Next Article in Journal
Syzygium oleosum (F.Muell.) B.Hyland (Myrtaceae), an Unexplored Australian Species: Anatomical and Micromorphological Study of Leafy Twigs, and Characterization and Biological Activity of Their Essential Oil
Previous Article in Journal
Edible Flowers as Bioactive Food Ingredients with Antidiabetic Potential: A Study on Paeonia officinalis L., Forsythia × intermedia, Gomphrena globosa L., and Clitoria ternatea L.
Previous Article in Special Issue
Advances in Genetic Diversity of Germplasm Resources, Origin and Evolution of Turnip Rape (Brassica rapa L.)
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Plants
Volume 14
Issue 16
10.3390/plants14162604
plants-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

by
Siyu Xiong
1,2,†,
Xiaochun Wei
1,2,†,
Wenjing Zhang
1,
Yanyan Zhao
1,
Shuangjuan Yang
1,
Henan Su
1,
Baoming Tian
1,2,
Fang Wei
1,2,
Xiaowei Zhang
1,2,* and
Yuxiang Yuan
1,*
1
Institute of Vegetables, Henan Academy of Agricultural Sciences, Graduate T&R Base of Zhengzhou University, Zhengzhou 450002, China
2
School of Agricultural Sciences, Zhengzhou University, Zhengzhou 450001, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Plants 2025, 14(16), 2604; https://doi.org/10.3390/plants14162604
Submission received: 28 July 2025 / Revised: 14 August 2025 / Accepted: 19 August 2025 / Published: 21 August 2025
(This article belongs to the Special Issue Epigenetic Regulation and Molecular Mechanisms in Brassica Crop Improvement)
Browse Figures
Versions Notes

Abstract

Ogura cytoplasmic male sterility (CMS) in Chinese cabbage (Brassica rapa) is characterized by complete pollen abortion, wherein stamens fail to produce viable pollen while pistils retain normal fertility. This maternally inherited trait is valuable for hybrid breeding. This study employed integrated analysis of miRNA, transcriptome, and degradome sequencing data aligned to the Chinese cabbage reference genome to elucidate the molecular function of bra-miR9569 in Ogura CMS pollen fertility and explore its associated pathways. Subsequently, a bra-miR9569 overexpression vector was constructed and transformed into Arabidopsis thaliana. Phenotypic characterization of transgenic Arabidopsis lines, combined with anther viability assessment and quantification of ATP content and reactive oxygen species (ROS) levels in Chinese cabbage, was performed to analyze the effects of bra-miR9569. Our findings demonstrate that mutation of the mitochondrial gene orf138 in Ogura CMS lines leads to upregulation of bra-miR9569. This microRNA negatively regulates the expression of the ATP-related gene AHA6, resulting in reduced H+-ATPase activity. The consequent energy deficiency triggers cellular content degradation, ultimately causing failure of pollen wall formation and pollen abortion.

1. Introduction

Male sterility (MS) is a phenomenon in which the female gametes of a plant develop normally, and the male gametes develop abnormally but can receive external normal pollen for fertilization and fruiting [1]. There are two types of MS in plants: cytoplasmic male sterility (CMS) and genic male sterility (GMS). CMS is co-regulated by mitochondrial genes coupled to nuclear genes [2], and it commonly occurs in higher plants. In CMS plants, mitochondrial and nuclear genes interact to control male specificity, fertility development, and restoration in plants. CMS is maternally inherited and widespread [3]. Under this system, stamen morphology generally shows short filaments and tiny wrinkled anthers [4], but female functional organs are normal and can receive foreign pollen for fertilization to produce offspring.
Chinese cabbage is the most consumed cruciferous crop in East Asia, and it has been domesticated for about 6000 years; there are currently several varieties of Chinese cabbage, including spring and autumn types [5]. The leaves of Chinese cabbage are popular for their antioxidant capacity as well as their rich nutritional value, such as fiber and vitamins [6,7,8]. Cytoplasmic male sterile lines have become an important production pathway for utilizing the hybrid advantage in vegetables. Ogura-type cytoplasmic male sterility (Ogura CMS) has the advantages of complete pollen abortion, easy transfer, and progeny sterility up to 100%, which are widely utilized to breed cruciferous crops [9,10]. Currently, Ogura CMS is widely used in the crossbreeding of cruciferous vegetables, including Chinese cabbage [11], but the mechanism of its MS is still unclear. Currently, CMS has been reported in a variety of plants, and many mitochondrial genes responsible for CMS have been identified [12,13], which is significant for the in-depth study of the molecular mechanisms regulating CMS and the simplification of crop breeding for high-quality production.
MicroRNAs (miRNAs) are endogenous small RNAs that are about 18–24 nucleotides in length and that negatively regulate the expression of target genes at the transcriptional or post-transcriptional level by degrading target genes or inhibiting translation and DNA methylation of target genes [14]. miRNAs play an important role in biological processes, such as developmental regulation, and biotic and abiotic stress responses in plants [15]. Some miRNAs affect pollen development through post-transcriptional regulation [16,17,18], especially in the development of microspores [19] and the sporophyte chorioallantoic layer, a single-celled layer encasing the anther chamber that plays a key role in pollen development [20,21].
During plant growth and development, the pollen tube is the fastest growing plant cell, and it needs to consume a large amount of energy during polarized growth, as ATP plays an important role [22]. Studies have shown that the plasma membrane H+-ATPase (AHAs) in Arabidopsis thaliana is the largest family of cation-transporting P-type ATPases found in plants [23,24]. It is also involved in membrane potential formation, stress resistance, and cell signaling, which are essential for normal plant growth and development. The structure, function, and mechanism of action of H+-ATPases have been extensively studied [25,26], and H+-ATPases are believed to be key factors in the formation and maintenance of the transmembrane H+ gradient [27]. Pollen development and pollen tube growth are processes with high energy demands [28]. H+-ATPases are required for normal pollen germination and pollen tube elongation, and this AHA enzyme functions redundantly in pollen tubes [29]. In Arabidopsis thaliana, AHA6, AHA8, and AHA9 are redundantly involved in the formation of the proton gradient and cell wall pattern in the pollen tube and are essential for polar growth of the pistil [30].
Mutations in the mitochondrial orf138 gene trigger programmed cell death (PCD) in autogamous cells, leading to abnormal enlargement and content degradation, which results in vacuolization of the three-cell stage and pollen abortion [31]. In maize, by strongly interacting with atp8 and atp9, the mitochondrial gene atp6C can disrupt the assembly of ATP synthase and reduce the activity of free hydrolase, leaving the demand for ATP unsatisfied during anther development, which increases reactive oxygen species (ROS) production, advances PCD in chorionic layer cells, and leads to pollen abortion [32]. In this study, we screened key differentially expressed miRNAs and target genes by sequencing the transcriptome, degradome, and miRNA library in Ogura CMS Chinese cabbage and maintainer lines with early and late pollen development and transformed Arabidopsis thaliana by constructing an OE-miR9569 overexpression vector. The mechanism by which bra-miR9569 is involved in CMS was further elaborated to provide new insights for hybrid seed production.

