Relationships among sporophytic and gametophytic traits of 27 subtropical montane moss species.

PREMISE: Moss sporophytes differ strongly in size and biomass partitioning, potentially reflecting reproductive and dispersal strategies. Understanding how sporophyte traits are coordinated is essential for understanding moss functioning and evolution. This study aimed to answer: (1) how the size and proportions of the sporophyte differ between moss species with and without a prominent central strand in the seta, (2) how anatomical and morphological traits of the seta are related, and (3) how sporophytic biomass relates to gametophytic biomass and nutrient concentrations. METHODS: We studied the relationships between seta anatomical and morphological traits, the biomass of seta, capsule, and gametophyte, and carbon, nitrogen, and phosphorus concentrations of 27 subtropical montane moss species. RESULTS: (1) Moss species with a prominent central strand in the seta had larger setae and heavier capsules than those without a prominent strand. (2) With increasing seta length, setae became thicker and more rounded for both groups, while in species with a prominent central strand, the ratio of transport-cell area to epidermal area decreased. (3) In both groups, mosses with greater gametophytic biomass tended to have heavier sporophytes, but nitrogen and phosphorus concentrations in the gametophyte were unrelated to sporophytic traits. CONCLUSIONS: Our study highlights that the central strand in the seta may have an important functional role and affect the allometry of moss sporophytes. The coordinated variations in sporophyte morphological and anatomical traits follow basic biomechanical principles of cylinder-like structures, and these traits relate only weakly to the gametophytic nutrient concentrations. Research on moss sporophyte functional traits and their relationships to gametophytes is still in its infancy but could provide important insights into their adaptative strategies.

The Ka /Ks and πa /πs Ratios under Different Models of Gametophytic and Sporophytic Selection.

Alternation of generations in plant life cycle provides a biological basis for natural selection occurring in either the gametophyte or the sporophyte phase or in both. Divergent biphasic selection could yield distinct evolutionary rates for phase-specific or pleiotropic genes. Here, we analyze models that deal with antagonistic and synergistic selection between alternative generations in terms of the ratio of nonsynonymous to synonymous divergence (Ka/Ks). Effects of biphasic selection are opposite under antagonistic selection but cumulative under synergistic selection for pleiotropic genes. Under the additive and comparable strengths of biphasic allelic selection, the absolute Ka/Ks for the gametophyte gene is equal to in outcrossing but smaller than, in a mixed mating system, that for the sporophyte gene under antagonistic selection. The same pattern is predicted for Ka/Ks under synergistic selection. Selfing reduces efficacy of gametophytic selection. Other processes, including pollen and seed flow and genetic drift, reduce selection efficacy. The polymorphism (πa) at a nonsynonymous site is affected by the joint effects of selfing with gametophytic or sporophytic selection. Likewise, the ratio of nonsynonymous to synonymous polymorphism (πa/πs) is also affected by the same joint effects. Gene flow and genetic drift have opposite effects on πa or πa/πs in interacting with gametophytic and sporophytic selection. We discuss implications of this theory for detecting natural selection in terms of Ka/Ks and for interpreting the evolutionary divergence among gametophyte-specific, sporophyte-specific, and pleiotropic genes.

The sporophyte-to-gametophyte transition: The haploid generation comes of age.

Flowering plants alternate between two multicellular generations: the diploid sporophyte and haploid gametophyte. Despite its small size, the gametophyte has significant impacts on plant genetics, evolution, and breeding. Each male pollen grain and female embryo sac is a multicellular organism with independent gene expression, a functioning metabolism, and specialized cell types. In this review, we describe recent progress in understanding the process in which the haploid genome takes over expression from its diploid parent - the sporophyte-to-gametophyte transition. The focus is on pollen, but similar concepts may also apply to the female gametophyte. Technological advances in single-cell genomics offer the opportunity to characterize haploid gene expression in unprecedented detail, positioning the field to make rapid progress.

Gametophyte phase of commercial kelps, the potential food supplements for essential fatty acids and n-3 polyunsaturated fatty acids.

