Brems1 mutation induced tapetum deficiency leading to male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

doi:10.21203/rs.3.rs-4489236/v1

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Brems1 mutation induced tapetum deficiency leading to male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

https://doi.org/10.21203/rs.3.rs-4489236/v1

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Male sterile lines are ideal for hybrid seed production in Chinese cabbage. Herein, the complete male sterile mutants M5026 and M5073 were obtained through ethyl methanesulfonate (EMS) mutagenesis in the cabbage double haploid line ‘FT’. Observation of paraffin sections showed that M5026 lacked the tapetum and had excessive microsporocytes. Transmission electron microscopy (TEM) revealed abnormal exine formation in M5026 microspores. Genetic analysis revealed a single recessive nuclear gene caused the male sterility phenotype of M5026. Using Mutmap sequencing and Kompetitive allele-specific PCR (KASP) identification and gene cloning, BraA10g029920.3.5C, encoding EMS1 (Excess microsporocytes 1), a leucine-rich repeat receptor-like protein kinase (LRR-RLK), was identified as the candidate gene of M5026 and named Brems1. A nonsynonymous G-to-A mutation in an exon of the Brems1 gene in M5026 resulted in the substitution of glycine with arginine. Employing Mutmap and cloning approaches, a C-to-T SNP was identified within Brems1 of M5073, resulting in premature translation termination. Both BrEMS1 and Brems1 were subcellularly localized at the cell membrane. qRT-PCR analysis indicated Brems1 exhibited the highest expression level in flower buds, while no expression was detected in roots. Transcriptomic analysis revealed that mutation in Brems1 reduced the expression levels of genes associated with the tapetum, pollen tube, and LRR-RLK family. These results suggested that Brems1 plays a critical role in pollen development and contributes to elucidating the molecular mechanisms underlying tapetum development and male sterility in Chinese cabbage.

Chinese cabbage

male sterility

Brems1

transcriptome

The mutation in Brems1 resulting in male sterility in Chinese cabbage were validated through two allelic mutations.

Male sterility in plants refers to the phenomenon in which the pistil develops normally while the stamen degenerates and fails to produce viable pollen. This is an effective method for utilizing hybrid vigor and hybrid breeding. Based on the inheritance pattern of sterility genes, male sterility can be classified into cytoplasmic male sterility and nuclear male sterility (Chen and Liu 2014). Cytoplasmic male sterility was typically caused by an interaction between mitochondrial or chloroplast genes and nuclear genes, and fertility could be restored by nuclear-encoded restorer genes (Yamagishi and Bhat 2014). Nuclear male sterility was controlled via the nuclear genome, where the molecular regulatory mechanisms of plant male sterility involved genes primarily participating in microspore development, tapetum formation, exine deposition, intine formation, and pollen wall rupture (Wilson et al. 2011).

The tapetum, located on the innermost layer of the anther wall, directly adjacent to the microspore mother cells, played a crucial role in the nutrient supply and regulating microsporogenesis. After cellular differentiation, the tapetum transformed into polarized secretory cells, synthesizing and secreting callose enzymes via programmed cell death to release uninucleate microspores, while also promoting pollen wall formation and serving as a significant source of nutrition for microspore development (Wu et al. 2000). Plants with mutations that lacked the tapetum or experienced genetic ablation of the tapetum were unable to produce pollen grains (Mariani et al. 1990). The Arabidopsis thaliana male-sterile mutants EMS1 (Zhao et al. 2002) and TPD1 (Yang et al. 2003), which lacked the tapetum and resulted in male infertility. Premature or delayed degeneration tapetum could also lead to male sterility. Dysfunction of the tapetum in the Arabidopsis thaliana male-sterile mutant DYT1 resulted in abnormal pollen development (Zhang et al. 2006). Several mutants with abnormal tapetum development have been identified in Chinese cabbage, including BrMS1 (Dong et al. 2022), BrACOS5 (Zou et al. 2023), BrGGL7 (Zhao et al. 2022), and BrABCG26 (Xu et al. 2024).