2. Results

2.1. Quality Control Analysis of Transcriptome Sequencing Data from Maintainer and Ogura CMS Chinese Cabbage Lines

Illumina sequencing was performed at the early (buds 1.5–3.5 mm in length) and late (buds > 5 mm in length) stages of pollen development in the Chinese cabbage maintainer line (Y231-330) and the Ogura CMS line (Tyms), with three biological replicates for each period. After sequencing quality control, 138.12 GB of clean data were retained, of which, ML1 (sample IDs: S07, S08, S09), the Chinese cabbage maintainer line at the early stage of pollen development, had an average of 77,421,534 clean reads; and ML2 (sample IDs: S01, S02, S03), at the late stage of pollen development, had an average of 78,679,234 clean reads. The Ogura CMS line had an average of 76,235,037 clean reads in the early stage of pollen development (CMS1, sample IDs: S10, S11, S12) and 75,917,886 clean reads in the late stage of pollen development (CMS2, sample IDs: S04, S05, S06). The GC content of the 12 samples ranged from 43.46 to 45.57%, and Q30 was greater than 95.5% for all samples. The minimum number of mapped reads localized to the reference genome of Chinese cabbage version 1.5 was 55,082,768, and the maximum number of mapped reads was 70,645,510, accounting for 72.23–88.59% of the clean reads (Table 1). These results showed that the sequencing data were of good quality and reliable and could be uniquely compared to the reference genome of Chinese cabbage. Three-dimensional principal component analysis (PCA) showed that the components were independent of each other, and the biological replicates were tightly clustered, indicating a high degree of independence between the samples and a strong correlation between the three replicates (Figure 1A). Using a false discovery rate (FDR) of 0.01 and a fold change criterion (Fold Change) of 2, the screen identified the presence of 3712 upregulated differentially expressed genes (DEGs) and 4036 downregulated DEGs in the early stages of pollen development in both lines. The number of up- and down-regulated DEGs identified in the late stages of pollen development in both lines were 2431 and 5258, respectively (Figure 1B).
The number of single nucleotide polymorphism (SNP) loci, proportion of transformation types, proportion of subversion types, and proportion of heterozygous SNP loci screened for each sample were counted (Table 2). The number of SNP loci was slightly higher during early pollen development than during late pollen development in both the maintainer line (Y231-330) and the Ogura CMS line (Tyms), and most of the SNP loci detected were located in the gene coding region. The percentages of the total number of SNP sites detected in the 12 samples were 57.37, 42.63, and 35.46% for conversion-, subversion-, and heterozygous SNP sites, respectively. CMS1 and CMS2 refer to the early and late stages of pollen development in sterile lines, while ML1 and ML2 refer to the early and late stages of pollen development in maintainer lines.

2.2. Quality Control of miRNA Sequencing Data from Anther Samples of Both Lines

To study the regulation of miRNA on the fertility of Ogura CMS, we constructed small RNA sequencing libraries from anthers of sterile and homozygous maintainer lines at different stages of pollen development and set up three biological replicates at two different stages for the two lines. Small RNA sequencing of 12 samples was completed, and 143.64 Mb of clean reads were obtained, with no less than 10.04 Mb of clean reads for each sample. The base quality values of Q30 were more than 96.11%, indicating that the sequencing data were of high quality and reliable (Table 3). After filtering the small RNA sequences without reads, the length distribution of small RNAs was 18–24 nt (Supplementary Material SA Figure S1). Most of the RNAs had sequence sizes of 19–24 nt, with most small RNAs in the library having 21 nt, followed by small RNAs of 24 nt in length.
miRNA sequencing data were statistically analyzed. The two lines had 325 commonly expressed miRNAs during early pollen development, with 12 miRNAs specifically expressed in the sterile line and only 7 miRNAs specifically expressed in the maintainer line (Figure 2A), whereas 235 commonly expressed miRNAs were found during late pollen development, with 66 miRNAs specifically expressed in the sterile line and 12 miRNAs specifically expressed in the maintainer line (Figure 2B).

2.3. Sequencing Analysis of the Degradome of Two Chinese Cabbage Lines

To identify the target genes of miRNAs obtained using miRNA sequencing, anther samples from different pollen development periods of the Ogura CMS line and its maintainer line were mixed, and a degradome library was constructed for late sequencing analysis. A total of 19.00 M clean tags were obtained by sequencing. The original tags obtained from sequencing, clean tags and cluster tags, were obtained after quality inspection, both of which were 47 nt in length, and a total of 18,995,289 clean data were obtained. The sequencing data from the samples were compared to the reference genome of Chinese cabbage (Brassica rapa v1.5). The comparison results are shown in Table 4. The p-value was set at less than 0.05, and miR9569 was detected to shear the target gene Bra013168 (AHA6) at locus 582 (Supplementary Material SA Figure S2).

2.4. Joint Analysis of miRNAs and Their Target Genes by miRNAomics

Differentially expressed miRNAs from miRNA and degradome sequencing of anthers of the Ogura CMS line and its isoforms at different pollen developmental stages were jointly analyzed with DEGs identified in transcriptome sequencing. The 22 genes regulated by novel_mir_403, novel_mir_448, novel_mir_51, and novel_mir_95 were differentially expressed miRNAs and DEGs (Supplementary Material SB). The NR annotations of these genes were sucrose transporter proteins, growth hormone response factors, cytochrome c biosynthesis proteins, cysteine proteases, bet v I allergenic family proteins, MYB-associated proteins, adenosine triphosphate conjugating, and H+-ATPase.

2.5. H+-ATPases Related Genes Are Significantly Downregulated During Anther Development in the Ogura CMS Sterile Line of Chinese Cabbage

This study utilized sequencing data from the miRNA, transcriptome, and degradome libraries of the Ogura CMS line and its isoform maintainer line (NCBI; CNGBdb). Screening statistics of genes downregulated in the maintainer and sterile lines during both periods revealed 1490 commonly expressed DEGs, with 3727 DEGs specifically expressed during the early stage of pollen development and 2591 genes expressed during the late stage of pollen development in both lines (Figure 3A). GO enrichment analysis of differential mRNAs commonly expressed in sterile and maintainer lines showed that they were mainly enriched in the entries of plasma membrane, proton translocation, and H+-ATPase activity (Figure 3B). Visualization of ATP-related gene expression in a heat map revealed that gene Bra013168 (AHA6) was significantly downregulated during pollen development in the sterile line (Figure 3C). AHA6 is an H+-ATPase that may be involved in energy metabolism, thereby affecting the formation of the pollen wall during pollen development and leading to pollen abortion. PPa3 is a gene that encodes a protein with inorganic pyrophosphorylase activity that promotes the hydrolysis of pyrophosphate to phosphoric acid, thereby participating in ATP synthesis. VHA-E2 is an H+-ATPase subunit, and VTAG3 is a V-ATPase G-subunit-like protein. AHA6 and AHA9 are both H+-ATPases. VHA-E2, VTAG3, AHA6, and AHA9 affect ATP generation by the binding of adenosine diphosphate to phosphoric acid, suggesting that a certain regulatory relationship between ATP and pollen septation may exist.
We preliminarily hypothesized the pathway mechanism of pollen abortion caused by the mutation of the mitochondrial gene orf138. The mutation of the mitochondrial gene orf138 in the Ogura CMS line caused the downregulation of ATP related genes, triggering the reduction of H+-ATPases and the lack of energy, which led to content degradation, failure to form the pollen wall, and pollen abortion.

2.6. Analysis of bra-miR9569 Targeting Relationship with Target Genes and Bioinformatics Analysis

To identify the targeting relationship between miR9569 and its target gene AHA6, this study analyzed the target location between miR9569 and its target gene AHA6 using psRNATarget and psRobot software and found that miR9569 cleaves to the target gene AHA6 at 582 bp of its sequence (Supplementary Material SD Figure S1). This anchors the targeting relationship between the two. Mature and precursor sequences of miR9569 were obtained from the miRBase database (Supplementary Material SC Table S1). Chromosomal localization of the miR9569 precursor and its target gene AHA6 revealed that both were located on chromosome A03. The results for the amplification of miR9569 precursor gene sequences are shown in Supplementary Material SD Figures S2 and S3. The secondary structure of the miR9569 precursor was predicted using RNAfold, and the red line shows the location of the sequence of its mature body (Supplementary Material SD Figure S4). Base conservation of the miR9569 mature body sequence was analyzed based on the software Weblogo and showed that miR9569 was highly conserved at bases 7, 9, 10, 12, and 15 (Supplementary Material SD Figure S5).

