Homologous genes of hub genes and their descriptions.
Table 3. Homologous genes of hub genes and their descriptions.
Gene NameHomologous GeneHomologous Gene DescriptionHomologous Gene Function
VvCESA4
(LOC100241197)		AtCESA4Cellulose synthase A4CesA is an enzyme that catalyzes the synthesis of cellulose, which is an important component of the pollen wall [49].
VvNPF2.11
(LOC100244038)		AtNPF2.11Major facilitator superfamily proteinThe AtNPF2.8/fst1 is specifically expressed in tapetum [48].
VvCER2
(LOC100245757)		AtCER2HXXXD-type acyl-transferase family proteincer2 cer2l2 Arabidopsis double mutant leads to male sterility [50].
VvMBF1C
(LOC100249249)		AtMBF1CMultiprotein bridging factor 1CMBF1c overexpression increased seed yield in Arabidopsis and Glycine max [54,55].
VvMYB101
(LOC100253438)		AtMYB101myb domain protein 101Arabidopsis triple mutant myb97 myb101 myb120 leads to defects in fertilization and partial plant male sterility [57].
VvCAJ1
(LOC100260760)		AtDJC77DNAJ heat shock N-terminal domain-containing proteinJ-domain protein J3 in Arabidopsis plays a role in integrating flowering signals [53].
VvHSP23.6
(LOC100253548)		TaSusHSP24.1 kDa heat shock protein, mitochondrial isoform X1Small heat shock protein gene (such as hsp23.5) is significantly down-regulated in male sterile lines of wheat BNS (Bainong sterility) [56].
VvNLTP6
(LOC100260175)		TansLTP3Non-specific lipid-transfer protein 3-likeThe three nsLTPs from the tobacco CMS line MSYY87 were significantly down-regulated [51]. Triticeae anther-expressed type III nsLTP with possible roles in pollen cell wall formation [52].
NtnsLTPNon-specific lipid-transfer protein-like
VvERF109
(LOC104879921)		BrERF109Ethylene-responsive transcription factor ERF109-likeSilencing BrERF109 can promote flavonoid biosynthesis [47], and flavonoid synthesis is crucial for the normal development of pollen.
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Ren, M.; Wang, Y.; Yi, S.; Chen, J.; Zhang, W.; Li, H.; Du, K.; Tao, J.; Zheng, H. Combined Transcriptome Analysis Reveals the Mechanism of ‘Shine Muscat’ Pollen Abortion Induced by CPPU and TDZ Treatment. Horticulturae 2025, 11, 549. https://doi.org/10.3390/horticulturae11050549

AMA Style

Ren M, Wang Y, Yi S, Chen J, Zhang W, Li H, Du K, Tao J, Zheng H. Combined Transcriptome Analysis Reveals the Mechanism of ‘Shine Muscat’ Pollen Abortion Induced by CPPU and TDZ Treatment. Horticulturae. 2025; 11(5):549. https://doi.org/10.3390/horticulturae11050549

Chicago/Turabian Style

Ren, Mengfan, Yixu Wang, Siyi Yi, Jingyi Chen, Wen Zhang, Haoran Li, Ke Du, Jianmin Tao, and Huan Zheng. 2025. "Combined Transcriptome Analysis Reveals the Mechanism of ‘Shine Muscat’ Pollen Abortion Induced by CPPU and TDZ Treatment" Horticulturae 11, no. 5: 549. https://doi.org/10.3390/horticulturae11050549

APA Style

Ren, M., Wang, Y., Yi, S., Chen, J., Zhang, W., Li, H., Du, K., Tao, J., & Zheng, H. (2025). Combined Transcriptome Analysis Reveals the Mechanism of ‘Shine Muscat’ Pollen Abortion Induced by CPPU and TDZ Treatment. Horticulturae, 11(5), 549. https://doi.org/10.3390/horticulturae11050549

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