The leucine-rich repeat receptor-like kinase (LRR-RLK) consisting of an intracellular domain with kinase activity, a transmembrane domain and extracellular domains responsible for ligand recognition (Shiu et al. 2004). Some of the LRR-RLK members family participated in plant stamen development. In Arabidopsis, the receptor-like kinase ERECTA mutant exhibited abnormal flower development in the form of absent anthers (Shpak et al. 2004); the triple mutant er-105, erl1-2, erl2-1, encoding an LRR-RLK, resulted in undifferentiated anthers, leading to male sterility (Hord et al. 2008); EMS1 gene encoded an LRR-RLK, and its mutant exhibited male sterility due to the absence of middle layer and tapetum cells (Zhao et al. 2002). In rice, a mutation in the LRR-RLK gene MSP1 resulted in absent of the middle and tapetum cell layers, leading to male sterility (Yang et al. 2016); In addition, a mutation in the ERECTA homologous gene, OsERL, resulted in significant defects in anther development (Liu et al. 2021b); and knocking down the expression level of the LRR-RLK family gene OsPEX1 using RNAi resulted in male sterility (Hang et al. 2021). In cotton, plants with RNAi of GhSERK1 exhibited reduced pollen viability (Shi et al. 2014), and editing the GhEMS1 gene using CRISPR/Cas9 produced, male-sterile mutants (Zhang et al. 2022). Currently, no reports detailing the involvement of members of the LRR-RLK family in the molecular mechanisms of male fertility in Chinese cabbage have been made.

Here, two male sterile mutants, M5026 and M5073, were obtained using ethyl methanesulfonate (EMS)-induced mutagenesis of ‘FT’. Cytological observations revealed that M5026 lacked tapetum, exhibited excessive microsporocytes production, and showed abnormal microspore exine morphology. Through MutMap, Kompetitive allele-specific PCR (KASP), and cloning sequencing, the predicted candidate gene was identified as BraA10g029920.3.5C (Brems1), encoding an LRR-RLK. Employing Mutmap and cloning approaches, a C-to-T SNP was identified within Brems1 of M5073, resulting in premature translation termination. BrEMS1 and Brems1 were subcellularly localized at the cell membrane. Brems1 exhibited high expression levels in buds. Transcriptomic analysis indicated that the mutation in Brems1 reduced the expression of genes associated with trichomes, pollen tubes and the LRR-RLK family. This study cloned the Brems1 gene in Chinese cabbage for the first time, contributing to elucidating the molecular mechanisms underlying tapetal development and male sterility in Chinese cabbage.

Plant materials

The DH line ‘FT’ of Chinese cabbage obtained via microspore culturing was used as the wild-type (Huang et al. 2016). The male-sterile mutants M5026 and M5073 were generated using EMS-induced mutagenesis of ‘FT’. Two parents were used to generate the F₁ and F₂ generations for genetic analysis. The F₂ generation was further used for gene mapping and KASP analysis.

Floral organ observation

Floral organ morphology (pistils, long stamens, short stamens, petals, sepals, and flower buds) of ‘FT’ and M5026 were observed at the reproductive growth stage using a stereomicroscope (Nikon SMZ800, Japan).

Pollen viability analysis

The pollen viabilities of ‘FT’ and M5026 were assessed using the 2,3,5-triphenyltetrazolium chloride (TTC) staining method. Anthers were collected from flower buds and placed on a glass slide. 50 µL of TTC (0.2%) staining solution was added to the slide, and a coverslip was placed on top. The slides were incubated at 37°C for 20–30 min. Pollen viability was observed using a fluorescence inverted microscope (OLYMPUS DP80, Japan).

Paraffin section observation

The buds at different developmental stages of ‘FT’ and M5026 were individually fixed in Formalin-Aceto-Alcohol (FAA) fixative for 12 hours, followed by dehydration using an ethanol solution gradient of 50%-100%. Subsequently, the samples were subjected to substitution with a mixed solution of ethanol and xylene (volume ratios = 3:1, 1:1, 1:3), and different mass ratios of xylene and paraffin were then impregnated into the samples. The samples were embedded in paraffin, and sections were cut using a microtome (Leica RM2016, Germany). After staining the anther sections with safranin and fast green, these sections were observed using an optical microscope (ECLIPSE80i, Nikon).

TEM observation

Buds of ‘FT’ and M5026 were fixed via immersion in a 1:1 mixture of glutaraldehyde and paraformaldehyde. The fixed samples were then stored in the dark at 4°C. The detailed steps for transmission electron microscopy (TEM) were conducted according to the previously described protocols (Ji et al. 2017).

Genetic analysis

Constructing F₁ and F₂ generations were constructed using parental strains M5026 and ‘FT’ for genetic analysis. The number of male fertile and sterile plants in the F₁ and F₂ generations was observed and statistically analyzed. The chi-square (χ²) test was employed to analyze the segregation ratio in the F₂ population.