2.7. qRT-PCR Validation of miR9569/AHA6 Regulation and Differential Expression Profile Reliability

miR9569 and its target gene, AHA6, were selected at different developmental periods in the Ogura CMS and maintainer lines and subjected to quantitative fluorescence analysis to verify the negative regulatory relationship between miRNAs and target genes. miR9569 expression increased significantly with pollen development in the Ogura CMS line, while the expression was lower in the maintainer line. The expression of its target gene, AHA6, was higher at the early stage of pollen development in the maintainer lines, and the expression of its target gene decreased with pollen development. It was not expressed in the sterile line. The fluorescence quantification results revealed that miR9569 negatively regulated the target gene AHA6 (Figure 4A,B).
One known miRNA (miR159a) and two new miRNAs (novel_miR_448, novel_miR_51) that were differentially expressed and six DEGs were selected to validate the reliability of the transcriptome sequencing data. The qRT-PCR measurements of these miRNAs (Figure 4C–E) and DEGs (Figure 4F–K) showed consistent expression trends with the sequencing FPKM (Fragments Per Kilobase of exon model per Million mapped fragments) values. These results showed that the selected and sequenced results had the same trend, demonstrating the reliability of the sequencing data.

2.8. Genetic Transformation of OE-miR9569 Arabidopsis Thaliana and Identification of Positive Seedlings

In this study, homologous recombination and golden gate seamless cloning were used for vector construction, and the recombinant plasmid OE-miR9569 was digested using EcoRV endonuclease. The recombinant plasmid fragments were of the correct size (Supplementary Material SE Table S1). The structure of the OE-miR9569 vector is shown in Supplementary Material SE Table S2. The constructed vector was subjected to agarose gel electrophoresis by colony PCR (Supplementary Material SE Table S3). Positive colony PCR products were sequenced, and the sequencing results were correct, indicating that the OE-miR9569 vector was successfully constructed.
The OE-miR9569 transgenic Arabidopsis seeds obtained by the flower dipping method were sown in 1/2MS medium containing genistein (G418). After about six weeks, DNA was extracted, and the antibiotic sequence fragment was amplified by PCR for the preliminary verification of positive seedlings. The presence of bands revealed that the vector was successfully transferred into plants (Figure 5A). After preliminary screening, RNA was extracted from the plants, and after designing primers for miR9569 expression detection, miR9569 and its target gene AHA6 were detected by fluorescence quantification. Compared with the wild-type plants, miR9569 expression in the OE-miR9569 transgenic plants was significantly increased (Figure 5B), and the expression of its target gene was significantly decreased (Figure 5C). Plants with significant changes in the expression of miR9569 and its target gene were selected. OE-miR9569-positive transgenic Arabidopsis plants were obtained, and the seeds were harvested and preserved after the pods matured.

2.9. Phenotypic Analysis Reveals miR9569 Regulates Pollen Dispersal and Maturation in Arabidopsis

To verify the function of miR9569, the OE-miR9569 Arabidopsis mutant and the wild type were phenotypically characterized in this study. There were no significant differences in inflorescence morphology size between wild-type Arabidopsis and OE-miR9569 transgenic Arabidopsis (Figure 6A,D). The anthers were then observed after removing their petals with forceps, and the amount of pollen dispersed by OE-miR9569 transgenic Arabidopsis thaliana was significantly reduced compared to that dispersed by the wild type during the flowering stage (Figure 6B,E). Observation of siliques at maturity in both materials revealed that OE-miR9569 transgenic Arabidopsis thaliana had smaller pods that were shriveled and not full compared to the wild type (Figure 6C,F). These studies indicate that miR9569 affects plant pollen fertility to a certain extent.

2.10. miR9569 Overexpression Compromises Pollen Viability and Anther ATP Levels via H+-ATPase Dysregulation

To further analyze the differences between OE-miR9569 transgenic Arabidopsis thaliana and the wild type, this study observed anther staining and identified the pollen viability of the OE-miR9569 Arabidopsis thaliana mutant and wild-type Arabidopsis thaliana. OE-miR9569 transgenic Arabidopsis thaliana had a certain degree of pollen inactivity compared with the wild type, and the whole stained anthers were transparent (Figure 7A,B). The inactivated part of pollen grains was not stained a purple–red color (Figure 7C,D), and the rate of pollen grain stainability was significantly reduced (Figure 7E). The ATP content of the anthers of both materials was measured, and it was found that the ATP content of the anthers of OE-miR9569 transgenic Arabidopsis thaliana was significantly decreased compared with that of the wild type (Figure 7F).
These findings suggest that miR9569 may affect pollen fertility by influencing H+-ATPases and that upregulated expression of miR9569 causes pollen inactivity or failure to form normal full oval pollen grains, reducing plant fertility.

2.11. Anther Cavitation and ROS Toxicity: Key Phenotypes of Pollen Sterility in miR9569-Overexpressing Arabidopsis

To observe the anthers and their internal structural status between the OE-miR9569 mutant and wild-type Arabidopsis thaliana, a scanning electron microscope was used to observe the anthers and pollen grains inside the anthers. There were no significant differences between their anthers at the unflowering stage (Figure 8A,D). Local magnification of the anthers revealed that wild-type Arabidopsis anthers were filled with a large number of full pollen grains (Figure 8B), whereas the local magnification of the internal parts of the OE-miR9569 transgenic Arabidopsis anthers showed empty chambers with few viable pollen grains (Figure 8E). The interior of wild-type Arabidopsis anthers was largely unstained, indicating that both had less internal ROS accumulation (Figure 8C), whereas the interior of OE-miR9569 transgenic Arabidopsis anthers was filled with green fluorescence, indicating that their anthers had more internal ROS accumulation (Figure 8F).
In summary, OE-miR9569 transgenic Arabidopsis thaliana has almost no production of normal pollen grains in some areas inside the anthers. Thus, it is hypothesized that the toxic effect of the large ROS accumulation inside its anthers reduces pollen fertility.

3. Discussion

In this study, we obtained high-quality sequencing results by sequencing the sRNA, degradome, and transcriptome of the Ogura CMS line and its homozygous maintainer line of Chinese cabbage. The transcriptome data were statistically analyzed, and the genes downregulated in the two periods in both maintainer and sterile lines were screened, revealing 1490 DEGs. GO enrichment analysis of these DEGs showed that they were mainly enriched in the plasma membrane, proton transport, and H+-ATPase activity, indicating that pollen development was closely related to H+-ATPases. Starch metabolism provides energy and a carbon skeleton for pollen development, germination, and pollen tube elongation and is decisive for pollen biological function and fertility. In barley, ATP-driven starch synthesis fuels pollen maturation [33,34], with ATP availability being rate-limiting for starch accumulation. Chili pepper CMS lines exhibit premature ATP hydrolysis during the tetrad stage, followed by undetectable H+-ATPase activity in uninucleate microspores, directly causing male sterility [35]. ATP is one of the main factors affecting pollen development and is crucial for pollen development, with large amounts of energy required for processes such as timely PCD of chorioallantoic cells and pollen wall formation [29].
In plant cells, mitochondria are important sites for ATP synthesis, and CMS expression is the result of subtle interactions between mitochondrial and nuclear genes [36]. Arabidopsis plasma membrane H+-ATPases (AHAs; notably AHA6/8/9) establish proton gradients that direct pollen tube tip growth through cell wall remodeling [30]. In maize, the mitochondrial gene atp6C is unable to meet the ATP requirement during anther development by interacting with atp8 and atp9, leading to pollen abortion [32]. We previously identified Brassica-specific miR9569 as a sterility-linked regulator that targets pollen-essential genes through its anther-specific overexpression in CMS lines [11]. Genome-wide analysis of the transporter protein genes expressed in the four developmental stages of the male gametophyte has shown that AHA6 is specifically or preferentially expressed relative to the sporophyte tissue [37]. The absence of AHAs leads to a decrease in energy, thus affecting pollen development [30], which is consistent with the results of the present experimental study.
This study established a mechanistic framework (Figure 9) in which the specific overexpression of miR9569 in anthers of the Chinese cabbage Ogura CMS line inhibits BrAHA6, a plasma membrane H+-ATPase that is crucial for pollen development. Key research findings indicate that miR9569-mediated downregulation of BrAHA6 disrupts the H+ electrochemical gradient across the plasma membrane, and H+-ATPase is a key factor in the formation and maintenance of the transmembrane H+ gradient [27]. The aberrant H+ gradient compromises secondary transport systems, ultimately affecting cellular energy homeostasis. Elevated ROS and ATP levels observed in miR9569-overexpressing (OE) lines reflect compensatory metabolic stress or failed energy utilization downstream of H+-ATPase dysfunction. Impaired proton pumping hinders ATP-dependent processes that are critical for fertility, impeding pollen wall formation and timely PCD of chorionic cells. Consequently, BrAHA6 suppression by miR9569 disrupts pollen wall patterning and tapetal PCD, directly leading to reduced pollen fertility. These findings underscore BrAHA6 as a pivotal node linking miRNA regulation to energy metabolism and structural development in pollen. In conclusion, this study provides theoretical guidance for the genetic breeding of Chinese cabbage through the study of the effect of miR9569 on pollen fertility.

