Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris)

doi:10.21203/rs.3.rs-3552806/v1

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Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris)

https://doi.org/10.21203/rs.3.rs-3552806/v1

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Pollen coat proteins (PCPs) are cysteine-rich small-molecule proteins, which exhibit high levels of polymorphism and are expressed in gametocytes. Previous investigations have revealed that PCP genes are involved in pollen wall synthesis, pollen-stigma recognition, pollen development and germination. However, gene expression and function of PCP family in pollen development is not well understood in Wucai (Brassica campestris L.). In this study, genome-wide identification and expression analysis of the BcPCPgene family members were conducted, including their physical and chemical properties, chromosome localization, phylogenetic tree, gene structure, and tertiary structure. A total of 20 BcPCP genes were identified and classified into three subfamilies with high homology to Arabidopsis thaliana. Furthermore, PCP members associated with male sterility were screened based on several transcriptome and proteome datasets, and BraA02g002400.3C was identified as a candidate gene. Gene sequence analysis revealed that this gene was homologous to GRP20 gene, and was named BcGRP20. Functional analysis of this gene showed that overexpression of BcGRP20 gene in the Arabidopsis grp20 mutant could restore anther fertility.

pollen coat proteins

male sterility

gene family

bioinformatics analysis

BcGRP20

This paper sheds light on the expression patterns of pollen coat protein (PCP) genes. BraA02g002400.3C (BcGRP20) is involved in pollen development in Wucai.

In angiosperms, male sterility (MS) is a common phenomenon that occurs due to the abnormally developed pollen or male gametes (Wang et al. 2014). Pollen development is a complex biological process, during which any abnormality can result in male sterility (Walbot and Egger 2016; Gómez et al. 2015; Tang et al. 2022). Recently, many male sterile related genes were identified, which shows high expression in the tapetum layer cells (Zhang and Yang 2014).

During pollen development, male gametophyte cells are enveloped by a unique cell wall consisting of two primary layers, with the outer wall layer being comprised of a complex biopolymer known as sporopollen (Zhou et al. 2012). The outer wall of mature pollen (pollen outer wall) is an extracellular matrix from the anther tapetum layer (Quilichini et al. 2015). After tapetum layer cell rupture, sporopollenin is deposited on the outermost surface of the pollen grain, filling the spaces and cavities of the outer wall (Yao et al. 2022). Pollen coat proteins (PCPs), the main components of the pollen outer wall and most of them are small cystine-rich peptides, play a crucial role in pollen-stigma recognition, adhesion and hydration, and pollen development and germination (Li et al. 2012; Lu et al. 2020). It was reported that an abnormal pollen coat protein can result in male sterility (Qiao et al. 2023).

According to the structure and functional characteristics, PCPs are classified into two major groups, PCP-A and PCP-B (Takayama et al. 2000; Liu et al. 2021a). PCP-A proteins are small, approximately 10 kDa in size, and basic cysteine-rich. Despite the considerable variation in their intermediate sequences, PCP-A shows high conservation of eight cysteine residues in the mature forms (Rejón et al. 2016). PCP-Bs are closely related to the embryonic developmental regulator ESF1 (Costa et al. 2014). Moreover, PCP-B genes are synergistically co-expressed with LUREs, which code the defensin-like cysteine-rich proteins (CRPs) that allow Arabidopsis pollen tubes to successfully germinate and enter the embryo sac (Takeuchi and Higashiyama 2012; Liu et al. 2021b).

Currently, most of the reported PCPs are concentrated on self- or insect-pollinated species, particularly Brassicaceae (Murphy 2006) and Arabidopsis (Mayfield et al. 2001; Wang et al. 2017). In Brassica napus, the first identified PCP protein, PCP7, also known as PCP-A1, is a 7-kDa small peptide from the pollen outer wall, encoded by a single transcribed gene, and most highly expressed during the tetrad period of pollen development (Hiscock et al. 1995). In B. oleracea, the S-locus cysteine-rich protein (SCR) located in the pollen coat is involved in the pollen self-incompatibility reaction, which leads to sterility if the plant fails to self-fertilize (Shiba et al. 2001). Recently, approximately 40 PCP gene members have been identified in the B. oleracea genome (Wang et al. 2023). In Arabidopsis, four PCP-B genes, namely AtPCP-Bα (AT5G61605), AtPCP-Bβ (AT2G29790), AtPCP-Bγ (AT2G16535), and AtPCP-Bδ (AT2G16505), have been identified (Wang et al. 2017). These genes are expressed during late pollen development. Loss of multiple PCP-Bs severely impairs pollen hydration, resulting in reduced pollen attachment and delayed pollen tube growth (Wang et al. 2017). Furthermore, the Arabidopsis grp17-1 mutant, which lacks oleosin protein, exhibits delayed pollen hydration (Mayfield and Preuss 2000). While cer3-8 mutant, lacks pollen outer wall lipids due to a defective epidermal wax biosynthesis, exhibits male sterility (Xu et al. 2020). These mutant lines with restored pollen coat lipids regained fertility.