Identification of the candidate gene

Candidate genes in M5026 and M5073 were identified using an improved Mutmap method. (Abe et al. 2012). A pool of male sterile mutants was constructed by collecting genomic DNA from 58 F₂ plants exhibiting male sterility. Re-sequencing was performed using NovaSeq 6000 platform (Illumina, San Diego, California, USA) following DNA extraction from ‘FT’ and male sterile mutant pool using a DNAsecure Plant Kit (Tiangen Biotech Ltd, Beijing, China). The sliding window method implemented in fastp (v0.20.0) was utilized for filtering raw data to obtain high-quality data (Liu et al. 2021a). Subsequently, these filtered high-quality data were aligned to the reference genome using bwa (0.7.12-r1039) (Li and Durbin 2010). Single-nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) were detected using the Genome Analysis Toolkit (GATK) (McKenna et al. 2010). ANNOVAR was employed for functional annotation (Wang et al. 2010). Circos was used to visualize the mapping of mutation information onto the genome (Krzywinski et al. 2009).

SNP genotype by KASP

A total of 70 F₂ male sterile individuals and two ‘FT’ plants were used as materials to conduct a KASP assay to detect SNP co-segregation and identify the Brems1 candidate gene. The thermal cycling conditions for KASP were set as described previously (Xi et al. 2018). The primers used in the KASP assay were listed in Table S1.

Cloning and sequencing of Brems1

The full-length DNA and coding sequence (CDS) of the Brems1 were amplified from ‘FT’, M5026 and M5073. The specific operational steps were conducted according to a previous study (Liu et al. 2021a). Sanger sequencing was performed by Sangon Biotech (Shanghai, China).

Bioinformatic characterization and phylogenetic analysis of the BrEMS1 protein

The full-length amino acid sequence of BrEMS1 was submitted to the SMART (https://smart. embl- heidelberg. de/) website for protein domain analysis. The three-dimensional protein structures of BrEMS1 and Brems1 were analyzed using SwissModel. A total of 1000 bootstrap replicates were established in MEGA6.0 software, and the neighbor-joining method was employed to construct the phylogenetic tree. DNAMAN v6.0 was used to perform multiple sequence alignment of the protein sequences.

Subcellular localization of BrEMS1 and Brems1

The CDS sequences of BrEMS1 and Brems1 were cloned from ‘FT’ and M5026, respectively, and inserted into the ProCAMV35S vector and transformed into Agrobacterium tumefaciens GV3101, and impregnated into tobacco leaf mesophyll cells at an optical density (OD) of 0.6–0.8. After 24 hours of dark treatment followed by 24 hours of light treatment, fluorescence signals were observed using a confocal laser scanning microscope (Leica Microsystems, Wetzlar, Germany). Green fluorescence protein (GFP) fluorescence signals were detected at a wavelength of 496 ~ 540 nm.

Quantitative Real-Time PCR (qRT-PCR)

The total RNA isolated from flowers, leaves, roots, buds, and stems of ‘FT’ and M5026 plants was subsequently reverse-transcribed into cDNA. qRT-PCR was performed using Ultra SYBR Green Mix (Kangwei Century, Beijing, China) on a QuantStudio 6 PCR system. Actin was selected as the internal control. The relative expression levels were calculated using the 2^−ΔΔCt method.

Transcriptome analysis

Buds from ‘FT’ and M5026 were individually sampled in triplicate, followed by RNA extraction. Subsequently, the RNA samples were subjected to rigorous quality control using an Agilent 2100 bioanalyzer, and libraries were constructed accordingly. The concentration of the libraries was first preliminarily quantified using a Qubit 2.0 Fluorometer, and then the insert size of the libraries was detected using an Agilent 2100 bioanalyzer. The effective concentrations of the libraries were accurately quantified via qRT-PCR to ensure the quality of the libraries. Once the libraries passed quality control, different libraries were pooled based on their effective concentrations and the desired amount of data for sequencing on the Illumina platform. These raw sequencing data were then filtered as follows: reads with adapters were removed; reads containing N (representing unknown bases) were removed; and low-quality reads (more than 50% of the bases with Qphred scores less than or equal to 5) were removed (Yan et al. 2013). These sequencing data were aligned to the reference genome Brara_Chiifu_V3.5 using HISAT2 software to obtain a rapid and precise mapping of the reads to the reference genome (Mortazavi et al. 2008). Based on the positional information of gene alignment on the reference genome, reads covering each gene within the start and end range were counted. Reads with alignment quality scores lower than 10, unpaired alignments, and reads aligned to multiple regions of the genome were filtered out. This analysis was conducted using the featureCounts tool within the subread software (Liao et al. 2014). After gene expression quantification was completed, a statistical analysis was conducted to identify genes that exhibited significant differences in expression levels across different conditions. The differential gene set was subjected to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using the clusterProfiler software.

The M5026 mutant displayed male sterility

The male sterility M5026 mutant was isolated from the EMS-induced mutant DH line ‘FT’. Pollen grains were absent on the anther surface of the M5026 mutant (Fig. 1a, b). Pollen viability analysis revealed that ‘FT’ produced viable pollen grains. In contrast, M5026 did not produce any pollen grains (Fig. 1c). Observations of the floral organs of M5026 and ‘FT’ plants revealed that the long stamens, short stamens, pistils, petals, and buds of M5026 mutant were all shorter than those of ‘FT’ (Fig. 1d-h), while the sepals did not different (Fig. 1i).