4. Materials and Methods

4.1. Experimental Materials

The Ogura CMS Chinese cabbage line (Tyms) and its maintainer line (Y231-330) selected for this study were provided by the Institute of Vegetables, Henan Academy of Agricultural Sciences (Zhengzhou, China), and were planted in Henan Modern Agricultural Research and Development Base (Yuanyang, China) [11]. Seeds were selected and sown in seed trays filled with substrate. During the germination period, the temperature was maintained at 25 °C. After transplanting to the field, timely control measures were taken against pests, diseases, and weeds to avoid the impact of non-natural factors on the experiment. Regular irrigation was carried out, and observations were made.

4.2. RNA Extraction and Library Construction

Total RNA was extracted from anther samples by using RNAiso Plus (Takara BioTechnology Co., Ltd., Dalian, China). The purity and concentration of total RNA were determined using a micro-volume spectrophotometer (Nano-500), and RNA integrity was assessed by 1.2% agarose gel electrophoresis. Total RNA samples that passed the test were separated into fragments using polyacrylamide gel electrophoresis, and small RNAs in the range of 18–30 nt were recovered using gel recovery. The small RNA libraries were obtained using reverse transcriptase, and libraries that passed the quality test were used in high-throughput sequencing with the HiSeq X-ten platform (Biomark Biotechnology Co., Ltd., Beijing, China). Raw reads were processed according to the base quality value [38] and nucleotide number to obtain high-quality sequences (clean reads). Bowtie software [39] was used to compare and process the clean reads to obtain the unannotated reads (including miRNAs), and sequence matching was performed with the corresponding reference genomes.

4.3. Construction of the cDNA Library and Data Processing

Sequencing of cDNA libraries using sequencing by synthesis (SBS) technology generates a large amount of high-quality data, which is referred to as raw data. The raw data generated in FASTQ format must be cleaned to remove low-quality reads and those containing adapters and poly-N sequences, resulting in clean reads. Clean reads were aligned against the Chinese cabbage reference genome v1.5 (http://39.100.233.196:82/download_genome/) (29 July 2025). The StringTie (1.3.1) software was used to calculate the FPKM values for mRNA and coding genes in each sample. The Bioconductor package EdgeR (1.10.1) [40] was used to identify differentially expressed genes, while Poisson distribution and empirical Bayesian methods were employed to reduce the coefficient of variation among transcripts, thereby enhancing the reliability of the analysis results. The screening criteria were a fold change (FC) ≥ 2 and a false discovery rate (FDR) < 0.05. All analyses were performed using software tools on the BMK Cloud platform (https://www.biocloud.net/) (29 July 2025).

4.4. Construction of the Degradome Library and Statistics for the Sequencing Results

Total RNA was extracted at different pollen development periods in Ogura CMS and maintainer Chinese cabbage lines and mixed into one sample in equal amounts to construct the degradome library. Clustering data were compared with the Rfam database [41] to obtain relevant annotation information, and the unannotated sequences were subjected to subsequent degradation site analysis. Degradation sites were detected by Cleaveland [42] software using the known miRNAs, miRNAs predicted from the small RNA analysis, and the sequence information of the transcripts of the corresponding species genes (Y231-330 and Tyms).

4.5. qRT-PCR Validation of miRNA and mRNA Sequencing Data

The target miRNAs were subjected to real-time fluorescence quantitative PCR (qRT-PCR) by adding poly-A at the 3′ end to verify the reliability of the miRNA sequencing data. miRNA reverse transcription was performed using a Takara Mir-XmiRNA First-Strand Synthesis Kit (Takara BioTechnology Co., Ltd., Dalian, China) and TB GreenTM qRT-PCR User Manual Reverse Transcription Kit (Takara BioTechnology Co., Ltd., Dalian, China). mRNA reverse transcription and qRT-PCR were performed using kits from Takara. Based on the mRNA sequences, Primer 5.0 was used to design the primers for mRNA fluorescence quantitative PCR. GAPDH screened in our laboratory was used as the internal reference primer, and primer synthesis was performed by Zhengzhou Sunya Biotechnology Co. (Zhengzhou, China). All qPT-PCR experiments were performed on the LightCycler® 480 Instrument II (F. Hoffmann-La Roche, Basel, Switzerland), and the expression levels of relevant genes for -miRNA and mRNA were analyzed using the 2−ΔΔCt method [43]. qRT-PCR-related primer sequences are listed in Supplementary Material SC Tables S2 and S3.

4.6. OE-miR9569 Arabidopsis Thaliana Transformation

The genetic transformation material used in this study was Arabidopsis thaliana (Columbia, 2n), provided by the Institute of Vegetables, Henan Academy of Agricultural Sciences (Zhengzhou, China). Agrobacterium tumefaciens (GV3101) and Escherichia coli (DH5α) were used as sensory cells by Weidi Biological Company (Shanghai, China); the vector interference and overexpression vectors used were the pBWA(V)KS-ccDB vector provided by Biorun Biological Company (Wuhan, China).
Arabidopsis thaliana was sown and incubated in a light incubator with 16 h of light (22 °C) and 8 h of dark (18 °C). The culture was then expanded using the flower dipping method with Agrobacterium tumefaciens and activated by shaking at 28 °C for 2 days. Arabidopsis thaliana was then infested by the flower dipping method. Agrobacterium spp. was expanded and activated by shaking at 28 °C for 2 days, transferred to centrifuge tubes, centrifuged at 6000 rpm for 10 min, and resuspended in osmotic solution (containing 5% sucrose, 0.02% SilwetL-77, and 1/4 MS (Murashige and Skoog) nutrient solution with Acetosyringone) to form Agrobacterium spp. Agrobacterium tumefaciens was resuspended in an osmotic solution (OD600 = 0.8). The siliques and open flowers of 6-week-old Arabidopsis plants were removed, and the unopened flower buds were removed with forceps. The Arabidopsis seedlings were inverted, and their inflorescences were immersed in the prepared suspension for 20–30 s to fully infest the inflorescences. After infestation, an appropriate amount of water was sprayed onto infested Arabidopsis, and the infested inflorescences were covered with black plastic film and grown in a dark box for 24 h. The treated Arabidopsis plants were placed in a light incubator (16 h of light at 22 °C, 8 h of dark at 18 °C) for normal growth. The infested inflorescences were re-infested after one week, and the infested inflorescences were harvested at maturity after about three weeks.

