Maize actin depolymerizing factor 1 (ZmADF1) negatively regulates pollen development.

The normal development of pollen grains and the completion of double fertilization in embryos are crucial for both the sexual reproduction of angiosperms and grain production. Actin depolymerizing factor (ADF) regulates growth, development, and responses to biotic and abiotic stress by binding to actin in plants. In this study, the function of the ZmADF1 gene was validated through bioinformatic analysis, subcellular localization, overexpression in maize and Arabidopsis, and knockout via CRISPR/Cas9. The amino acid sequence of ZmADF1 exhibited high conservation and a similar tertiary structure to that of ADF homologs. Subcellular localization analysis revealed that ZmADF1 is localized mainly to the nucleus and cytoplasm. The ZmADF1 gene was specifically expressed in maize pollen, and overexpression of the ZmADF1 gene decreased the number of pollen grains in the anthers of transgenic Arabidopsis plants. The germination rate of pollen and the empty seed shell rate in the fruit pods of the overexpressing plants were significantly greater than those in the wild-type (WT) plants. In maize, the pollen viability of the knockout lines was significantly greater than that of both the WT and the overexpressing lines. Our results confirmed that the ZmADF1 gene was specifically expressed in pollen and negatively regulated pollen quantity, vigor, germination rate, and seed setting rate. This study provides insights into ADF gene function and possible pathways for improving high-yield maize breeding.

Maize sterility gene DRP1 encodes a desiccation-related protein that is critical for Ubisch bodies and pollen exine development.

Desiccation tolerance is a remarkable feature of pollen, seeds, and resurrection-type plants. Exposure to desiccation stress can cause sporophytic defects, resulting in male sterility. Here, we report the novel maize sterility gene DRP1 (Desiccation-Related Protein 1), which was identified by bulked-segregant analysis sequencing and encodes a desiccation-related protein. Loss of function of DRP1 results in abnormal Ubisch bodies, defective tectum of the pollen exine, and complete male sterility. Our results suggest that DRP1 may facilitate anther dehydration to maintain appropriate water status. DRP1 is a secretory protein that is specifically expressed in the tapetum and microspore from the tetrad to the uninucleate microspore stage. Differentially expressed genes in drp1 are enriched in Gene Ontology terms for pollen exine formation, polysaccharide catabolic process, extracellular region, and response to heat. In addition, DRP1 is a target of selection that appears to have played an important role in the spread of maize from tropical/subtropical to temperate regions. Taken together, our results suggest that DRP1 encodes a desiccation-related protein whose loss of function causes male sterility. Our findings provide a potential genetic resource that may be used to design crops for heterosis utilization.

Editing melon eIF4E associates with virus resistance and male sterility.

The cap-binding protein eIF4E, through its interaction with eIF4G, constitutes the core of the eIF4F complex, which plays a key role in the circularization of mRNAs and their subsequent cap-dependent translation. In addition to its fundamental role in mRNA translation initiation, other functions have been described or suggested for eIF4E, including acting as a proviral factor and participating in sexual development. We used CRISPR/Cas9 genome editing to generate melon eif4e knockout mutant lines. Editing worked efficiently in melon, as we obtained transformed plants with a single-nucleotide deletion in homozygosis in the first eIF4E exon already in a T0 generation. Edited and non-transgenic plants of a segregating F2 generation were inoculated with Moroccan watermelon mosaic virus (MWMV); homozygous mutant plants showed virus resistance, while heterozygous and non-mutant plants were infected, in agreement with our previous results with plants silenced in eIF4E. Interestingly, all homozygous edited plants of the T0 and F2 generations showed a male sterility phenotype, while crossing with wild-type plants restored fertility, displaying a perfect correlation between the segregation of the male sterility phenotype and the segregation of the eif4e mutation. Morphological comparative analysis of melon male flowers along consecutive developmental stages showed postmeiotic abnormal development for both microsporocytes and tapetum, with clear differences in the timing of tapetum degradation in the mutant versus wild-type. An RNA-Seq analysis identified critical genes in pollen development that were down-regulated in flowers of eif4e/eif4e plants, and suggested that eIF4E-specific mRNA translation initiation is a limiting factor for male gametes formation in melon.

Mutation of glucose-methanol-choline oxidoreductase leads to thermosensitive genic male sterility in rice and Arabidopsis.

Thermosensitive genic male sterility (TGMS) lines serve as the major genetic resource for two-line hybrid breeding in rice. However, their unstable sterility under occasional low temperatures in summer highly limits their application. In this study, we identified a novel rice TGMS line, ostms18, of cultivar ZH11 (Oryza sativa ssp. japonica). ostms18 sterility is more stable in summer than the TGMS line carrying the widely used locus tms5 in the ZH11 genetic background, suggesting its potential application for rice breeding. The ostms18 TGMS trait is caused by the point mutation from Gly to Ser in a glucose-methanol-choline (GMC) oxidoreductase; knockout of the oxidoreductase was previously reported to cause complete male sterility. Cellular analysis revealed the pollen wall of ostms18 to be defective, leading to aborted pollen under high temperature. Further analysis showed that the tapetal transcription factor OsMS188 directly regulates OsTMS18 for pollen wall formation. Under low temperature, the flawed pollen wall in ostms18 is sufficient to protect its microspore, allowing for development of functional pollen and restoring fertility. We identified the orthologous gene in Arabidopsis. Although mutants for the gene were fertile under normal conditions (24°C), fertility was significantly reduced under high temperature (28°C), exhibiting a TGMS trait. A cellular mechanism integrated with genetic mutations and different plant species for fertility restoration of TGMS lines is proposed.

Gametophyte genome activation occurs at pollen mitosis I in maize.

Flowering plants alternate between multicellular haploid (gametophyte) and diploid (sporophyte) generations. Pollen actively transcribes its haploid genome, providing phenotypic diversity even among pollen grains from a single plant. In this study, we used allele-specific RNA sequencing of single pollen precursors to follow the shift to haploid expression in maize pollen. We observed widespread biallelic expression for 11 days after meiosis, indicating that transcripts synthesized by the diploid sporophyte persist long into the haploid phase. Subsequently, there was a rapid and global conversion to monoallelic expression at pollen mitosis I, driven by active new transcription from the haploid genome. Genes showed evidence of increased purifying selection if they were expressed after (but not before) pollen mitosis I. This work establishes the timing during which haploid selection may act in pollen.

Comprehensive analysis of LIM gene family in wheat reveals the involvement of TaLIM2 in pollen development.

LIM domain proteins were involved in organizing the cytoskeleton, adjusting the metabolism and gene expression, some of them were specific express in pollen. LIM gene family in plants were studied in sunflower, tobacco, foxtail millet, rape, rice and Arabidopsis thaliana, however, it has not been investigated in wheat to date. In the present study, we totally characterized 29 TaLIM genes through genome-wide analysis, which were divided into two categories and five subclasses according to phylogenetic analysis. RNA-Seq analysis indicated the expression patterns of TaLIM genes have specific temporal and spatial characteristics, especially TaLIM2 was highly expressed in fertility anthers. Phenotypic and cytological of BSMV: TaLIM2 showed that it had defects in the later stage of pollen development and germination, which further testified that TaLIM2 was closely related to fertility conversion. These findings will be useful for functional analysis of LIM genes in wheat fertility and contribute to hybrid wheat breeding.

Patterns of gene expression in pollen of cotton (Gossypium hirsutum) indicate downregulation as a feature of thermotolerance.

Reproductive performance in plants is impaired as maximum temperatures consistently approach 40°C. However, the timing of heatwaves critically affects their impact. We studied the molecular responses during pollen maturation in cotton to investigate the vulnerability to high temperature. Tetrads (TEs), uninucleate and binucleate microspores, and mature pollen were subjected to SWATH-MS and RNA-seq analyses after exposure to 38/28°C (day/night) for 5 days. The results indicated that molecular signatures were downregulated progressively in response to heat during pollen development. This was even more evident in leaves, where three-quarters of differentially changed proteins decreased in abundance during heat. Functional analysis showed that translation of genes increased in TEs after exposure to heat; however, the reverse pattern was observed in mature pollen and leaves. For example, proteins involved in transport were highly abundant in TEs whereas in later stages of pollen formation and leaves, heat suppressed synthesis of proteins involved in cell-to-cell communication. Moreover, a large number of heat shock proteins were identified in heat-affected TEs, but these proteins were less abundant in mature pollen and leaves. We speculate that the sensitivity of TE cells to heat is related to high rates of translation targeted to pathways that might not be essential for thermotolerance. Molecular signatures during stages of pollen development after heatwaves could provide markers for future genetic improvement.

The miRNome function transitions from regulating developmental genes to transposable elements during pollen maturation.

Animal and plant microRNAs (miRNAs) are essential for the spatio-temporal regulation of development. Together with this role, plant miRNAs have been proposed to target transposable elements (TEs) and stimulate the production of epigenetically active small interfering RNAs. This activity is evident in the plant male gamete containing structure, the male gametophyte or pollen grain. How the dual role of plant miRNAs, regulating both genes and TEs, is integrated during pollen development and which mRNAs are regulated by miRNAs in this cell type at a genome-wide scale are unknown. Here, we provide a detailed analysis of miRNA dynamics and activity during pollen development in Arabidopsis thaliana using small RNA and degradome parallel analysis of RNA end high-throughput sequencing. Furthermore, we uncover miRNAs loaded into the two main active Argonaute (AGO) proteins in the uninuclear and mature pollen grain, AGO1 and AGO5. Our results indicate that the developmental progression from microspore to mature pollen grain is characterized by a transition from miRNAs targeting developmental genes to miRNAs regulating TE activity.

How Cysteine Protease Gene PtCP5 Affects Seed Germination by Mobilizing Storage Proteins in Populus trichocarpa.

In higher plants, seed storage proteins are deposited in protein storage vacuoles (PSVs) and degraded by protease, especially cysteine proteases, as a source of nitrogen for seed germination. In this study, a cathepsin B-like cysteine protease PtCP5, which is important for seed germination and pollen development, was first cloned in Populus trichocarpa. The GUS staining of the ProPtCP5-GUS reporter line showed that PtCP5 is expressed in the roots, stems, leaves, flowers, siliques and seeds of Arabidopsis. We reveal that PtCP5 is present in plasma membrane and co-localizes with the plasma membrane marker REM1.3. Both seed germination and early seedling development are slower in OX-PtCP5 transgenic Arabidopsis when compared with the wild-type. Further analysis revealed that, when stained with toluidine blue, the observed storage protein accumulation was lower in OX-PtCP5 than in the wild-type. Our results also show that the number of abnormal pollen grains is higher and the germination rate of pollen is lower in OX-PtCP5 than in the wild-type. These results indicate that PtCP5 is an important factor in mobilizing storage proteins and that the proper expression of PtCP5 is necessary for both pollen and seed maturation and germination. This study sheds further light on the biological functions of cysteine proteases and provides further reference for seed development research on woody plants.

Dynamic changes in primexine during the tetrad stage of pollen development.

Formation of pollen wall exine is preceded by the development of several transient layers of extracellular materials deposited on the surface of developing pollen grains. One such layer is primexine (PE), a thin, ephemeral structure that is present only for a short period of time and is difficult to visualize and study. Recent genetic studies suggested that PE is a key factor in the formation of exine, making it critical to understand its composition and the dynamics of its formation. In this study, we used high-pressure frozen/freeze-substituted samples of developing Arabidopsis (Arabidopsis thaliana) pollen for a detailed transmission electron microscopy analysis of the PE ultrastructure throughout the tetrad stage of pollen development. We also analyzed anthers from wild-type Arabidopsis and three mutants defective in PE formation by immunofluorescence, carefully tracing several carbohydrate epitopes in PE and nearby anther tissues during the tetrad and the early free-microspore stages. Our analyses revealed likely sites where these carbohydrates are produced and showed that the distribution of these carbohydrates in PE changes significantly during the tetrad stage. We also identified tools for staging tetrads and demonstrate that components of PE undergo changes resembling phase separation. Our results indicate that PE behaves like a much more dynamic structure than has been previously appreciated and clearly show that Arabidopsis PE creates a scaffolding pattern for formation of reticulate exine.

Transcriptome and Metabolome Analyses Provide Insights into the Stomium Degeneration Mechanism in Lily.

Lily (Lilium spp.) is a widely cultivated horticultural crop that has high ornamental and commercial value but also the serious problem of pollen pollution. However, mechanisms of anther dehiscence in lily remain largely unknown. In this study, the morphological characteristics of the stomium zone (SZ) from different developmental stages of 'Siberia' lily anthers were investigated. In addition, transcriptomic and metabolomic data were analyzed to identify the differentially expressed genes (DEGs) and secondary metabolites involved in stomium degeneration. According to morphological observations, SZ lysis occurred when flower buds were 6-8 cm in length and was completed in 9 cm. Transcriptomic analysis identified the genes involved in SZ degeneration, including those associated with hormone signal transduction, cell structure, reactive oxygen species (ROS), and transcription factors. A weighted co-expression network showed strong correlations between transcription factors. In addition, TUNEL (TdT-mediated dUTP nick-end labeling) assays showed that programmed cell death was important during anther SZ degeneration. Jasmonates might also have key roles in anther dehiscence by affecting the expression of the genes involved in pectin lysis, water transport, and cysteine protease. Collectively, the results of this study improve our understanding of anther dehiscence in lily and provide a data platform from which the molecular mechanisms of SZ degeneration can be revealed.

Arabidopsis antiporter CHX23 and auxin transporter PIN8 coordinately regulate pollen growth.

Both the antiporter CHX23 (Cation/Proton Exchangers 23) and auxin transporter PIN8 (PIN-FORMED 8) are localized in the ER and regulate pollen growth in Arabidopsis. But how these two proteins regulate pollen growth remains to be studied. Here, we report that CHX23 and PIN8 act coordinately in regulating pollen growth. The chx23 mutant was reduced in pollen growth and normally shaped pollen grains, and complementation with CHX23 restored both pollen growth and normal pollen morphology. NAA treatments showed that CHX23 was crucial for pollen auxin homeostasis. The pin8 chx23 double mutant was decreased in pollen growth and normal pollen grains, indicating the joint effort of CHX23 and PIN8 in pollen growth. In vivo germination assay showed that CHX23 and PIN8 were involved in the early stage of pollen growth. CHX23 and PIN8 also function collaboratively in maintaining pollen auxin homeostasis. PIN8 depends on CHX23 in regulating pollen morphology and response to NAA treatments. CHX23 co-localized with PIN8, but there was no physical interaction. KCl and NaCl treatments showed that pollen growth of chx23 was reduced less than Col-0; pin8 chx23 was reduced less than chx23 and pin8. Together, CHX23 may regulate PIN8 function and hence pollen growth through controlling K+ and Na+ homeostasis mediated by its transport activity.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S-locus containing just three genes. We report that the S-locus gene, BAHD, is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

Sheet-like clay nanoparticles deliver RNA into developing pollen to efficiently silence a target gene.

Topical application of double-stranded RNA (dsRNA) can induce RNA interference (RNAi) and modify traits in plants without genetic modification. However, delivering dsRNA into plant cells remains challenging. Using developing tomato (Solanum lycopersicum) pollen as a model plant cell system, we demonstrate that layered double hydroxide (LDH) nanoparticles up to 50 nm in diameter are readily internalized, particularly by early bicellular pollen, in both energy-dependent and energy-independent manners and without physical or chemical aids. More importantly, these LDH nanoparticles efficiently deliver dsRNA into tomato pollen within 2-4 h of incubation, resulting in an 89% decrease in transgene reporter mRNA levels in early bicellular pollen 3-d post-treatment, compared with a 37% decrease induced by the same dose of naked dsRNA. The target gene silencing is dependent on the LDH particle size, the dsRNA dose, the LDH-dsRNA complexing ratio, and the treatment time. Our findings indicate that LDH nanoparticles are an effective nonviral vector for the effective delivery of dsRNA and other biomolecules into plant cells.

Loss of THIN EXINE2 disrupts multiple processes in the mechanism of pollen exine formation.

Exine, the sporopollenin-based outer layer of the pollen wall, forms through an unusual mechanism involving interactions between two anther cell types: developing pollen and tapetum. How sporopollenin precursors and other components required for exine formation are delivered from tapetum to pollen and assemble on the pollen surface is still largely unclear. Here, we characterized an Arabidopsis (Arabidopsis thaliana) mutant, thin exine2 (tex2), which develops pollen with abnormally thin exine. The TEX2 gene (also known as REPRESSOR OF CYTOKININ DEFICIENCY1 (ROCK1)) encodes a putative nucleotide-sugar transporter localized to the endoplasmic reticulum. Tapetal expression of TEX2 is sufficient for proper exine development. Loss of TEX2 leads to the formation of abnormal primexine, lack of primary exine elements, and subsequent failure of sporopollenin to correctly assemble into exine structures. Using immunohistochemistry, we investigated the carbohydrate composition of the tex2 primexine and found it accumulates increased amounts of arabinogalactans. Tapetum in tex2 accumulates prominent metabolic inclusions which depend on the sporopollenin polyketide biosynthesis and transport and likely correspond to a sporopollenin-like material. Even though such inclusions have not been previously reported, we show mutations in one of the known sporopollenin biosynthesis genes, LAP5/PKSB, but not in its paralog LAP6/PKSA, also lead to accumulation of similar inclusions, suggesting separate roles for the two paralogs. Finally, we show tex2 tapetal inclusions, as well as synthetic lethality in the double mutants of TEX2 and other exine genes, could be used as reporters when investigating genetic relationships between genes involved in exine formation.

A Novel Lateral Organ Boundary-domain Factor CmLBD2 Positively Regulates Pollen Development by Activating CmACOS5 in Chrysanthemum morifolium.

Male sterility, as a common reproductive characteristic in plants, plays an important role in breeding, in which pollen abortion is a key factor leading to male sterility. Here, based on a low expression level gene CmACOS5 in transcriptome of pollen abortive chrysanthemum, a new transcription factor CmLBD2 of the Lateral Organ Boundaries Domain family, which could bind the promoter of CmACOS5 by yeast one-hybrid library was screened. This study revealed the origin and expression pattern of CmLBD2 in chrysanthemum and verified the functions of two genes in pollen development by transgenic means. Inhibiting the expression of CmACOS5 or CmLBD2 can lead to a large reduction in pollen and even abortion in chrysanthemum. Using yeast one-/two-hybrid, electrophoretic mobility shift assays, and luciferase reporter assays, it was verified that CmLBD2 directly binds to the promoter of CmACOS5. These results suggest that LBD2 is a novel, key transcription factor regulating pollen development. This result will provide a new research background for enriching the function of LBD family proteins and also lay a new foundation for the breeding of male sterile lines and the mechanism of pollen development.

ZmFAR1 and ZmABCG26 Regulated by microRNA Are Essential for Lipid Metabolism in Maize Anther.

The function and regulation of lipid metabolic genes are essential for plant male reproduction. However, expression regulation of lipid metabolic genic male sterility (GMS) genes by noncoding RNAs is largely unclear. Here, we systematically predicted the microRNA regulators of 34 maize white brown complex members in ATP-binding cassette transporter G subfamily (WBC/ABCG) genes using transcriptome analysis. Results indicate that the ZmABCG26 transcript was predicted to be targeted by zma-miR164h-5p, and their expression levels were negatively correlated in maize B73 and Oh43 genetic backgrounds based on both transcriptome data and qRT-PCR experiments. CRISPR/Cas9-induced gene mutagenesis was performed on ZmABCG26 and another lipid metabolic gene, ZmFAR1. DNA sequencing, phenotypic, and cytological observations demonstrated that both ZmABCG26 and ZmFAR1 are GMS genes in maize. Notably, ZmABCG26 proteins are localized in the endoplasmic reticulum (ER), chloroplast/plastid, and plasma membrane. Furthermore, ZmFAR1 shows catalytic activities to three CoA substrates in vitro with the activity order of C12:0-CoA > C16:0-CoA > C18:0-CoA, and its four key amino acid sites were critical to its catalytic activities. Lipidomics analysis revealed decreased cutin amounts and increased wax contents in anthers of both zmabcg26 and zmfar1 GMS mutants. A more detailed analysis exhibited differential changes in 54 monomer contents between wild type and mutants, as well as between zmabcg26 and zmfar1. These findings will promote a deeper understanding of miRNA-regulated lipid metabolic genes and the functional diversity of lipid metabolic genes, contributing to lipid biosynthesis in maize anthers. Additionally, cosegregating molecular markers for ZmABCG26 and ZmFAR1 were developed to facilitate the breeding of male sterile lines.

Jasmonic Acid Plays a Pivotal Role in Pollen Development and Fertility Regulation in Different Types of P(T)GMS Rice Lines.

Two-line hybrid rice systems represent a new technical approach to utilizing the advantages of rice hybrids. However, the mechanism underlying the male sterile-line fertility transition in rice remains unclear. Peiai 64S (PA64S) is a photoperiod- and thermo-sensitive genic male sterile (PTGMS) line in which male sterility manifests at an average temperature above 23.5 °C under long-day (LD) conditions. Nongken 58S (NK58S) is a LD-sensitive genic male sterile (PGMS) rice that is sterile under LD conditions (above 13.75 h-day). In contrast, D52S is a short-day (SD)-PGMS line that manifests male sterility under SD conditions (below 13.5 h-day). In this study, we obtained fertile and sterile plants from all three lines and performed transcriptome analyses on the anthers of the plants. Gene ontology (GO) analysis suggested that the differentially expressed genes identified were significantly enriched in common terms involved in the response to jasmonic acid (JA) and in JA biosynthesis. On the basis of the biochemical and molecular validation of dynamic, tissue-specific changes in JA, indole-3-acetic acid (IAA) levels, gibberellin (GA) levels, and JA biosynthetic enzyme activities and expression, we proposed that JA could play a pivotal role in viable pollen production through its initial upregulation, constant fluctuation and leaf-spikelet signaling under certain fertility-inducing conditions. Furthermore, we also sprayed methyl jasmonate (MEJA) and salicylhydroxamic acid (SHAM) on the plants, thereby achieving fertility reversal in the PGMS lines NK58S and D52S, with 12.91-63.53% pollen fertility changes. Through qPCR and enzyme activity analyses, we identified two key enzymes-allene oxide synthase (AOS) and allene oxide cyclase (AOC)-that were produced and upregulated by 20-500-fold in PGMS in response to spraying; the activities of these enzymes reversed pollen fertility by influencing the JA biosynthetic pathway. These results provide a new understanding of hormone interactions and networks in male-sterile rice based on the role of JA that will help us to better understand the potential regulatory mechanisms of fertility development in rice in the future.

Differential gene expression patterns during gametophyte development provide insights into sex differentiation in the dioicous kelp Saccharina japonica.

BACKGROUND: In brown algae, dioicy is the prevalent sexual system, and phenotypic differences between male and female gametophytes have been found in many dioicous species. Saccharina japonica show remarkable sexual dimorphism in gametophytes before gametogenesis. A higher level of phenotypic differentiation was also found in female and male gametes after gametogenesis. However, the patterns of differential gene expression throughout gametophyte development and how these changes might relate to sex-specific fitness at the gamete stage in S. japonica are not well known. RESULTS: In this study, differences in gene expression between male and female gametophytes in different developmental stages were investigated using comparative transcriptome analysis. Among the 20,151 genes expressed in the haploid gametophyte generation, 37.53% were sex-biased. The abundance of sex-biased genes in mature gametophytes was much higher than that in immature gametophytes, and more male-biased than female-biased genes were observed in the mature stage. The predicted functions of most sex-biased genes were closely related to the sex-specific characteristics of gametes, including cell wall biosynthesis, sperm motility, and sperm and egg recognition. In addition, 51 genes were specifically expressed in males in both stages, showing great potential as candidate male sex-determining region (SDR) genes. CONCLUSIONS: This study describes a thorough investigation into differential gene expression between male and female gametophytes in the dioicous kelp S. japonica. A large number of sex-biased genes in mature gametophytes may be associated with the divergence of phenotypic traits and physiological functions between female gametes (eggs) and male gametes (sperm) during sexual differentiation. These genes may mainly come from new sex-biased genes that have recently evolved in the S. japonica lineage. The duplication of sex-biased genes was detected, which may increase the number of sex-biased genes after gametogenesis in S. japonica to some extent. The excess of male-biased genes over female-biased genes in the mature stage may reflect the different levels of sexual selection across sexes. This study deepens our understanding of the regulation of sex development and differentiation in the dioicous kelp S. japonica.