Wucai (Brassica campestris L. syn. B. rapa L. ssp. chinensis var. rosularis Tsen), a non-heading Chinese cabbage variant, is an essential vegetable specie in the Brassicaceae family in China (Chen et al. 2019b). Due to its fresh taste and rich nutrient, Wucai is widely cultivated, particularly in the Jianghuai basin of China (Chen et al. 2018). So far, the function of the PCPs in Wucai is poorly understood. In this study, we aim to identify the BcPCP gene family using bioinformatics tools, and screen for those associated with fertility based on transcriptome and proteome data. Subsequently, the functions of key genes were explored through quantitative real-time PCR (qRT-PCR) and genetic transformation. These findings will serve as a foundation for a better understanding of the molecular mechanisms underlying the regulation of male sterility in Wucai, and provide insights into the roles of key PCP genes.

Plant materials and growth conditions

Wucai cytoplasmic male sterile (CMS) line 14A and its maintainer 14B were utilized as the plant materials in this experiment (Fig. 1). During the full-blooming period, samples of roots, stems, leaves, buds, and flowers were gathered from both fertile and sterile lines. Buds at different stages of development were categorized into four stages: microsporocyte (< 1 mm), tetrad (1–2 mm), microspore (2–4 mm), and mature pollen (> 4 mm), according to our previous report (Zhang et al. 2019; Tang et al. 2022). All samples were stored at -80℃. Arabidopsis mutant grp20 (At5g07560, SALK_124861C) and its wild-type Col-1 were sown in 1/2 MS culture medium, while tobacco (Nicotiana benthamiana L.) seeds and one-week-old Arabidopsis seedlings were cultured in a substrate and maintained in an incubator with a light cycle of 16 h/25°C for light and 8 h/18°C for dark.

Morphological and microscopy observation

Inflorescence and flower images were captured utilizing a digital camera (EOS550D, Canon, Japan). The morphology of the anther and filament was visualized using a stereomicroscope (SZX10, Olympus, Japan). For analysis of bud development at various stages, paraffin sections were prepared according to our previous reports (Zhang et al. 2019; Tang et al. 2022).

Genome-wide identification of PCP family in Brassica species

The genomes and their corresponding annotation data of B. rapa (Syn. B. campestris; V3.0; AA), B. juncea (V1.0, BB), and B. oleracea (V1.0, CC) were downloaded from the Brassica database (BRAD; http://brassicadb.cn). Arabidopsis thaliana PCP gene family members were obtained from the Arabidopsis Information Resource (TAIR; https://www.arabidopsis.org/). Homologous genes in B. rapa, B. juncea, and B. oleracea were retrieved using the BLASTP strategy, respectively, with parameters setting e-value < le-10 and coverage ratio ≥ 60%. The candidate genes were cross-validated using various databases based on previous reports (Chen et al. 2020b; Wang et al. 2023). The confirmed sequences of BcPCPs, BjPCPs, and BoPCPs were used to construct a phylogenetic tree along with the AtPCP sequences using the MEGA (V7.0) and Evolview (http://www.evolgenius.info/evolview/#/login) software.