4.7. Identification of Pollen Viability

Plant pollen grain viability was determined by Alexander staining. Viable pollen grains stained with Alexander’s stain were a distinct purple–red color, while pollen that was not stained with Alexander’s stain had abnormal coloring. Alexander staining was performed using an appropriate amount of Alexander staining solution placed in a 200 µL EP tube. The mature anthers were removed with tweezers under the stereo microscope (Toup Optoelectronics Technology Co., Ltd., Hangzhou, China) and placed into the centrifuge tube containing the Alexander staining solution. They were stained for 1 h under light protection, and the stained anthers were rinsed with water. They were placed on slides (with an appropriate amount of water added to the surface), adjusted, and covered with a coverslip. The slides were then observed, and images were acquired after microscopic examination of the film.

4.8. Determination of the ATP Content and ROS Content

We selected Arabidopsis thaliana in full bloom as the experimental material. The ATP content of the anthers of transgenic Arabidopsis thaliana was determined using a reagent kit (Grace Biotechnology Co., Suzhou, China). To determine the active oxygen content, the floral organs, including petals, calyx, and stigma, of transgenic Arabidopsis thaliana were removed with tweezers, and the anthers were placed in 1.5 mL EP tubes with freshly prepared MES-KCl buffer. The material was completely immersed, and the liquid in the centrifuge tubes was carefully removed with a syringe after continuous immersion for 30 min. The dye H2DCFDA was added to the centrifuge tubes for a final concentration of 50 µM for 1 h. The dye was aspirated with a syringe. The samples were rinsed two or three times with MES-KCl buffer solution and soaked in MES-KCl buffer for 15 min. The samples were removed and placed on slides, prepared (not pressed) with a drop of MES-KCl buffer, and then viewed and photographed using the FITC channel of a confocal microscope (LSM880, ZEISS).

5. Conclusions

In this study, we sequenced the Ogura CMS and maintainer lines of Chinese cabbage at the early and late stages of pollen development. After quality control analysis, the sequencing data of miRNAs, transcriptomes, and degradomes were highly reliable and verified to be consistent with the sequencing results obtained by qRT-PCR. Through gene differential expression, target prediction, and functional enrichment analysis, we identified a regulatory relationship between miR9569 and the differentially expressed gene AHA6, and constructed a miR9569 overexpression vector, which was then transformed into Arabidopsis thaliana. Comparative analysis of wild-type and OE-miR9569 transgenic Arabidopsis thaliana was performed by phenotypic observation, Alexander’s staining, microscopic observation, and the ATP quantification were used to functionally validate the role of miR9569 in pollen development. The pollen of OE-miR9569 Arabidopsis thaliana at anthesis was significantly different from that of the wild type. Anther viability and ATP content were significantly lower than that of wild-type Arabidopsis thaliana. The ROS content was significantly higher than that of the wild type, as observed by confocal microscopy, and defective exine patterning were produced, as observed by scanning electron microscopy.
In summary, the mitochondrial gene orf138 mutation regulates the downregulation of miR9569, which targets the ATP related gene, AHA6, blocking downstream H+-ATPases. The lack of energy leads to the obstruction of the normal formation of the pollen wall and ultimately causes pollen abortion.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/plants14162604/s1, Supplementary Material SA. Figure S1. Small RNA sequence length statistics; Figure S2. miR9569 shear target gene Bra013168 (AHA6). Supplementary Material SB. Differentially expressed miRNAs targeting differentially expressed mRNAs during different developmental periods in pollen of Ogura CMS and maintained lines of Chinese cabbage. Supplementary Material SC. miR9569 matrices sequences and precursor sequences and other related information; qRT-PCR related primer information. Supplementary Material SD. Bioinformatics analysis of miR9569. Figure S1. miR9569 target gene structure analysis and target location prediction; Figure S2. Chromosomal localization of pre-miR9569 and its target genes; lanes 1, 2, 3, and 4: maintenance (Y231-330) and sterile (Tyms) lines were used as background genes, respectively; Figure S3. Secondary structure prediction of miR9569 precursor; Figure S4. Fragment of miR9569 precursor sequence obtained; Figure S5. miR9569 mature sequence base conservation analysis. Supplementary Material SE. The information of OE-miR9569 vector. Figure S1. OE-miR9569 recombinant plasmid verification gel electrophoresis map; Figure S2. A schematic diagram of the construction of the OE-miR9569 vector; Figure S3. OE-miR9569 colony PCR gel electrophoresis.

Author Contributions

Y.Y. and X.Z. conceived the study. F.W. and X.W. designed the experiments. S.Y. and H.S. performed the experiments and analyzed the data. W.Z., Y.Z. and B.T. participated in the qRT-PCR and phenotype identification. X.W. and S.X. drafted the manuscript. X.W. revised and finalized the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by Zhongyuan Sci-Tech Innovation Leading Talents (244200510041); Superior Discipline Cultivation of Sci-Tech Research and Development Project of Joint Fundation in Henan Province (242301420127); China Agricultural Research System (CARS-25-G15); Key science and technology program of Henan Academy of Agricultural Sciences (2024666033).

Data Availability Statement

The original contributions presented in the study are publicly available. The transcriptome data can be found here: National Center for Biotechnology Information (NCBI) BioProject database under accession number PRJNA657160. The miRNAs and degradome data can be found here: China National GeneBank DataBase (CNGBdb) under accession numbers CNP0006167 and CNP0006176.