For heteromorphic algae with alternating generations, the thallus and gametophyte phases are different morphologies in free-living life history. The thalli are popular used as traditional vegetables and herbal drugs, whereas the gametophyte phases are little involved. To better understand the functional lipids in the gametophyte phase of three commercial kelps, Saccharina japonica, Undaria pinnatifida, and Costaria costata, the contents of total lipids (TLs), fatty acid (FA) profiles, and transcriptomic analysis were performed. For the studied kelps, the TL contents in gametophyte phase were always almost twice more than those in the thallus, and the kelp species, their life stage, and the gender were critical factors affecting lipid accumulation. The gametophyte phases of U. pinnatifida and C. costata were rich in essential FA C18:2 n - 6 and C18:3 n - 3. The S. japonica gametophyte phase contained abundant C20:5 n - 3 and C18:4 n - 3, possessed an ideal ratio of n - 6/n - 3 polyunsaturated fatty acid below 1.0, and was supported by the transcriptome data which showed that the key sjD12/15 (n - 3) gene of gametophyte partially upregulated than sporophyte. The results suggested that S. japonica gametophyte phase was the worthiest of further development and utilization as a functional food. PRACTICAL APPLICATION: It is the first report on the fatty acid characteristics of three gametophyte phases of Saccharina japonica, Undaria pinnatifida, and Costaria costata and find that the S. japonica was worthy of further development and utilization as a functional food owing to its satisfactory fatty acid composition.

Single-nucleus sequencing deciphers developmental trajectories in rice pistils.

Angiosperms possess a life cycle with an alternation of sporophyte and gametophyte generations, which happens in plant organs like pistils. Rice pistils contain ovules and receive pollen for successful fertilization to produce grains. The cellular expression profile in rice pistils is largely unknown. Here, we show a cell census of rice pistils before fertilization through the use of droplet-based single-nucleus RNA sequencing. The ab initio marker identification validated by in situ hybridization assists with cell-type annotation, revealing cell heterogeneity between ovule- and carpel-originated cells. A comparison of 1N (gametophyte) and 2N (sporophyte) nuclei identifies the developmental path of germ cells in ovules with typical resetting of pluripotency before the sporophyte-gametophyte transition, while trajectory analysis of carpel-originated cells suggests previously neglected features of epidermis specification and style function. These findings gain a systems-level view of cellular differentiation and development of rice pistils before flowering and lay a foundation for understanding female reproductive development in plants.

BAG6-A from Fragaria viridis pollen modulates gametophyte development in diploid strawberry.

Male and female gametophyte development processes are essential steps in the life cycles of all land plants. Here, we characterized a gene, FviBAG6-A, screened from the Fragaria viridis (2 n = 2x=14) pollen cDNA library and physically interacted with S-RNase. Ubiquitinated of Sa-RNase might be determined by the interaction of FviBAG6-A in the ubiquitin-proteasome system during fertilization. We found that overexpression of FviBAG6-A in Arabidopsis caused shorter silique length, and decreased silique number. Moreover, overexpression of FviBAG6-A in Fragaria vesca (2 n = 2x=14) led to a greatly reduced seed number, with nearly 80% of the seeds aborted. Analyses of paraffin sections and reactive oxygen species (ROS) content revealed that the majority of severe pollen defects were likely due to the early degradation of the tapetum and middle layer as a result of ROS accumulation and abnormal development of the uninucleate megaspore mother. Moreover, the FviBAG6-A interact with the E3 ligase SIZ1 and contribute to the SUMOylation of FviBAG6-A , which may be induced by the high level of ROS content, further promoting gametophyte abortion in strawberry transgenic lines. This study characterized the FviBAG6-A and reveals its novel function in gametophyte development.

RUVBL proteins are involved in plant gametophyte development.

The proper development of male and female gametophytes is critical for successful sexual reproduction and requires a carefully regulated series of events orchestrated by a suite of various proteins. RUVBL1 and RUVBL2, plant orthologues of human Pontin and Reptin, respectively, belong to the evolutionarily highly conserved AAA+ family linked to a wide range of cellular processes. Previously, we found that RUVBL1 and RUVBL2A mutations are homozygous lethal in Arabidopsis. Here, we report that RUVBL1 and RUVBL2A play roles in reproductive development. We show that mutant plants produce embryo sacs with an abnormal structure or with various numbers of nuclei. Although pollen grains of heterozygous mutant plants exhibit reduced viability and reduced pollen tube growth in vitro, some of the ruvbl pollen tubes are capable of targeting ovules in vivo. Similarly, some ruvbl ovules retain the ability to attract wild-type pollen tubes but fail to develop further. The activity of the RUVBL1 and RUVBL2A promoters was observed in the embryo sac, pollen grains, and tapetum cells and, for RUVBL2A, also in developing ovules. In summary, we show that the RUVBL proteins are essential for the proper development of both male and particularly female gametophytes in Arabidopsis.

A single silk- and multiple pollen-expressed PMEs at the Ga1 locus modulate maize unilateral cross-incompatibility.