Bioinformatics analysis of BcPCPs

TBtools (Chen et al. 2020a) was used to determine the chromosomal location of BcPCP genes and to analyze collinearity between BcPCPs and AtPCPs. The Expasy ProtParam tool (https://web.expasy.org/protparam/) was employed to calculate the theoretical isoelectric point (pI), amino acid length, and molecular weight (MW) of BcPCP proteins. Gene structures information, cis-acting elements of BcPCP promoters, conserved domains and motifs, and protein tertiary structures of BcPCPs were obtained from the Gene Structure Display Server (GSDS; http://gsds.gao-lab.org/), PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/), Conserved Domain Database (NCBI-CDD; www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi), Multiple Em for Motif Elicitation tool (MEME; http://meme-suite.org/tools/meme), and SWISSMODEL (www.swissmodel.expasy.org/) software, respectively. The Protein-Protein Interaction (PPI) network was analyzed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database (https://string-db.org). The KaKs_Calculator (V2.0) software was utilized to calculate the non-synonymous (Ka) and synonymous (Ks) rates between duplicated BcPCPs.

Expression analysis of BcPCP gene family based on transcriptome and proteome

For the anther abortion occurs from the tetrad stage, the transcriptome and proteome analyses of the tetrad stage buds from 14A (CMS-Wucai) and 14B lines were carried out by the GENE DENOVO (Guangzhou, China). Their raw data have been deposited in the GSA (CRA011131) and iprox (IPX0006795000) database, respectively. RNA-seq data from different tissues (Tong et al. 2013) and various male sterile lines, including three GMS lines (GMS-AB01, GMS-AB03, and GMS-Wucai) (Zhou et al. 2017; Liu et al. 2016; Wang et al. 2021), two male sterile mutants (ftms and msm) (Tan et al. 2018; Huang et al. 2020), and four CMS lines (CMS-pol, CMS-ogu1, CMS-ogu2, and CMS-turnip) (Chen et al. 2018; Lin et al. 2019; Wei et al. 2020), were obtained from the NCBI database. Additionally, the proteome of Wucai GMS was analyzed (Tang et al. 2022). The raw datasets were reanalyzed according to our previous report (Chen et al. 2020b). The transcript levels and protein abundance were represented by Reads Per Kilobase of exon model per Million mapped reads (FPKM) values and count values, respectively. The differentially expressed genes (DEGs) and proteins (DEPs) indicated expression differences between the male sterile and fertile lines.

Quantitative real-time PCR analysis (qRT-PCR)

Total RNA was extracted from different samples utilizing the FastPure Plant Total RNA Isolation Kit (Ambion, USA), and cDNA was synthesized using the PrimeScript™ RT Reagent Kit (TaKaRa, Japan). qRT-PCR was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme, Japan). The BcActin gene was employed as the internal reference gene (Tang et al. 2022; Chen et al. 2018) The specific primers used in this study are listed in Table S1. Three biological replicates for each sample and three technical repeats for each gene were conducted. The relative expression level of the gene was calculated using the 2^−∆∆CT method (Livak and Schmittgen 2001).

BcGRP20 gene function analysis

The cDNA sequence of BraA02g002400.3C (BcGRP20, another name BcOlnB1) was obtained via homologous cloning from Wucai. The BcGRP20 gene was then inserted into the pCAMBIA2300-GFP vector for subcellular localization in tobacco leaves, and the resulting images were processed using laser scanning confocal microscopy (LSCM; Leica TCS SP5). The recombinant plant expression vector, p1305-BcGRP20, was constructed and transformed into Arabidopsis mutant grp20 via inflorescence infiltration using Agrobacterium strain GV3101. Transgenic Arabidopsis seeds were collected and grown in MS medium (pH = 5.8) supplemented with 50 mg/L hygromycin. Positive seedlings were selected and identified by PCR with the primers (LB + BP, LB + PR), and the T₃ generation was used for morphological and gene expression analyses.

Morphological and cytological analyses of Wucai male sterile line

At the vegetative growth stage, no discernable differences were observed between the male sterile and fertile lines. During the reproductive growth period, the inflorescence, flowers, petals, and stigmas of the sterile and fertile lines appeared morphologically similar. Nonetheless, the sterile line exhibited degenerated stamens, evidenced by shrunken anthers, absence of pollen production, and shorter filaments in comparison to the fertile line (Fig. 1). Furthermore, cytological examination revealed that anther abortion occurred at the tetrad stage, in which the tapetum developed abnormally (Fig. 1).

Identification and phylogenetic analysis of BcPCPs

A total of 20 PCP genes were identified in the B. rapa genome (Table S2), with protein length ranging from 108 to 2,047 AA, pI ranging from 2.75 to 11.56, and protein MW ranging from 7.9 to 203.3 kDa. The subcellular localization predictions revealed that BcPCP members target different cellular compartments, such as cytoplasm, cell wall, chloroplast, cell membrane, and nucleus (Table S2). Gene mapping analysis displayed that BcPCP members were unevenly distributed on 8 out of 10 chromosomes, with four tandem duplications (Fig. S1).

Furthermore, we identified 18 and 16 PCP members in the genomes of B. juncea (BB) and B. oleracea (CC), respectively. Phylogenetic analysis of BcPCPs, BjPCPs, BoPCPs, and AtPCPs was performed, which clustered these members into three groups, designated as Groups A, B, and C (Fig. 2). The three groups included GDSL esterase/lipase (Group A), cysteine-rich repeat secretory protein (Group B), and tapetal oleosin (Group C), respectively. All these protein types have been reported to be involved in plant reproductive growth (Dong et al. 2016; Takeuchi 2021; Lévesque-Lemay et al. 2016), thus it is postulated that BcPCPs might also play a role in regulating the fertility of Wucai.

Gene conservative motif and structure analyses of BcPCPs

We conducted a conservative motifs analysis of BcPCP family members and identified ten distinct motifs. Among the 20 BcPCP members, ten contained motif 6 at the N-terminus and motif 3 at the C-terminus, six possessed motif 10 and motif 5 at the N-terminus, and two had motif 9 at the N-terminus and motif 8 at the C-terminus. However, BraA07g014240.3C exhibited no conserved motif (Fig. 3A). These three types were consistent with their phylogenetic tree results and tertiary structures (Fig. S2), indicating that these homologous pairs of proteins may have similar functions and roles.

Conserved domain analysis suggested that more than half of the family members contained stress-antifungal structural domains (Fig. 3B). In addition, BraA02g023130.3C and BraA02g002300.3C only contained the SGNH plant lipase-like structural domain, while BraA02g023140.3C only contained the SGNH hydrolase superfamily structural domain (Fig. 3B). Furthermore, we performed a gene structure assay and found that BcPCP gene sequences contained 0 to 6 introns (Fig. 3C). These results demonstrated that the more similar the structures were, the closer the related members were, as supported by the phylogenetic results of BcPCP members (Fig. 3).

Synteny and Ka/Ks analyses of BcPCPs

To investigate the evolutionary of the BcPCP gene family, we conducted a collinearity analysis between B. rapa and A. thaliana. Our analysis revealed eight collinear pairs of PCP genes (Fig. S3), indicating the conservation of these genes during evolution. Interestingly, each AtPCP gene corresponded to one to three BcPCP homologous genes, suggesting the potential functional redundancy of these genes. To understand the selective pressure and functional differentiation of BcPCP genes during evolution, we calculated the Ka/Ks value of duplicated gene pairs among BcPCPs. These results showed that all Ka/Ks values of BcPCP duplicated gene pairs were less than 1 (Table S3), indicating that the BcPCP gene family had been subjected to purifying selection during evolution.

Gene expression analysis of BcPCPs in different tissues

To explore the potential functions of BcPCP genes, we analyzed their expression patterns in different tissues using RNA-Seq data (Tong et al. 2013). The results revealed that most BcPCP genes exhibited low expression levels in various tissues, and some genes had significantly different expression levels among root, stem, leaf, flower, and silique (Fig. S4). Specifically, BraA02g023130.3C and BraA03g040060.3C exhibited variable expression levels across different tissues, while BraA03g040040.3C was specifically expressed in flower, and BraA05g025240.3C, BraA05g025270.3C, BraA08g028990.3C, and BraA10g029710.3C were specifically expressed in silique. To validate these results, we performed qRT-PCR using Wucai roots, stems, leaves, buds, and flowers. Our qRT-PCR results showed that most of BcPCPs had higher expression levels in the buds and flowers and lower levels in the roots, stems, and leaves (Fig. 4). These results suggested that the BcPCP gene family may be involved in reproductive development, and specific genes within the family may have distinct functions in different tissues.

Gene analysis of BcPCPs in male sterile lines

To gain insights into the role of BcPCPs in male gametophyte development, we reanalyzed several transcriptome datasets of B. rapa male sterility. The results revealed that BcPCPs were significantly downregulated in various male sterile lines (Fig. 5A). Moreover, the transcriptome analyses of Wucai GMS and CMS lines also showed a downregulation of BcPCPs in male sterile lines (Fig. 5B,C). Additionally, we identified five BcPCP genes in the ten male sterile materials analyzed (Fig. 5D), indicating their potential association with male sterility. To further investigate the expression of BcPCP genes at the protein level, we analyzed two proteomic datasets of Wucai, and 11 BcPCP proteins were found to have low abundance in both GMS and CMS materials (Fig. 5B,C,S5). Integration of transcriptome and proteome analyses led to the identification of three BcPCP genes (BraA02g002400.3C, BraA02g023130.3C, and BraA04g002300.3C) that exhibited differential expression at both transcriptional and protein levels (Fig. 5E), indicating their crucial role in male gametophyte development.

Expression analysis of BcPCPs during pollen development

To investigate the expression patterns of BcPCP genes during pollen development, qRT-PCR was performed using different developmental stages of the fertile line (Fig. 6). The results showed that BraA02g002380.3C, BraA03g003060.3C, and BraA08g028990.3C had the highest expression levels in the microsporocyte period, while BraA02g002400.3C, BraA03g014690.3C, BraA10g029710.3C, and BraA10g029720.3C were the most highly expressed in the microspore period. The remaining genes were most highly expressed in the mature pollen period, and their expression increased with flower maturity. These results suggested that BcPCP gene family might play an important role in pollen development.

Furthermore, we examined the expression levels of three genes (BraA02g002400.3C, BraA02g023130.3C, and BraA04g002300.3C) in the male sterile line, and found that their highest expression level occurred in the microspore period (Fig. 7A). However, compared with the fertile line, all their expression levels exhibited significant decrease during the pollen development, which was consistent with their transcript levels and protein abundance obtained from transcriptome and proteome analyses (Fig. 7B). Notably, the expression level of BraA02g002400.3C decreased to its lowest level from the tetrad period, which is the microspore abortion period, suggesting that the abnormal expression of this gene might lead to male sterility in Wucai.

Functional analysis of BcGRP20 (BraA02g002400.3C) gene

The 450-bp ORF sequences of BraA02g002400.3C was homologously cloned from Wucai (Fig. S6A), and found to be highly similar to the GRP20 protein sequences of A. thaliana and other Brassica crops (Fig. S6B, S7). This gene was named BcGRP20 and identified as a member of the PCP-B class proteins. The subcellular localization of BcGRP20 was determined using the 35S::GRP20-GFP vector (Fig. 8A), which was found to be concentrated on the cell membrane in transiently expressed tobacco levels (Fig. 8B). Therefore, BcGRP20 was identified as a membrane protein.

To investigate the function of BcGRP20 in pollen development, a PPI network was analyzed, which showed that key genes for male sterility (Table S4), MS1 and AGL84, may interact with BcGRP20 (Fig. 9A). MS1 and AGL84 were downregulated in male sterile line based on transcriptome analysis (Fig. 9A), and qRT-PCR was used to confirm their expression levels (Fig. 9B). Furthermore, genetic complementation was performed on the grp20 mutant (Fig. S8), which showed semi-sterile pollen compared to wild type (Col-0) plant (Fig. 10A,B). However, transgenic lines expressing BcGRP20 exhibited fertility similar to Col-0 line, and all pollen gains were restored to fertility (Fig. 10C). Gene expression analysis of transgenic lines showed that BcGRP20 and AtAPO3 were over-expressed, and AtMS1 and AtAGL84 expression levels were restored (Fig. 10D). These results suggested that BcGRP20 plays a critical role in pollen development.

Plant anther development can be roughly divided into four stages, including microspore mother cell formation, tetrad formation (meiosis), microspore (early, middle and late), and mature pollen stages (Quilichini et al. 2014). Pollen dysplasia or anther-blocked dehiscence during these stages can cause anther and/or pollen abortion, leading to male sterility. Previous cytological studies on several different GMS and CMS materials have demonstrated that male sterility is directly related to abnormally developed tapetum cells (Huang et al. 2020; Zhou et al. 2017; Liu et al. 2016; Chen et al. 2019a; Tan et al. 2018). During the normal development of anthers, the tapetal cells degrade at the tetrad and microspore stages and are involved in the formation of the outer pollen wall and tepal (Quilichini et al. 2014). In this experiment, we conducted a cytological examination on Wucai ogu CMS line 14A and its maintainer 14B, revealing abnormalities in tapetum layer development that caused anther abortion (Fig. 1H). The tapetal cells showed vacuolation, resulting in abnormal pollen outer wall formation and failure to release microspores from tetrads. These findings were consistent with our previous reports (Zhang et al. 2019; Wang et al. 2021; Tang et al. 2022).

In male sterility research, numerous genes associated with tapetum PCD, pollen coat formation, and anther dehiscence have been discovered (Jiang 2020; Shi et al. 2021). The pollen outer wall is comprised of small molecule proteins, including CRPs, SCR/SP11, and PCPs, with the latter being highly polymorphic, gametophyte-specific, and cysteine-rich (Bashir et al. 2013). It has been demonstrated that the pollen outer wall proteins contain male determinants that can either be recognized or not by stigma papillae cells, thus affecting the adhesion process between pollen and stigma (Doughty et al. 2000). They are involved in signal transduction, resistance to adversity, and pollen germination in plants (Seki et al. 2002). Additionally, the pollen coat proteins play a crucial role in protecting pollen from external interference, pollen-stigma specific recognition and other important processes (Abhinandan et al. 2022; Wang et al. 2017).

In this study, we identified 20 BcPCP members (Table S2) based on 11 AtPCP genes from B. rapa genome, which underwent triploid duplication (Wang et al. 2011). The number of BcPCP genes was less than the theoretical number 30, suggesting that some BcPCP genes have been lost during evolution. Conserved structural domains and motifs of the BcPCP gene family were classified into three types, which was consistent with the three groups of their phylogenetic tree (Fig. 2,3). Furthermore, the protein tertiary structure prediction results suggested that the BcPCP gene family can be basically divided into three categories (Fig. S2), which was consistent with their evolutionary tree classification (Fig. 2), indicating that the same type of protein of this gene family may have similar functions. Analysis of the conserved structural motifs of the BcPCPs shows that most proteins contain multiple conserved motifs, except for BraA07g014240.3C, which has no conserved motif (Fig. 3), indicating it may be potentially important and deserves further study.

Phylogenetic analysis revealed that BcPCP gene family contains three groups, GDSL esterase/lipase, cysteine-rich repeat secretory protein, and tapetum layer oleosin protein (Fig. 2). Previous study has reported that GDSL esterases in Zea mays are involved in anther cuticle formation, synthesis of the pollen outer wall, and ultimately maize fertility (An et al. 2019). Cysteine-rich proteins (CRPs) are involved in the regulation of plant growth, development, and reproduction (Doughty et al. 2000). In self-incompatible species, the S-locus rich cysteine protein (SCR/SP11) acts as a male determinant cluster and interacts with the S receptor kinase (SRK) to trigger pollen rejection through targeted degradation of the basal affinity factor EXO70A1 (Takasaki et al. 2000; Doughty et al. 2000; Samuel et al. 2009). Huang et al (Huang et al. 2013) confirmed that Arabidopsis oleosin can enhance pollen viability. All these three proteins are related to plant reproductive growth, so it was speculated that BcPCPs also affect the fertility of Wucai.

The qRT-PCR results of BcPCP genes in Wucai showed that all members were abundantly expressed in buds and flowers, and most of them have highest expression in buds and traceless expression in roots and leaves (Fig. 4), suggesting that these BcPCP members may be involved in the regulation of fertility in Wucai. To further investigate the relationship between the BcPCP gene family and male sterility, ten transcriptome datasets and two proteome datasets of several male sterility materials were comparatively analyzed. It revealed that three BcPCP genes were downregulated at both transcriptional and protein levels in male sterility (Fig. 5). Research shows that the pollen coat protein, oleosin-domain protein GRP17, is required for the rapid initiation of hydration on the stigma (Mayfield and Preuss 2000), and is in a chromosome 5 cluster with GRP20 (a sixth oleosin) (Mayfield et al. 2001). BraA04g002300.3C homologous gene in Arabidopsis, EXL4, plays an important role in the efficient hydration of pollen (Updegraff et al. 2009). The expression analysis of these three homologous genes in sterile and fertile lines uncovered their similar expression trends during anther development, among which BraA02g002400.3C and BraA04g002300.3C had the highest levels in the microspore stage (Fig. 6,7A). However, their expression levels in the sterile line were significantly lower than those in the fertile line at the microspore and mature pollen stages (Fig. 7B). These results demonstrated that these three genes were involved in the formation of the pollen wall and their abnormal expression might be a cause of male sterility.

During the anther development, the BcGRP20 (BraA02g002400.3C) gene expression had the lowest level from the tetrad period (Fig. 7B), which is the anther abortion period (Fig. 1H). We cloned this gene and found that it was located on the cell membrane (Fig. 8). Its protein sequences had a repetitive motif of three alanine and proline residues (AAAP) in C-terminal (Fig. S6), which is present in all T-oleosin expressed in pollen (Cao et al. 2015). It was reported that the grp20 mutant exhibited defects in the organization of tapetosome, and reduced levels of oleagin and flavonoids in the outer pollen wall compared to the wild type (Huang et al. 2013). We transformed the BcGRP20 gene into grp20 mutant and the transgenic plants exhibited complete restoration of pollen fertility (Fig. 10A-C). Furthermore, expression levels of the co-expression genes, AtMS1, and AtAGL84, were restored, and AtAPO3 was even upregulated in transgenic lines comparing to wild type (Fig. 10D). MS1 is considered to be a critical controlling factor for the anther and pollen development, and it is specifically expressed in the tapetum during the tetrad and microspore stages and is required for pollen wall formation (Zhou et al. 2017; Lu et al. 2020). AGL belongs to the type I gene in the MADS-box gene family, and it has been reported that transcript of AGL84 is preferentially expressed in pollen grains (Pina et al. 2005). These results suggested that BcGRP20 plays an important role in anther development and its abnormal function can lead to pollen abortion.

In this study, we identified 20 BcPCP genes in B. rapa genome, which could be categorized into three subgroups: GDSL esterase/lipase, cysteine-rich repeat secretory protein, and tapetal oleosin. The qRT-PCR analysis revealed that the expression levels of BcPCP members varied significantly in different tissues, with BraA02g002400.3C (BcGRP20) exhibiting the highest expression in the floral tissue of fertile Wucai plants and downregulated in sterile materials. Gene function analysis showed that BcGRP20 is localized on the cell membrane, and ectopic expression of BcGRP20 gene can restore the pollen fertility in Arabidopsis grp20 mutant.

Author Contribution

G. C. and X. T. designed the research. X. P., Y. W., and X. L. carried out the experiments. G. C., Y. W., and X. L. wrote the manuscript. W. W., and A. K. polished the language and helped to draft the manuscript. D. Y., Y. W., L. Z., C. W., L. Y., and J. H. helped to draft the manuscript. All authors have read and approved the final manuscript.

Acknowledgments:
This work was supported by the Key Projects of Natural Science Research Funds of Anhui Colleges (2022AH050913); Anhui Provincial Natural Science Foundation (2308085MC96); Hefei Natural Science Foundation (202343); Anhui Provincial Program on Major Basic Research Project (2023z04020005); National Natural Science Foundation of China (31801853); Visiting Scholar Project of China Scholarship Council (202008775006).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement:
Some data that support the findings were derived from the following resources available in the public domain: GEO (GSE43245, GSE125485, GSE77427, GSE95242, GSE125485), SRA (PRJNA471024, PRJNA291315, PRJNA505114, and SUB8933024), and iProX (PXD029175). Some data will be available in GSA (CRA011131) and iprox (IPX0006795000) following an embargo from the date of publication to allow for commercialization of research findings.
Conflicts of Interest: The authors declare no conflict of interest.

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Genome-wide identification, transcript profiling and functional analyses of PCP gene family in Wucai (Brassica campestris)

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Abstract

Figures

Key Message

Introduction

Materials and methods

Plant materials and growth conditions

Morphological and microscopy observation

Genome-wide identification of PCP family in Brassica species

Bioinformatics analysis of BcPCPs

Expression analysis of BcPCP gene family based on transcriptome and proteome

Quantitative real-time PCR analysis (qRT-PCR)

BcGRP20 gene function analysis

Results

Morphological and cytological analyses of Wucai male sterile line

Identification and phylogenetic analysis of BcPCPs

Gene conservative motif and structure analyses of BcPCPs

Synteny and Ka/Ks analyses of BcPCPs

Gene expression analysis of BcPCPs in different tissues

Gene analysis of BcPCPs in male sterile lines

Expression analysis of BcPCPs during pollen development

Functional analysis of BcGRP20 (BraA02g002400.3C) gene

Discussion

Conclusions

Declarations

References

Supplementary Files

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