Acknowledgments

We thank Yuanlin Zhang, School of Agricultural Sciences, Zhengzhou University, for the pre-experiment and data collection for the improvement of this manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Chen, L.; Liu, Y.-G. Male Sterility and Fertility Restoration in Crops. Annu. Rev. Plant Biol. 2014, 65, 579–606. [Google Scholar] [CrossRef]
  2. Vedel, F.; Pla, M.; Vitart, V.; Gutierres, S.; Chétrit, P.; Paepe, R.d. Molecular basis of nuclear and cytoplasmic male sterility in higher plants. Plant Physiol. Biochem. 1994, 32, 601–608. [Google Scholar]
  3. Wang, F.; Feng, C.; O’Connell, M.A.; Stewart, J.M.; Zhang, J. RFLP analysis of mitochondrial DNA in two cytoplasmic male sterility systems (CMS-D2 and CMS-D8) of cotton. Euphytica 2010, 172, 93–99. [Google Scholar] [CrossRef]
  4. Dong, X.; Kim, W.K.; Lim, Y.-P.; Kim, Y.-K.; Hur, Y. Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes. Plant Sci. 2013, 199–200, 7–17. [Google Scholar] [CrossRef]
  5. Su, T.; Wang, W.; Li, P.; Zhang, B.; Li, P.; Xin, X.; Sun, H.; Yu, Y.; Zhang, D.; Zhao, X. A genomic variation map provides insights into the genetic basis of spring Chinese cabbage (Brassica rapa ssp. pekinensis) selection. Mol. Plant 2018, 11, 1360–1376. [Google Scholar] [CrossRef]
  6. Pokluda, R. Nutritional quality of Chinese cabbage from integrated culture. Hortic. Sci. 2008, 35, 145–150. [Google Scholar] [CrossRef]
  7. Seong, G.-U.; Hwang, I.-W.; Chung, S.-K. Antioxidant capacities and polyphenolics of Chinese cabbage (Brassica rapa L. ssp. Pekinensis) leaves. Food Chem. 2016, 199, 612–618. [Google Scholar] [CrossRef] [PubMed]
  8. Watanabe, M.; Musumi, K.; Ayugase, J. Carotenoid pigment composition, polyphenol content, and antioxidant activities of extracts from orange-colored Chinese cabbage. LWT—Food Sci. Technol. 2011, 44, 1971–1975. [Google Scholar] [CrossRef]
  9. Ren, W.; Si, J.; Chen, L.; Fang, Z.; Zhuang, M.; Lv, H.; Wang, Y.; Ji, J.; Yu, H.; Zhang, Y. Mechanism and utilization of ogura cytoplasmic male sterility in cruciferae crops. Int. J. Mol. Sci. 2022, 23, 9099. [Google Scholar] [CrossRef]
  10. Lin, S.; Miao, Y.; Su, S.; Xu, J.; Jin, L.; Sun, D.; Peng, R.; Huang, L.; Cao, J. Comprehensive analysis of Ogura cytoplasmic male sterility-related genes in turnip (Brassica rapa ssp. rapifera) using RNA sequencing analysis and bioinformatics. PLoS ONE 2019, 14, e0218029. [Google Scholar] [CrossRef] [PubMed]
  11. Wei, X.; Zhang, X.; Yao, Q.; Yuan, Y.; Li, X.; Wei, F.; Zhao, Y.; Zhang, Q.; Wang, Z.; Jiang, W.; et al. The miRNAs and their regulatory networks responsible for pollen abortion in Ogura-CMS Chinese cabbage revealed by high-throughput sequencing of miRNAs, degradomes, and transcriptomes. Front. Plant Sci. 2015, 6, 894. [Google Scholar] [CrossRef]
  12. Chase, C.D. Cytoplasmic male sterility: A window to the world of plant mitochondrial-nuclear interactions. Trends Genet. 2007, 23, 81–90. [Google Scholar] [CrossRef]
  13. Takatsuka, A.; Kazama, T.; Toriyama, K. Cytoplasmic Male Sterility-Associated Mitochondrial Gene orf312 Derived from Rice (Oryza sativa L.) Cultivar Tadukan. Rice 2020, 14, 46. [Google Scholar] [CrossRef]
  14. Rogers, K.; Chen, X. Biogenesis, Turnover, and Mode of Action of Plant MicroRNAs. Plant Cell 2013, 25, 2383–2399. [Google Scholar] [CrossRef] [PubMed]
  15. Sun, G. MicroRNAs and their diverse functions in plants. Plant Mol. Biol. 2012, 80, 17–36. [Google Scholar] [CrossRef] [PubMed]
  16. Grant-Downton, R.; Le Trionnaire, G.; Schmid, R.; Rodriguez-Enriquez, J.; Hafidh, S.; Mehdi, S.; Twell, D.; Dickinson, H. MicroRNA and tasiRNA diversity in mature pollen of Arabidopsis thaliana. BMC Genom. 2009, 10, 643. [Google Scholar] [CrossRef] [PubMed]
  17. Jiang, J.; Lv, M.; Liang, Y.; Ma, Z.; Cao, J. Identification of novel and conserved miRNAs involved in pollen development in Brassica campestris ssp. chinensis by high-throughput sequencing and degradome analysis. BMC Genom. 2014, 15, 146. [Google Scholar] [CrossRef]
  18. Li, H.; Wang, Y.; Wu, M.; Li, L.; Jin, C.; Zhang, Q.; Chen, C.; Song, W.; Wang, C. Small RNA sequencing reveals differential miRNA expression in the early development of broccoli (Brassica oleracea var. italica) pollen. Front. Plant Sci. 2017, 8, 404. [Google Scholar] [CrossRef]
  19. Chambers, C.; Shuai, B. Profiling microRNA expression in Arabidopsis pollen using microRNA array and real-time PCR. BMC Plant Biol. 2009, 9, 87. [Google Scholar] [CrossRef]
  20. Quilichini, T.D.; Friedmann, M.C.; Samuels, A.L.; Douglas, C.J. ATP-binding cassette transporter G26 is required for male fertility and pollen exine formation in Arabidopsis. Plant Physiol. 2010, 154, 678–690. [Google Scholar] [CrossRef]
  21. Mizelle, M.; Sethi, R.; Ashton, M.; Jemen, W. Development of the pollen grain and tapetum of wheat (Triticum aestivum) in untreated plants and plants treated with chemical hybridizing agent RH0007. Sex. Plant Reprod. 1989, 2, 231–253. [Google Scholar] [CrossRef]
  22. Liu, J.; Lim, S.-L.; Zhong, J.Y.; Lim, B.L. Bioenergetics of pollen tube growth in Arabidopsis thaliana revealed by ratiometric genetically encoded biosensors. Nat. Commun. 2022, 13, 7822. [Google Scholar] [CrossRef]
  23. Harper, J.F.; Manney, L.; Sussman, M.R. The plasma membrane H+-ATPase gene family in Arabidopsis: Genomic sequence of AHA10 which is expressed primarily in developing seeds. Mol. Gen. Genet. MGG 1994, 244, 572–587. [Google Scholar] [CrossRef]
  24. Haruta, M.; Gray, W.M.; Sussman, M.R. Regulation of the plasma membrane proton pump (H+-ATPase) by phosphorylation. Curr. Opin. Plant Biol. 2015, 28, 68–75. [Google Scholar] [CrossRef]
  25. Morsomme, P.; Boutry, M. The plant plasma membrane H+-ATPase: Structure, function and regulation. BBA Biomembr. 2000, 1465, 1–16. [Google Scholar] [CrossRef]
  26. Palmgren, M. Plant Plasma Membrane H+-ATPases: Powerhouses for Nutrient Uptake. Annu. Rev. Plant Physiol. Plant Mol. Biol. 2001, 52, 817–845. [Google Scholar] [CrossRef]
  27. Duby, G.; Boutry, M. The plant plasma membrane proton pump ATPase: A highly regulated P-type ATPase with multiple physiological roles. Pflügers Arch. Eur. J. Physiol. 2009, 457, 645–655. [Google Scholar] [CrossRef] [PubMed]
  28. Selinski, J.; Scheibe, R. Pollen tube growth: Where does the energy come from? Plant Signal. Behav. 2014, 9, e977200. [Google Scholar] [CrossRef]
  29. Hoffmann, R.D.; Portes, M.T.; Olsen, L.I.; Damineli, D.S.C.; Hayashi, M.; Nunes, C.O.; Pedersen, J.T.; Lima, P.T.; Campos, C.; Feijó, J.A.; et al. Plasma membrane H+-ATPases sustain pollen tube growth and fertilization. Nat. Commun. 2020, 11, 2395. [Google Scholar] [CrossRef] [PubMed]
  30. Chen, W.; Jia, P.-F.; Yang, W.-C.; Li, H.-J. Plasma membrane H+-ATPases-mediated cytosolic proton gradient regulates pollen tube growth. J. Integr. Plant Biol. 2020, 62, 1817–1822. [Google Scholar] [CrossRef]