The F₁ and F₂ generations were constructed using the parental strains M5026 and ‘FT’ to conduct genetic analysis. All F₁ plants showed the male fertile phenotype. The F₂ population had 581 male fertile and 187 male sterile plants, a ratio of 3:1 (χ² = 0.17) (Table 1). A single recessive nuclear gene, Brems1, regulated the male sterility phenotype in M5026.

Table 1

Genetic analysis in *M5026.*
Generations	Total	Male fertility	Male sterile	Segregation ratio	χ²	χ²_0.05
P₁ (‘FT’)	50	50	0
P₂ (M5026)	50	0	50
F₁ (P₂× P₁)	20	20	0
F₁’(P₁× P₂)	20	20	0
F₂	768	581	187	3.10:1	0.17	3.841

Table 2

Candidate SNP information.
Chromosome	Position	WT	MUT	Snpindex	Region	MutationType	GeneID
A10	19,022,513	C	T	1	intronic	/	BraA10g029920.3.5C
A10	19,087,850	C	T	1	exonic	nonsynonymous SNV	BraA10g030050.3.5C
A10	19,417,177	C	T	1	exonic	synonymous SNV	BraA10g030900.3.5C

Tapetum deficiency, excessive microsporocytes, and abnormal exine in the Mutant M5026

To further elucidate the cytological characteristics of the sterile mutant, we employed paraffin sectioning to examine the anthers of ‘FT’ and M5026 at different developmental stages. Based on the established categorization of Arabidopsis anther developmental stages (Sanders et al. 1999), we selected eight anther developmental stages of anther development in both ‘FT’ and M5026 for analysis. During stage 4 (S4) of anther development, no differences were observed between ‘FT’ (Fig. 2a) and M5026 (Fig. 2e). At stage 5 (S5), ‘FT’ developed the tapetum characterized by rectangular-shaped cells forming a layer outside the microsporocytes (Fig. 2b), however, M5026 lacked the tapetum and exhibited excess microsporocytes (Fig. 2f). At stage 6 (S6), the microsporocytes of M5026 exhibited morphological abnormalities characterized by concavity (Fig. 2g). During stages 7–12 (S7-12), the microsporocytes of ‘FT’ sequentially formed tetrads, microspores, and pollen grains (Fig. 2d, i-l). In contrast, M5026 continued to lack the tapetum, and its microsporocytes underwent continuous degradation, ultimately failing to produce pollen grains (Fig. 2h, m-p).

TEM was utilized to observe further ‘FT’ and M5026. The microspore exine structure of ‘FT’ was intact, with well-defined bacula and tectum morphology (Fig. 3a, b), while the microspore of M5026 exhibited an abnormal exine structure (Fig. 3c, d).

Identification of candidate gene contributing to the M5026 male sterility phenotype.

The adapted MutMap approach was employed to isolate candidate genes. Sequencing data filtering was performed to obtain high-quality data. The wild-type and F₂ mutant pool yielded 175,935,758 (95.25%) and 177,453,976 (96.23%) high quality dates, respectively. Of these, 98.98% and 99.21% could be mapped to the Brara_Chiifu_V3.5 reference genome. SNPs were detected and filtered using the GATK software, resulting in a total of 732,795 SNPs. Subsequently, SNP-index calculation was conducted, followed by plotting the distribution of the SNP-index across the chromosome (Fig. 4a). In the case of selecting regions with SNP-index values exceeding the 95th percentile as candidate regions associated with the M5026 male sterility trait, the candidate gene, Brems1, was mapped to the region of chromosome A10 from 18,400,000 to 20,725,698. A total of 30 SNPs were found in the candidate interval, among which three have SNP-index values of 1 and were located within genes, namely SNP19,022,513, SNP19,087,850, and SNP19,417,177. SNP19,022,513 was located in an intron, SNP19,087,850 was a nonsynonymous mutation located in an exon, and SNP19,417,177 was a synonymous mutation located in an exon.

To validate the causal SNP for male sterility, KASP genotyping was performed on three SNPs using two ‘FT’ and seventy F₂ male sterile individuals. The results showed that only SNP 19,022,513 co-segregated with the male sterility phenotype, with all ‘FT’ plants being C:C and all mutant phenotype plants being T:T. Recombination was identified at SNP 19,087,850, with one C:C and one C:T genotype observed among the mutant phenotype plants. Similarly, at SNP 19,022,513, recombination was also detected, revealing one C:C and two C:T genotypes in the plants exhibiting the mutant phenotype (Fig. 4b). SNP 19,022,513 was located on BraA10g029920.3.5C. Its Arabidopsis ortholog is EMS1 (AT5G07280), which encodes EXCESS MICROSPOROCYTES1 (EMS1), an LRR-RLK that controls the fate of somatic and germ cells in Arabidopsis anthers.

Cloning and sequence analysis of Brems1

The DNA and cDNA sequences of BraA10g029920.3.5C were cloned from M5026 and ‘FT’, respectively. The results revealed that BraA10g029920.3.5C has a full length of 3699 bp, comprising of a single exon. A non-synonymous mutation was identified at position 3439 bp, where a G to A transition occurred, changing GGG to AGG, resulting in the substitution of glycine (Gly) with arginine (Arg) (Fig. 5a).

The gene sequence of BraA10g029920.3.5C cloned from our experimental materials exhibited differences compared with the reference genome Brara_Chiifu_V3.5. In the reference genome, the full length of BraA10g029920.3.5C was 4613 bp (Fig. 5b1), which was 911 bp longer than in ‘FT’ and M5026, with the additional length located at the gene’s 5’ end of the gene (Fig. 5b2). BraA10g029920.3.5C in the reference genome contained three exons and two introns (Fig. 5b1), while those in ‘FT’ and M5026 had only one exon and no introns (Fig. 5b2). The mutation site in M5026 was located in the second intron of BraA10g029920.3.5C in the reference genome and in the exon of ‘FT’ and M5026 (Fig. 5b). The EMS1 mutation in Arabidopsis thaliana resulted in male sterility (Zhao et al. 2002). Hence, we presumed BraA10g029920.3.5C was the candidate gene for Brems1.

Identification of the allelic material M5073

A male sterile mutant M5073 was induced by EMS mutagenesis in ‘FT’ (Fig. S1a). M5026 and M5073 were crossed with ‘FT’ to generate F₁ hybrids, respectively. Allelism tests were performed by crossing these two F₁ generations. The observed ratio of male fertility to sterility in the progeny was 3:1 (χ² = 1.405) (Table S2), indicating that the male sterility traits in M5026 and M5073 were controlled by a single allelic gene. Subsequently, Mutmap method was employed to isolate candidate genes of M5073. Regions with SNP index values exceeding the 95th percentile were selected as candidate intervals, a SNP with an SNP-index value of 1 was identified at position 19,024,244 bp on chromosome A10, located within the Brems1 gene (Fig. S1b). Cloning of the Brems1 gene from M5073 revealed a C-to-T mutation at position 1708 bp, converting CAG to TAG, resulting in the substitution of glutamine for a stop codon and premature translation termination (Fig. S1c). These results indicated that the mutation in Brems1 caused the male sterile phenotype observed in M5026 and M5073, confirming its role in stamen development.

Phylogenetic and structural analysis of BrEMS1

To further investigate the role of BrEMS1 in male fertility of Chinese cabbage, the structure of the BrEMS1 protein was analyzed. The BrEMS1 protein comprised 1233 amino acids, with a theoretical isoelectric point of 6.62 and a molecular weight of 133.2 kDa. It harbored a serine or threonine-specific kinase subfamily (S_TKc) along with 17 LRR structural domains (Fig. 6a). The mutation in M5026 occurred at the 1147th amino acid of the BrEMS1 protein, where Gly was substituted with Arg, located within the S_TKc domain. The SwissModel tool was employed to predict the protein structures of both BrEMS1 and Brems1, revealing that the mutation in BrEMS1 altered the three-dimensional structure of the protein (Fig. 6b). The evolutionary relationships among BrEMS1 homologous sequences across different species were elucidated through phylogenetic analysis, demonstrating its high homology among multiple crops in the Brassicaceae family (Fig. 6c). The S_TKc domain was highly conserved across multiple crops in the Brassicaceae family (Fig. 6d).

Expression pattern analysis of Brems1

To elucidate the expression pattern of Brems1, we conducted a subcellular localization and fluorescence quantitative analysis of Brems1. BrEMS1-GFP and Brems1-GFP were introduced into tobacco and their co-localization with the cell membrane marker (Pip2a-RFP) was analyzed. The results demonstrated that the GFP fluorescence of both BrEMS1-GFP and Brems1-GFP was localized on the cell membrane (Fig. 7a), indicating that the mutation of BrEMS1 did not affect on its subcellular localization.

The relative transcription levels of Brems1 in different organs of ‘FT’ and M5026 were assessed using qRT-PCR. qRT-PCR analysis revealed no significant differences in the relative abundances of Brems1 transcripts among the flowers, leaves, roots, buds, and stems of ‘FT’ and M5026 plants. Notably, Brems1 expression was highest in buds, followed by flowers, leaves, and stems, while expression was absent in roots (Fig. 7b).

Brems1 affects the transcription levels of genes associated with the tapetum and pollen tube

To elucidate the molecular mechanisms underlying Brems1 regulation of male sterility, transcriptomic analysis was conducted on the buds of ‘FT’ and M5026. ‘FT’ and M5026 obtained totals of 136,535,602 and 145,985,440 clean reads, respectively, with 88.88% and 87.99% mapped to the reference genome for gene expression analysis. A total of 3510 differentially expressed genes (DEGs) were identified (padi < = 0.05, log2FoldChange > = 1), with 3203 upregulated DEGs and 307 downregulated DEGs in M5026 compared with ‘FT’ (Fig. 8a).

DEGs associated with male sterility included genes involved in both the tapetum and pollen tube. Significantly downregulated tapetum-related genes were identified among the DEGs, including BCP1 (BraA09g039020.3.5C, BraA08g025680.3.5C and BraA07g012900.3.5C) and AMS (BraA03g043930.3.5C and BraA07g004250.3.5C) (Fig. 8a). Among the DEGs associated with pollen tube function, LRX8 (BraA01g035230.3.5C and BraA03g039100.3.5C), LRX9 (BraA08g003810.3.5C), LRX10 (BraA07g005090.3.5C and BraA03g059520.3.5C), and LRX11 (BraA01g004310.3.5C, BraA08g016780.3.5C and BraA03g043440.3.5C) exhibited significant downregulation (Fig. 8a). Brems1 belongs to the LRR-RLK family, and we identified DEGs within this family, including PRK1 (BraA08g009650.3.5C), PRK2A (BraA07g006840.3.5C), PRK3 (BraA06g022820.3.5C), PRK5 (BraA05g019360.3.5C and BraA08g002990.3.5C), and PRK8 (BraA07g030150.3.5C), which all exhibited significant downregulation (Fig. 8a). Mutations in Brems1 potentially affected the expression levels of genes associated with the tapetum, pollen tube and LRR-RLK family in Chinese cabbage.

The KEGG pathway enrichment analysis of these DEGs revealed significant enrichment in multiple metabolic pathways, including arginine and proline metabolism (brp00330), cyanoamino acid metabolism (brp00460), arachidonic acid metabolism (brp00590), nitrogen metabolism (brp00910), amino sugar and nucleotide sugar metabolism (brp00520), galactose metabolism (brp00052), starch and sucrose metabolism (brp00500), taurine and hypotaurine metabolism (brp00430), glycerolipid metabolism (brp00561), ascorbate and aldarate metabolism (brp00053), inositol phosphate metabolism (brp00562) and glycerophospholipid metabolism (brp00564), (Fig. 8b). Therefore, the pollen sterility in M5026 might have influenced its metabolism.

The utilization of male sterile lines has provided a crucial tool for breeding and producing hybrid crop varieties. Moreover, male sterile materials can also be employed to investigate genes involved in anther development. In this study, we identified two male sterile mutants, M5026 and M5073, in Chinese cabbage and cloned the mutant gene Brems1 (BraA10g029920.3.5C). Brems1 encoded an LRR-RLK. According to our knowledge, this study represents the first successful cloning of the EMS1 homologous gene in the genus Brassica, contributing to elucidating the molecular mechanisms underlying male sterility in Chinese cabbage.

The abnormal development of the tapetal was a primary cause of male sterility in plants (Wilson et al. 2011). The tapetal functioned as the innermost layer of the anther wall and directly contacted the microsporocytes to provide them with nutrients and generated enzymes that degraded sporopollenin to facilitate microspore release (Ma et al. 2015). Additionally, it regulated the formation of the pollen exine by secreting sporopollenin precursors (Zou et al. 2023). Persistent expansion of tapetum cells and abnormal aggregation of microsporocytes in the GMS line ‘AB01’ in Chinese cabbage led to male sterility (Zhou et al. 2017). The vacuolization anomaly of tapetum cells in the male-sterile mutant ftms in Chinese cabbage resulted in anther abortion (Tan et al. 2019). The programmed cell death of tapetum cells in the male-sterile mutant msm2 in Chinese cabbage was delayed, leading to an inability to produce pollen (Dong et al. 2022). The abnormal development of the tapetum in Chinese cabbage msm1 resulted in male sterility (Zou et al. 2023). The abnormal enlargement and vacuolization of tapetum cells in the Chinese cabbage mutant ftms1 resulted in infertility (Zhao et al. 2022). The abnormal degradation of the tapetum in the male-sterile mutant msm3 of Chinese cabbage led to complete stamen degeneration (Xu et al. 2024). In this study, the mutant M5026, lacking the tapetum, resulted in complete stamen sterility.

The exine is a multilayered structure precisely assembled from sporopollenin, exhibiting characteristics such as radiation protection, high-temperature resistance, and resistance to degradation (Hou et al. 2023), and abnormalities in the exine structure led to anther abortion. In the Arabidopsis thaliana male-sterile mutant ms1, exine formation was aberrant with limited sporopollenin deposition (Vizcay-Barrena and Wilson 2006). The rice male-sterile mutant osms1 displayed aberrant exine morphology, presenting a bilayered structure with an absence of bacula (Yang et al. 2019). Exine thinning in the rice mutant ms7-6007 results in male sterility (An et al. 2020). Severe exine deficiency was observed in the Chinese cabbage male sterile mutant msm1 (Zou et al. 2023), and the abnormal exine of the Chinese cabbage mutant msm2 resulted in anther abortion (Dong et al. 2022). The tapetum provided molecules for exine formation, known as exine proteins and callose, which constitute the proteinaceous coat of pollen. The abnormal microspore exine structure in the mutant M5026 investigated in this study led to male sterility, possibly due to the absenct tapetum.

Receptor protein kinases (RPKs) constituted a transmembrane protein family that played crucial roles in cellular signaling pathways in both animals and plants (Becraft and PW 1998). RPKs were typically composed of three structural domains: the extracellular domain, the transmembrane domain, and the cytosolic protein kinase domain (Walker 1994). Based on the presumed ligand-binding domain structures, RPKs in plants were classified into five families: wall-associated kinases (WAKs), the lectin-like RPK family, the CR4-like RPK family, the S-domain RPK family, and the LRR-RPK family (Mccarty and Chory 2000). LRR-RPK family genes were implicated in defense responses and diverse growth and developmental processes, including nodulation (Gresshoff et al. 2003), plant transpiration (Masle et al. 2005), organ shape and size regulation (Xu et al. 2008), cell fate determination and patterning in anther development (Jia et al. 2008), organ abscission (Kumpf et al. 2013), steroid hormone signaling (Santiago et al. 2013), nitrogen acquisition (Tabata et al. 2014), regulation of root and shoot meristem size (Shinohara et al. 2016), defense responses (Zorzatto et al. 2015), and pollen tube reception (Takeuchi and Higashiyama 2016). In this study, the causative gene Brems1 of the male-sterile mutant encoded an LRR-RLK belonging to the LRR-RLK family, resulting in defective anther development.

In conclusion, this study identified two male sterile mutants through EMS-induced mutagenesis. M5026 exhibited an absence of the tapetal layer, an abundance of micropylar cells, and aberrant microspore exine structure. The LRR-RLK gene, Brems1, homologous to the Arabidopsis male sterility gene EMS1, was responsible for male sterility in M5026 and M5073. A SNP G to A mutation was observed in the S_TKc domain of Brems1 in M5026. A C-to-T SNP was identified within Brems1 of M5073, resulting in premature translation termination. The present study provided insights into the molecular mechanisms of male sterility in Chinese cabbage.

Figure legends

Figure 1 Morphological characteristics of M5026 and ‘FT’. a M5026 and ‘FT’ at the flowering stage. Bar, 1 cm. b The flowers of M5026 and ‘FT’. Bar, 3 mm. c Pollen viability analysis of M5026 and ‘FT’. Bar, 50 µm. d - i The floral organs of M5026 and ‘FT’. d, pistils. e, long stamens. f, short stamens. g, petals. h, buds. i, sepals. Bars, 3mm.

Figure 2 Paraffin sections during anther development in ‘FT’ (a-d, i-l) and M5026 (e-h, m-p). Bar = 50 µm. E, epidermis; PT, precursors of tapetal cells; PPT, putative precursors of tapetal cells; T, tapetal layer; MS, microsporocytes; dMS, degraded microsporocytes; Tds, tetrads; Msp, microspore; PG, pollen grain; Se, septum.

Figure 3 TEM observation of microspores in ‘FT’ and M5026. a Microspore of ‘FT’. b Localized magnification of microspore in ‘FT’. c Microspore of M5026. d Localized magnification of microspore in M5026. Bar = 1 µm. Msp, microspore; MS, microsporocytes; Ex, exine; ba, bacula; te, tectum; In, Intine; ab-Ex, abnormal exine.

Figure 4 SNP-index distribution plot and KASP genotyping. a SNP-index distribution plot. The red line represented the mean SNP-index within the window, the pink line denotes the threshold line corresponding to the 99th percentile, and the orange line indicated the threshold line corresponding to the 95th percentile. b KASP genotyping of SNPs. C:C corresponds to red dots, C:T to green dots, and T:T to blue dots.

Figure 5 The cloning and gene structure of BraA10g029920.3.5C. a The alignment of cloned gene sequences surrounding the mutation site in ‘FT’ and M5026. The red box highlighted the mutation site. b The gene structure of BraA10g029920.3.5C in the reference genome Brara_Chiifu_V3.5, ‘FT’, and M5026. b1 The gene structure of BraA10g029920.3.5C in the reference genome Brara_Chiifu_V3.5. b2 The gene structure of BraA10g029920.3.5C in ‘FT’, and M5026. The red arrow highlighted the mutation site.

Figure 6 Protein analysis of BrEMS1. a Protein domain analysis of BrEMS1. The red arrow indicated the mutation site. b Analysis of the three-dimensional structure of the BrEMS1 protein. b1 The overall three-dimensional structure of the BrEMS1 protein. b2 The local three-dimensional structure of BrEMS1 protein around the mutation site. b3 The overall three-dimensional structure of the Brems1 protein. b4 The local three-dimensional structure of BrEMS1 protein around the mutation site. c The phylogenetic tree of BrEMS1. d Amino acid sequence alignment of the S_TKc domain.

Figure 7 Subcellular localization and qRT-PCR of ‘FT’ and M5026. a The subcellular localization of BrEMS1 and Brems1. b qRT-PCR analysis of Brems1 in different organs of ‘FT’ and M5026.

Figure 8 Transcriptome analysis of buds in ‘FT’ and M5026. a Transcriptional levels of DEGs associated with the tapetum, pollen tube and LRR-RLK family. b KEGG enrichment analysis of DEGs. The red asterisk indicated metabolic pathways.

Fig. S1 The allelic material M5037 of M5026. a M5037 and ‘FT’ at the flowering stage. Bar, 1 cm. b The flowers of M5037 and ‘FT’. Bar, 3 mm. c SNP-index distribution plot. The red line represented the mean SNP-index within the window, the pink line denotes the threshold line corresponding to the 99th percentile, and the orange line indicated the threshold line corresponding to the 95th percentile. d The alignment of cloned gene sequences surrounding the mutation site in ‘FT’ and M5037. The red box highlighted the mutation site.

Conflict of interest

All the authors declare that they have no competing interests.

Authors’ contributions

CL and LW analyzed the data. CL drafted the manuscript. CL, LW and DZ participated in the creation of materials and performed the experiments. CT and ZL directed the whole study including designing experiments and revising the manuscript.

Acknowledgements

This work was supported by the Earmarked Fund for CARS-23-A02, the National Natural Science Foundation of China (32272736 and 31972406) and the Key Research and Development Program of Liaoning (2021JH2/10200003).

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and its additional fles. The Illumina RNA-Seq datasets and Mutmap data are available in the the Sequence Read Archives (SRA) of the National Center for Biotechnology Information NCBI under BioProject ID: PRJNA1105001 andPRJNA1105243. Genomic sequences and gene annotation information of B.rapa are downloaded online at http://brassicadb.cn.

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Fig.S1.tif
Fig. S1 The allelic material M5037 of M5026. a M5037 and ‘FT’ at the flowering stage. Bar, 1 cm. b The flowers of M5037 and ‘FT’. Bar, 3 mm. c SNP-index distribution plot. The red line represented the mean SNP-index within the window, the pink line denotes the threshold line corresponding to the 99th percentile, and the orange line indicated the threshold line corresponding to the 95th percentile. d The alignment of cloned gene sequences surrounding the mutation site in ‘FT’ and M5037. The red box highlighted the mutation site.
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Brems1 mutation induced tapetum deficiency leading to male sterility in Chinese cabbage (Brassica rapa L. ssp. pekinensis)

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Abstract

Figures

Key Message

Introduction

Materials and methods

Plant materials

Floral organ observation

Pollen viability analysis

Paraffin section observation

TEM observation

Genetic analysis

Identification of the candidate gene

SNP genotype by KASP

Cloning and sequencing of Brems1

Bioinformatic characterization and phylogenetic analysis of the BrEMS1 protein

Subcellular localization of BrEMS1 and Brems1

Quantitative Real-Time PCR (qRT-PCR)

Transcriptome analysis

Results

The M5026 mutant displayed male sterility

Tapetum deficiency, excessive microsporocytes, and abnormal exine in the Mutant M5026

Cloning and sequence analysis of Brems1

Phylogenetic and structural analysis of BrEMS1

Expression pattern analysis of Brems1

Brems1 affects the transcription levels of genes associated with the tapetum and pollen tube

Discussion

Declarations

Conflict of interest

Authors’ contributions

Acknowledgements

Availability of data and materials

References

Supplementary Files

Status:

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