The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross-incompatibility (UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene (PME) (ZmGa1F) and eight pollen-expressed PMEs (ZmGa1P and ZmGa1PL1-7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.

Methods for characterizing pollen fitness in Cannabis sativa L.

Pollen grains are male gametophytes, an ephemeral haploid generation of plants, that commonly engage in competition for a limited supply of ovules. Since variation in reproductive capabilities among male gametophytes may influence the direction and pace of evolution in populations, we must be able to quantify the relative fitness of gametophytes from different sires. To explore this, we estimated the relative fitness of groups of male gametophytes in a dioecious, wind-pollinated model system, Cannabis sativa, by characterizing the non-abortion rate (measured via chemical staining) and viability (measured via in vitro germination) of pollen from multiple sires. Pollen viability quickly declined within two weeks of anther dehiscence, and pollen stored under freezer conditions did not germinate regardless of storage time. In contrast, pollen non-abortion rates declined slowly and persisted longer than the lifetime of a sporophyte plant under both room temperature and freezer conditions. Pollen samples that underwent both viability and non-abortion rate analysis displayed no significant correlation, implying that researchers cannot predict pollen viability from non-abortion rates, nor infer male gametophytic fitness from a single measure. Our work demonstrates two independent, differential approaches to measure proxies of male fitness in C. sativa.

Sporophytic control of pollen meiotic progression is mediated by tapetum expression of ABORTED MICROSPORES.

Pollen development is dependent on the tapetum, a sporophytic anther cell layer surrounding the microspores that functions in pollen wall formation but is also essential for meiosis-associated development. There is clear evidence of crosstalk and co-regulation between the tapetum and microspores, but how this is achieved is currently not characterized. ABORTED MICROSPORES (AMS), a tapetum transcription factor, is important for pollen wall formation, but also has an undefined role in early pollen development. We conducted a detailed investigation of chromosome behaviour, cytokinesis, radial microtubule array (RMA) organization, and callose formation in the ams mutant. Early meiosis initiates normally in ams, shows delayed progression after the pachytene stage, and then fails during late meiosis, with disorganized RMA, defective cytokinesis, abnormal callose formation, and microspore degeneration, alongside abnormal tapetum development. Here, we show that selected meiosis-associated genes are directly repressed by AMS, and that AMS is essential for late meiosis progression. Our findings indicate that AMS has a dual function in tapetum-meiocyte crosstalk by playing an important regulatory role during late meiosis, in addition to its previously characterized role in pollen wall formation. AMS is critical for RMA organization, callose deposition, and therefore cytokinesis, and is involved in the crosstalk between the gametophyte and sporophytic tissues, which enables synchronous development of tapetum and microspores.

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.

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.

Heteromannans are the predominant hemicelluloses in the gametophytic stem of the umbrella moss Hypnodendron menziesii and occur in the walls of all cell types.

MAIN CONCLUSION: Heteromannans are the predominant hemicelluloses in the gametophytic stem of the moss Hypnodendron menziesii and occur in the walls of all cell types Little is known about the cell-wall polysaccharides of mosses. Monosaccharide analysis of cell walls isolated from the stem of the umbrella moss Hypnodendron menziesii was consistent with heteromannans, probably galactoglucomannans, being the predominant hemicellulosic polysaccharides in the walls. Immunofluorescence and immunogold microscopy with the monoclonal antibody LM21, specific for heteromannans, showed that these polysaccharides were present in the walls of all stem cell types. These cell types, except the hydroids, have secondary walls. Experiments in which sections were pre-treated with 0.1 M sodium carbonate and with the enzyme pectate lyase indicated that the heteromannans have O-acetyl groups that limit LM21 binding and the cell walls contain pectic homogalacturonan that masks detection of heteromannans using LM21. Therefore, to fully detect heteromannans in the cell walls, it was essential to use these pre-treatments to remove the O-acetyl groups from the heteromannans and pectic homogalacturonan from the cell walls. Fluorescence microscopy experiments with a second monoclonal antibody, LM22, also specific for heteromannans, showed similar results, but the binding was considerably weaker than with LM21, possibly as a result of subtle structural differences in the epitopes of the two antibodies. Although heteromannans occur abundantly in the cell walls of many species in basal lineages of tracheophytes, prior to the present study, research on the distribution of these polysaccharides in the walls of different cell types in mosses was confined to the model species Physcomitrium patens.

ESCRT components ISTL1 andLIP5 are required for tapetal function and pollen viability.

Pollen wall assembly is crucial for pollen development and plant fertility. The durable biopolymer sporopollenin and the constituents of the tryphine coat are delivered to developing pollen grains by the highly coordinated secretory activity of the surrounding tapetal cells. The role of membrane trafficking in this process, however, is largely unknown. In this study, we used Arabidopsis thaliana to characterize the role of two late-acting endosomal sorting complex required for transport (ESCRT) components, ISTL1 and LIP5, in tapetal function. Plants lacking ISTL1 and LIP5 form pollen with aberrant exine patterns, leading to partial pollen lethality. We found that ISTL1 and LIP5 are required for exocytosis of plasma membrane and secreted proteins in the tapetal cells at the free microspore stage, contributing to pollen wall development and tryphine deposition. Whereas the ESCRT machinery is well known for its role in endosomal trafficking, the function of ISTL1 and LIP5 in exocytosis is not a typical ESCRT function. The istl1 lip5 double mutants also show reduced intralumenal vesicle concatenation in multivesicular endosomes in both tapetal cells and developing pollen grains as well as morphological defects in early endosomes/trans-Golgi networks, suggesting that late ESCRT components function in the early endosomal pathway and exocytosis.

The rice VCS1 is identified as a molecular tool to mark and visualize the vegetative cell of pollen.

Cell-type-specific markers are valuable tools to reveal developmental processes underlying cell differentiation during plant reproduction. Here we report the pollen vegetative cell marker gene VCS1 (Vegetative Cell Specific 1) of rice (Oryza sativa japonica). VCS1 was expressed specifically in late pollen and was predicted to encode a small FAF domain-containing protein of 205 amino acid residues (aa). The expression of reporter fusion proteins showed that VCS1 was exclusively targeted to the vegetative nucleus of pollen. Upon pollen germination, VCS1 lost vegetative nucleus localization, and appeared diffused in the vegetative cytoplasm of pollen grain but not in the pollen tube. T-DNA insertional mutation which disrupted the carboxyl-terminal 21 aa of VCS1 did not affect plant vegetative growth and pollen development, while destruction of VCS1 by CRISPR/Cas9 only moderately affect pollen viability. VCS1 is evolutionally conserved in monocots but appeared absent in dicotyledons. This study reveals a molecular tool for visualizing the vegetative cell of rice and possible other monocots, which has potential values in the genetic engineering of male-sterile lines.

Evaluation of the S-locus in Prunus domestica, characterization, phylogeny and 3D modelling.

Self-compatibility has become the primary objective of most prune (Prunus domestica) breeding programs in order to avoid the problems related to the gametophytic self-incompatibility (GSI) system present in this crop. GSI is typically under the control of a specific locus., known as the S-locus., which contains at least two genes. The first gene encodes glycoproteins with RNase activity in the pistils., and the second is an SFB gene expressed in the pollen. There is limited information on genetics of SI/SC in prune and in comparison., with other Prunus species, cloning., sequencing and discovery of different S-alleles is very scarce. Clear information about S-alleles can be used for molecular identification and characterization of the S-haplotypes. We determined the S-alleles of 36 cultivars and selections using primers that revealed 17 new alleles. In addition, our study describes for the first time the association and design of a molecular marker for self-compatibility in P. domestica. Our phylogenetic tree showed that the S-alleles are spread across the phylogeny, suggesting that like previous alleles detected in the Rosaceae., they were of trans-specific origin. We provide for the first time 3D models for the P. domestica SI RNase alleles as well as in other Prunus species, including P. salicina (Japanese plum), P. avium (cherry), P. armeniaca (apricot), P. cerasifera and P. spinosa.

The aquatic carnivorous plant Aldrovanda vesiculosa (Droseraceae) exhibits altered developmental stages in male gametophyte.

Aldrovanda vesiculosa (Droseraceae) is a rare aquatic carnivorous plant, distributed in Europe, Asia, Africa, and Australia. Aldrovanda populations can flower prolifically under favourable conditions, but seed set is very limited. We studied the structure of Aldrovanda pollen collected from flowers in different developmental stages (opened and non-opened anthers) from both European and Australian populations to elucidate pollination traits and the basis of poor seed set on the basis of microscopic observation of pollen and anther structure. Microscopic analyses of Aldrovanda pollen showed that this plant has pollen arranged in tetrads like other species in the Droseraceae family. In hydrated pollen, cytoplasmic protrusions originate from pores located along the equatorial wall of monads, and can develop into pollen tubes. Interestingly, pollen development from microspores occurs in open anthers, suggesting a delay of the developmental stages. In addition, pollen development displays altered sperm cell formation and precocious pollen germination. Precocious germination may characterize recalcitrant pollen, which naturally do not undergo dehydration before anthesis and remain partially hydrated, particularly in aquatic and wetland plants. These alterations of male gametophyte development could affect fertilization processes, and be the reason for the low reproductive capability of Aldrovanda observed both in the field and in cultures. Generally, reduced pollen longevity and very quick germination are considered an adaptation to aquatic or wet environments.

COPII genes SEC31A/B are essential for gametogenesis and interchangeable in pollen development in Arabidopsis.

In eukaryotes, coat protein complex II (COPII) vesicles mediate anterograde traffic from the endoplasmic reticulum to the Golgi apparatus. Compared to yeasts, plants have multiple COPII coat proteins; however, the functional diversity among them is less well understood. SEC31A and SEC31B are outer coat proteins found in COPII vesicles in Arabidopsis. In this study, we explored the function of SEC31A and compared it with that of SEC31B from various perspectives. SEC31A was widely expressed, but at a significantly lower level than SEC31B. SEC31A-mCherry and SEC31B-GFP exhibited a high co-localization rate in pollen, but a lower rate in growing pollen tubes. The sec31a single mutant exhibited normal growth. SEC31A expression driven by the SEC31B promoter rescued the pollen abortion and infertility observed in sec31b. A sec31asec31b double mutant was unavailable due to lethality of the sec31asec31b gametophyte. Transmission electron microscopy revealed that one quarter of male gametogenesis was arrested at the uninuclear microspore stage, while confocal laser scanning microscopy showed that 1/4 female gametophyte development was suspended at the functional megaspore stage in sec31a-1/+sec31b-3/+ plants. Our study highlights the essential role of SEC31A/B in gametogenesis and their interchangeable functions in pollen development.

Isolation of the sperm and egg cells in wild rice (Oryza officinalis) as a mechanism for crop improvement.

KEY MESSAGE: Sperm cells can be isolated from the mature pollen grains of medicinal wild rice (Oryza officinalis) using an osmotic shock method, and the viable egg cells can be isolated by enzymatic digestion and mechanical dissection steps. Favorable alleles for rice breeding have been identified in natural cultivars and wild rice by association analysis of known functional genes with target trait performance. Transferring these genes from wild rice into cultivated rice varieties is one of the important objectives for rice breeders. The isolation of the sperm and egg cells of wild and cultivated rice is a prerequisite for the in vitro hybridization of distantly related cultivated rice and wild rice lines. Here, we provide a technical approach for isolating the sperm and egg cells of wild rice (Oryza officinalis). In this method, sperm cells were isolated from the mature pollen grains of medicinal wild rice (O. officinalis) according to an osmotic shock method. Additionally, viable O. officinalis egg cells were isolated following enzymatic digestion and mechanical dissection steps. Specifically, ovules were digested in an enzymatic solution containing pectinase and cellulase for 30 min, after which the ovule was cut into two halves. Three egg apparatus cells were released by gently applying pressure to the micropylar end. Generally, six or seven egg cells could be isolated from 20 ovules in 60 min. The same method was used to isolate zygotes from flowers at 24 h after pollination. This technology solved the difficulty of isolating sperm and egg cells in O. officinalis and allowed the isolated sperm and egg cells to be combined by in vitro hybridization of distantly related wild and cultivated rice lines.

The ScRALF3 secreted peptide is involved in sporophyte to gametophyte signalling and affects pollen mitosis I.

Signalling events through small peptides are essential in multiple aspects of plant reproduction. The ScRALF3 Solanum chacoense Rapid Alkalinization Factor (RALF) peptide was previously shown to regulate multiple aspects of cell-cell communication between the surrounding sporophytic tissue and the female gametophyte during ovule development. We analysed the global expression pattern of ScRALF3 with GUS reporter gene under control of the ScRALF3 promoter and validated it with in situ hybridisation. To better understand the role of ScRALF3 we used three different RNA interference (RNAi) lines that reduced the expression of ScRALF3 during pollen development. Both expression methods showed the presence of ScRALF3 in different tissues, including stigma, style, vascular tissues and during stamen development. Down-regulation of ScRALF3 expression through RNAi showed drastic defects in early stages of pollen development, mainly on the first mitosis. These results suggest that the ScRALF3 secreted peptide regulates the transition from sporogenesis to gametogenesis in both male and female gametophytes.