  31. Wei, X.; Zhang, Y.; Zhao, Y.; Chen, W.; Nath, U.K.; Yang, S.; Su, H.; Wang, Z.; Zhang, W.; Tian, B.; et al. Mitotic Pollen Abnormalities Linked to Ogura Cytoplasmic Male Sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis). J. Integr. Agric. 2024, 24, 1092–1107. [Google Scholar] [CrossRef]
  32. Yang, H.; Xue, Y.; Li, B.; Lin, Y.; Li, H.; Guo, Z.; Li, W.; Fu, Z.; Ding, D.; Tang, J. The chimeric gene atp6c confers cytoplasmic male sterility in maize by impairing the assembly of mitochondrial ATP synthase complex. Mol. Plant 2022, 15, 872–886. [Google Scholar] [CrossRef] [PubMed]
  33. Amanda, D.; Frey, F.P.; Neumann, U.; Przybyl, M.; Šimura, J.; Zhang, Y.; Chen, Z.; Gallavotti, A.; Fernie, A.R.; Ljung, K.; et al. Auxin boosts energy generation pathways to fuel pollen maturation in barley. Curr. Biol. 2022, 32, 1798–1811.e1798. [Google Scholar] [CrossRef] [PubMed]
  34. Chen, L.; Dong, X.; Yang, H.; Chai, Y.; Xia, Y.; Tian, L.; Qu, L.Q. Cytosolic disproportionating enzyme2 is essential for pollen germination and pollen tube elongation in rice. Plant Physiol. 2022, 191, 96–109. [Google Scholar] [CrossRef]
  35. Ji, J.-J.; Huang, W.; Li, D.-W.; Yin, Y.-X.; Chai, W.-G.; Gong, Z.-H. A CMS-related gene, ψatp6-2, causes increased ATP hydrolysis activity of the mitochondrial F1 Fo-ATP synthase and induces male sterility in pepper (Capsicum annuum L.). Plant Mol. Biol. Report. 2014, 32, 888–899. [Google Scholar] [CrossRef]
  36. Linke, B.; Börner, T. Mitochondrial effects on flower and pollen development. Mitochondrion 2005, 5, 389–402. [Google Scholar] [CrossRef]
  37. Bock, K.W.; Honys, D.; Ward, J.M.; Padmanaban, S.; Nawrocki, E.P.; Hirschi, K.D.; Twell, D.; Sze, H. Integrating Membrane Transport with Male Gametophyte Development and Function through Transcriptomics. Plant Physiol. 2006, 140, 1151–1168. [Google Scholar] [CrossRef]
  38. Ewing, B.; Green, P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998, 8, 175–185. [Google Scholar] [CrossRef]
  39. Langmead, B. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. J. Genome Biol. 2009, 25, 423–433. [Google Scholar] [CrossRef] [PubMed]
  40. Love, M.I.; Huber, W.; Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014, 15, 550. [Google Scholar] [CrossRef]
  41. Burge, S.W.; Daub, J.; Eberhardt, R.; Tate, J.; Barquist, L.; Nawrocki, E.P.; Eddy, S.R.; Gardner, P.P.; Bateman, A. Rfam 11.0: 10 years of RNA families. Nucleic Acids Res. 2012, 41, D226–D232. [Google Scholar] [CrossRef] [PubMed]
  42. Addo-Quaye, C.; Miller, W.; Axtell, M.J. CleaveLand: A pipeline for using degradome data to find cleaved small RNA targets. Bioinformatics 2009, 25, 130–131. [Google Scholar] [CrossRef] [PubMed]
  43. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef] [PubMed]
Plants 14 02604 g001
Figure 1. Sample principal component analysis and statistics of differentially expressed mRNAs. (A) Sample principal component analysis; (B) statistics of the number of differentially expressed genes in ML1 vs. CMS1 and ML2 vs. CMS2.
Figure 1. Sample principal component analysis and statistics of differentially expressed mRNAs. (A) Sample principal component analysis; (B) statistics of the number of differentially expressed genes in ML1 vs. CMS1 and ML2 vs. CMS2.
Plants 14 02604 g001
Plants 14 02604 g002
Figure 2. Statistics of the number of differential miRNAs at two periods of pollen development in the Ogura CMS line and maintainer line. (A) Number of differential miRNAs at the early stage of pollen development in maintainer and sterile lines; (B) number of differential miRNAs at the late stage of pollen development in maintainer and sterile lines.
Figure 2. Statistics of the number of differential miRNAs at two periods of pollen development in the Ogura CMS line and maintainer line. (A) Number of differential miRNAs at the early stage of pollen development in maintainer and sterile lines; (B) number of differential miRNAs at the late stage of pollen development in maintainer and sterile lines.
Plants 14 02604 g002
Plants 14 02604 g003
Figure 3. Intergroup comparison of downregulated differentially expressed genes of the Ogura CMS and maintainer lines. (A) Number of differentially expressed and commonly expressed genes between the two groups; (B) GO enrichment analysis of downregulated commonly expressed genes between the two groups; (C) heatmap of H+-ATPases differentially expressed genes in the Ogura CMS and maintainer lines of Chinese cabbage.
Figure 3. Intergroup comparison of downregulated differentially expressed genes of the Ogura CMS and maintainer lines. (A) Number of differentially expressed and commonly expressed genes between the two groups; (B) GO enrichment analysis of downregulated commonly expressed genes between the two groups; (C) heatmap of H+-ATPases differentially expressed genes in the Ogura CMS and maintainer lines of Chinese cabbage.
Plants 14 02604 g003
Plants 14 02604 g004
Figure 4. qRT-PCR validation of pollen fertility-related miRNAs and mRNAs. (A,B) qRT-PCR validation of bra-miR9569 and its target gene, BrAHA6. (C–E) qRT-PCR validation of known miRNA (miR159a) and novel miRNAs (novel_mir_448, novel_mir_51). (F–K) qRT-PCR verification of six differentially expressed genes related to ATP pathway.
Figure 4. qRT-PCR validation of pollen fertility-related miRNAs and mRNAs. (A,B) qRT-PCR validation of bra-miR9569 and its target gene, BrAHA6. (C–E) qRT-PCR validation of known miRNA (miR159a) and novel miRNAs (novel_mir_448, novel_mir_51). (F–K) qRT-PCR verification of six differentially expressed genes related to ATP pathway.
Plants 14 02604 g004
Plants 14 02604 g005
Figure 5. Identification results of OE-miR9569-positive plants. (A) Identification of eukaryotic resistant antibiotics in the T1 generation of OE-miR9569. (B) Fluorescence quantification of miR9569 expression in the T2 generation of OE-miR9569 plants. (C) Fluorescence quantification of AHA6 expression in the target gene in the T2 generation of OE-miR9569 plants. One-way ANOVA using multiple tests (* means p < 0.05; ** means p < 0.01; *** means p < 0.001; **** means p < 0.0001).
Figure 5. Identification results of OE-miR9569-positive plants. (A) Identification of eukaryotic resistant antibiotics in the T1 generation of OE-miR9569. (B) Fluorescence quantification of miR9569 expression in the T2 generation of OE-miR9569 plants. (C) Fluorescence quantification of AHA6 expression in the target gene in the T2 generation of OE-miR9569 plants. One-way ANOVA using multiple tests (* means p < 0.05; ** means p < 0.01; *** means p < 0.001; **** means p < 0.0001).
Plants 14 02604 g005
Plants 14 02604 g006
Figure 6. Observation of phenotypic characteristics of wild-type and OE-miR9569 mutant Arabidopsis thaliana. (A,D) Observation of wild-type and miR9569 mutant inflorescences. (B,E) Observation of anthers and stigmas at anthesis of wild-type and miR9569 mutant. (C,F) Observation of wild-type and miR9569 mutant siliques. Scale bar = 0.5 mm (A,D), 0.4 mm (B,E), 1 cm (C,F).
Figure 6. Observation of phenotypic characteristics of wild-type and OE-miR9569 mutant Arabidopsis thaliana. (A,D) Observation of wild-type and miR9569 mutant inflorescences. (B,E) Observation of anthers and stigmas at anthesis of wild-type and miR9569 mutant. (C,F) Observation of wild-type and miR9569 mutant siliques. Scale bar = 0.5 mm (A,D), 0.4 mm (B,E), 1 cm (C,F).
Plants 14 02604 g006
Plants 14 02604 g007
Figure 7. Identification of pollen viability and determination of ATP content in wild-type and OE-miR9569 mutants. (A,B) Alexander rectification staining of anthers, scale bar = 200 μm. (C,D) Alexander staining of pollen grains, scale bar = 100 μm. (E) Stainability statistics (%). (F) ATP content determination (µmol/g). One-way ANOVA (* means p < 0.05; *** means p < 0.001).
Figure 7. Identification of pollen viability and determination of ATP content in wild-type and OE-miR9569 mutants. (A,B) Alexander rectification staining of anthers, scale bar = 200 μm. (C,D) Alexander staining of pollen grains, scale bar = 100 μm. (E) Stainability statistics (%). (F) ATP content determination (µmol/g). One-way ANOVA (* means p < 0.05; *** means p < 0.001).
Plants 14 02604 g007
Plants 14 02604 g008
Figure 8. Scanning electron microscopy observation of the ultrastructure and identification of reactive oxygen species in wild-type and OE-miR9569 anthers. (A,D) Scanning electron microscopy observation of wild-type and OE-miR9569 anthers at the unopened stage. (B) Wild-type Arabidopsis anthers with normal elliptical full pollen grains. (E) Partial local magnification of the mature anthers of OE-miR9569 at the unopened stage, which appeared to be empty chambers. Scale bar = 200 μm (A,D), 50 μm (B,E). (C,F) Confocal microscopy observation of reactive oxygen species staining results; scale bar = 200 μm.
Figure 8. Scanning electron microscopy observation of the ultrastructure and identification of reactive oxygen species in wild-type and OE-miR9569 anthers. (A,D) Scanning electron microscopy observation of wild-type and OE-miR9569 anthers at the unopened stage. (B) Wild-type Arabidopsis anthers with normal elliptical full pollen grains. (E) Partial local magnification of the mature anthers of OE-miR9569 at the unopened stage, which appeared to be empty chambers. Scale bar = 200 μm (A,D), 50 μm (B,E). (C,F) Confocal microscopy observation of reactive oxygen species staining results; scale bar = 200 μm.
Plants 14 02604 g008
Plants 14 02604 g009
Figure 9. The miR9569 target, the BrAHA6 gene, regulates the pollen fertility pathway in Chinese cabbage.
Figure 9. The miR9569 target, the BrAHA6 gene, regulates the pollen fertility pathway in Chinese cabbage.
Plants 14 02604 g009
Table 1. Statistics of transcriptome sequencing data evaluation of anther samples from two lines.
Table 1. Statistics of transcriptome sequencing data evaluation of anther samples from two lines.
Sample IDSamplesClean ReadsGC (%)Q30 (%)Mapped Reads
S07ML1-168,768,53243.8595.9951,673,715 (75.14%)
S08ML1-285,298,78444.4796.0364,307,383 (75.39%)
S09ML1-378,197,28845.1996.0260,180,391 (76.96%)
ML1-average77,421,53544.5096.0158,720,496 (75.83%)
S10CMS1-173,943,44043.4695.8052,303,161 (70.73%)
S11CMS1-280,664,30645.5796.1658,978,727 (73.12%)
S12CMS1-374,097,36645.3895.9953,966,421 (72.83%)
CMS1-average76,235,03744.8095.9855,082,768 (72.23%)
S01ML2-183,770,77445.1995.6477,887,958 (89.76%)
S02ML2-277,257,46844.9096.0169,431,461 (89.87%)
S03ML2-375,009,46044.9395.7464,617,111 (86.15%)
ML2-average78,679,23445.0195.8070,645,510 (88.59%)
S04CMS2-177,760,42845.0795.9960,702,156 (78.06%)
S05CMS2-269,712,21444.6996.0652,983,713 (76.00%)
S06CMS2-380,281,01645.4295.9663,100,530 (78.60%)
CMS2-average75,917,88645.0696.0058,928,800 (77.55%)
Table 2. SNP (single nucleotide polymorphism) locus statistics of anther samples from two lines.
Table 2. SNP (single nucleotide polymorphism) locus statistics of anther samples from two lines.
Sample IDSampleSNP NumberGenic SNPsTransitionTransversionHeterozygosity
S07ML1-1216,700170,19757.24%42.76%29.14%
S08ML1-2237,398184,12457.04%42.96%29.68%
S09ML1-3208,136166,03657.31%42.69%29.24%
ML1-average220,745173,45257.20%42.80%29.35%
S10CMS1-1227,465179,71357.09%42.91%45.29%
S11CMS1-2230,515182,97357.25%42.75%44.86%
S12CMS1-3225,547179,23357.32%42.68%44.34%
CMS1-average227,842180,64057.22%42.78%44.83%
S01ML2-1176,283140,12257.69%42.31%28.57%
S02ML2-2172,702137,36357.68%42.32%28.27%
S03ML2-3173,190138,32257.66%42.34%28.53%
ML2-average174,058138,60257.68%42.32%28.46%
S04CMS2-1199,387157,59757.35%42.65%39.67%
S05CMS2-2188,110148,74057.42%42.58%38.77%
S06CMS2-3197,367156,19957.38%42.62%39.18%
CMS2-average194,955154,17957.38%42.62%39.21%
Table 3. Sample miRNA sequencing data assessment statistics.
Table 3. Sample miRNA sequencing data assessment statistics.
SamplesSample IDRaw_ReadsContaining
‘N’ Reads		Length < 18Length > 30Clean ReadsQ30 (%)
ML1S0715,893,2771443,955,8831,122,05110,815,19996.87
ML1S0820,103,763785,518,1881,542,87713,042,62096.11
ML1S0919,280,153703,545,5532,318,05813,416,47296.73
CMS1S1015,264,422932,935,708907,13111,421,49096.70
CMS1S1141,575,0416722,906,8121,512,06617,156,09696.59
CMS1S1215,261,1481161,457,7232,035,91111,767,39896.36
ML2S0113,467,807422,319,292911,33810,237,13597.48
ML2S0217,142,925414,425,026868,52911,849,32997.04
ML2S0313,400,65440850,5861,322,76011,227,26897.27
CMS2S0420,246,0381119,026,1321,174,82210,044,97396.86
CMS2S0520,265,5191117,341,5081,495,00511,428,89596.93
CMS2S0621,509,3851249,318,264960,14211,230,85596.88
Table 4. Sequence comparison results of the sample sequence data with the reference genome.
Table 4. Sequence comparison results of the sample sequence data with the reference genome.
TypeTag NumberPercent
Total6,944,263100%
Mapped4,618,21066.50%
Perfect map3,657,82079.20%
Imperfect map960,39020.80%
Unmapped2,326,05332.50%
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Xiong, S.; Wei, X.; Zhang, W.; Zhao, Y.; Yang, S.; Su, H.; Tian, B.; Wei, F.; Zhang, X.; Yuan, Y. bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Plants 2025, 14, 2604. https://doi.org/10.3390/plants14162604

AMA Style

Xiong S, Wei X, Zhang W, Zhao Y, Yang S, Su H, Tian B, Wei F, Zhang X, Yuan Y. bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Plants. 2025; 14(16):2604. https://doi.org/10.3390/plants14162604

Chicago/Turabian Style

Xiong, Siyu, Xiaochun Wei, Wenjing Zhang, Yanyan Zhao, Shuangjuan Yang, Henan Su, Baoming Tian, Fang Wei, Xiaowei Zhang, and Yuxiang Yuan. 2025. "bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)" Plants 14, no. 16: 2604. https://doi.org/10.3390/plants14162604

APA Style

Xiong, S., Wei, X., Zhang, W., Zhao, Y., Yang, S., Su, H., Tian, B., Wei, F., Zhang, X., & Yuan, Y. (2025). bra-miR9569 Targets the BrAHA6 Gene to Negatively Regulate H+-ATPases, Affecting Pollen Fertility in Chinese Cabbage (Brassica rapa L. ssp. pekinensis). Plants, 14(16), 2604. https://doi.org/10.3390/plants14162604

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop