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.

AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube guidance.

Reproductive isolation is a prerequisite to form and maintain a new species. Multiple prezygotic and postzygotic reproductive isolation barriers have been reported in plants. In the model plant, Arabidopsis thaliana conspecific pollen tube precedence controlled by AtLURE1/PRK6-mediated signaling has been recently reported as a major prezygotic reproductive isolation barrier. By accelerating emergence of own pollen tubes from the transmitting tract, A. thaliana ovules promote self-fertilization and thus prevent fertilization by a different species. Taking advantage of a septuple atlure1null mutant, we now report on the role of AtLURE1/PRK6-mediated signaling for micropylar pollen tube guidance. Compared with wild-type (WT) ovules, atlure1null ovules displayed remarkably reduced micropylar pollen tube attraction efficiencies in modified semi-in vivo A. thaliana ovule targeting assays. However, when prk6 mutant pollen tubes were applied, atlure1null ovules showed micropylar attraction efficiencies comparable to that of WT ovules. These findings indicate that AtLURE1/PRK6-mediated signaling regulates micropylar pollen tube attraction in addition to promoting emergence of own pollen tubes from the transmitting tract. Moreover, semi-in vivo ovule targeting competition assays with the same amount of pollen grains from both A. thaliana and Arabidopsis lyrata showed that A. thaliana WT and xiuqiu mutant ovules are mainly targeted by own pollen tubes and that atlure1null mutant ovules are also entered to a large extent by A. lyrata pollen tubes. Taken together, we report that AtLURE1/PRK6-mediated signaling promotes conspecific micropylar pollen tube attraction representing an additional prezygotic isolation barrier.

Anther and ovule development of Clematis serratifolia (Ranunculaceae)-with new formation types in megaspore and nucellus.

Morphological indices of vegetative organs or reproductive organs, which are often used to analyze the evolution and classify Clematis, indicate that Clematis serratifolia and C. glauca could be related members at similar evolutionary levels. However, this assumption differs with phylogenetic studies based on genetics. Embryonic characteristics, which are more stable, are commonly used to estimate the phylogeny and evolution of angiosperms. We studied the microsporogenesis, microgametogenesis, megasporogenesis and macrogametogenesis development of C. serratifolia, and compared the early embryological characteristics among C. serratifolia, C. serratifolia and other Clematis species reported to provide a reference for the taxonomy of the genus Clematis. Our results showed that C. serratifolia and C. glauca differ in megaspore formation and nucellus types suggesting that they have originated from different ancestors. The differences among Clematis were mainly found in the type of the anther wall development, tapetum, pollen grains, megaspore formation and nucellus types.

miR167 limits anther growth to potentiate anther dehiscence.

In flowering plants, anther dehiscence and pollen release are essential for sexual reproduction. Anthers dehisce after cell wall degradation weakens stomium cell junctions in each anther locule, and desiccation creates mechanical forces that open the locules. Either effect or both together may break stomium cell junctions. The microRNA miR167 negatively regulates ARF6 and ARF8, which encode auxin response transcription factors. Arabidopsis mARF6 or mARF8 plants with mutated miR167 target sites have defective anther dehiscence and ovule development. Null mir167a mutations recapitulated mARF6 and mARF8 anther and ovule phenotypes, indicating that MIR167a is the main miR167 precursor gene that delimits ARF6 and ARF8 expression in these organs. Anthers of mir167a or mARF6/8 plants overexpressed genes encoding cell wall loosening functions associated with cell expansion, and grew larger than wild-type anthers did starting at flower stage 11. Experimental desiccation enabled dehiscence of miR167-deficient anthers, indicating competence to dehisce. Conversely, high humidity conditions delayed anther dehiscence in wild-type flowers. These results support a model in which miR167-mediated anther growth arrest permits anther dehiscence. Without miR167 regulation, excess anther growth delays dehiscence by prolonging desiccation.

Sex change in kiwifruit (Actinidia chinensis Planch.): a developmental framework for the bisexual to unisexual floral transition.

KEY MESSAGE: The developmental morphology of male and female kiwifruit flowers is tracked to delimit a framework of events to aid the study of divergence in floral gene expression. The transition from hermaphrodite to unisexual development of kiwifruit (Actinidia chinensis Planch) flowers has been reported previously, but differences in gene expression controlling sexual development for this species have not been associated with the major developmental changes occurring within pistils. We investigated the key stages in male and female flower development to define the point at which meristematic activities diverge in the two sexes. A combination of scanning electron microscopy and light microscopy was used to investigate pistil development from the earliest stages. We identified seven distinct stages characterized by differences in ovary size and shape, macrosporogenesis, ovule primordium development, anther locule lengthening, microspore wall thickening, and pollen degeneration. Sex differences were evident from the initial stage of development, with a laterally compacted gynoecium in male flowers. However, the key developmental stage, at which tissue differentiation clearly deviated between the two sexes, was stage 3, when flowers were 3.5 to 4.5 mm in length at approximately 10 d from initiation of stamen development. At this stage, male flowers lacked evident carpel meristem development as denoted by a lack of ovule primordium formation. Pollen degeneration in female flowers, probably driven by programmed cell death, occurred at the late stage 6, while the final stage 7 was represented by pollen release. As the seven developmental stages are associated with specific morphological differences, including flower size, the scheme suggested here can provide the required framework for the future study of gene expression during the regulation of flower development in this crop species.

The Solanum chacoense Fertilization-Related Kinase 3 (ScFRK3) is involved in male and female gametophyte development.

BACKGROUND: The Fertilization-related kinases (FRK) form a class that belongs to the MEKK subfamily of plant MAPKKKs. It was recently shown that FRK class kinases expanded during angiosperm evolution, reaching their maximum numbers in the lineage leading to solanaceous species and culminating in the Solanum genus where they account for more than 40% of the total MEKKs. The first members studied, ScFRK1 and ScFRK2 were shown to play a pivotal role in gametophyte development in the wild potato species Solanum chacoense. RESULTS: ScFRK3 is also involved in gametophyte development. ScFRK3 is expressed in developing pollen and young ovules, reaching its highest level immediately after meiosis and during the mitosis steps in both gametophytes. Hence, three independent lines of ScFRK3 RNAi mutant plants showed decreased number of seeds per fruit. We also observed an important number of degenerated embryo sac in mature ovary. Analysis of ovule development showed that most embryo sac did not enter mitosis I in ScFRK3 RNAi mutant plants. Severe lethality was also observed during male gametophyte development, pollen being arrested before mitosis I, as observed in the female gametophyte. Obvious defects in vegetative organs were not observed, emphasizing the reproductive roles of the FRK class kinases. To isolate MAP kinases acting downstream of ScFRK3, a de novo S. chacoense transcriptome from male and female reproductive organs was assembled. Of the five ScMKKs and 16 ScMPKs retrieved, only the ScMKK3 interacted with ScFRK3, while only the ScMPK13 interacted with ScMKK3, leading to an apparent single three-tiered canonical MAP kinase cascade combination involving ScFRK3-ScMKK3-ScMPK13. CONCLUSIONS: The ScFRK3 MAPKKK is involved in a signaling cascade that regulates both male and female gamete development, and most probably act upstream of ScMKK3 and ScMPK13.

An Essential Role for miRNA167 in Maternal Control of Embryonic and Seed Development.

Maternal cells play a critical role in ensuring the normal development of embryos, endosperms, and seeds. Mutations that disrupt the maternal control of embryogenesis and seed development are difficult to identify. Here, we completely deleted four MICRORNA167 (MIR167) genes in Arabidopsis (Arabidopsis thaliana) using a clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein9 (Cas9) genome-editing technology. We found that plants with a deletion of MIR167A phenocopied plants overexpressing miRNA167-resistant versions of Auxin Response Factor6 (ARF6) or ARF8, two miRNA167 targets. Both the mir167a mutant and the ARF overexpression lines were defective in anther dehiscence and ovule development. Serendipitously, we found that the mir167a (♀) × wild type (♂) crosses failed to produce normal embryos and endosperms, despite the findings that embryos with either mir167a+/- or mir167a-/- genotypes developed normally when mir167a+/- plants were self-pollinated, revealing a central role of MIR167A in maternal control of seed development. The mir167a phenotype is 100% penetrant, providing a great genetic tool for studying the roles of miRNAs and auxin in maternal control. Moreover, we found that mir167a mutants flowered significantly later than wild-type plants, a phenotype that was not observed in the ARF overexpression lines. We show that the reproductive defects of mir167a mutants were suppressed by a decrease of activities of ARF6, ARF8, or both. Our results clearly demonstrate that MIR167A is the predominant MIR167 member in regulating Arabidopsis reproduction and that MIR167A acts as a maternal gene that functions largely through ARF6 and ARF8.

Comparative development of staminate and pistillate flowers in the dioecious cactus Opuntia robusta.

KEY MESSAGE: PCD role in unisexual flowers. The developmental processes underlying the transition from hermaphroditism to unisexuality are key to understanding variation and evolution of floral structure and function. A detailed examination of the cytological and histological patterns involved in pollen and ovule development of staminate and pistillate flowers in the dioecious Opuntia robusta was undertaken, and the potential involvement of programmed cell death in the abortion of the sex whorls was explored. Flowers initiated development as hermaphrodites and became functionally unisexual by anthesis. Female individuals have pistillate flowers with a conspicuous stigma, functional ovary, collapsed stamens and no pollen grains. Male individuals have staminate flowers, with large yellow anthers, abundant pollen grains, underdeveloped stigma, style and an ovary that rarely produced ovules. In pistillate flowers, anther abortion resulted from the premature degradation of the tapetum by PCD, followed by irregular deposition of callose wall around the microsporocytes, and finally by microspore degradation. In staminate flowers, the stigma could support pollen germination; however, the ovaries were reduced, with evidence of placental arrest and ovule abortion through PCD, when ovules were present. We demonstrate that PCD is recruited in both pistillate and staminate flower development; however, it occurs at different times of floral development. This study contributes to the understanding of the nature of the O. robusta breeding system and identifies developmental landmarks that contribute to sexual determination in Cactaceae.

Low temperature-induced aberrations in male and female reproductive organ development cause flower abortion in chickpea.

Chickpea (Cicer arietinum L.) is susceptible to low temperature (LT) at reproductive stage. LT causes flower abortion and delays pod set in chickpea until terminal drought becomes an issue, thereby decreasing yield potential. In chickpea, flower and anther/pollen development as well as LT-induced abnormalities on anther and pollen development are described inadequately. In the present manuscript, we report flower development stages, anther development stages, and aberrations in male gamete formation in chickpea under LT. Flower length was linearly correlated to flower and anther stages and can be used to predict these stages in chickpea. LT affected male gamete development in a flower/anther age-dependent manner where outcome ranged from no pollen formation to pollen sterility or no anther dehiscence to delayed dehiscence. In anthers, LT inhibited microsporogenesis, microgametogenesis, tapetum degeneration, breakage of septum and stomium, and induced pollen sterility. Whereas disruption of male function was the prime cause of abortion in flowers below vacuolated pollen stage, flower abortion was due to a combination of male and female reproductive functions in flowers with mature pollen. The study will help in elucidating mechanisms governing flower development, anther and pollen development, and tolerance/susceptibility to LT.

Arabinogalactan proteins: Distribution during the development of male and female gametophytes.

Arabinogalactan proteins (AGPs), i.e. a subfamily of hydroxyproline-rich proteins (HRGPs), are widely distributed in the plant kingdom. For many years, AGPs have been connected with the multiple phases of plant reproduction and developmental processes. Currently, extensive knowledge is available about their various functions, i.e. involvement in pollen grain formation, initiation of pollen grain germination, pollen tube guidance in the transmission tissue of pistil and ovule nucellus, and function as a signaling molecule during cell-cell communication. Although many studies have been performed, the mechanism of action, the heterogeneous molecule structure, and the connection with other extracellular matrix components have not been sufficiently explained. The aim of this work was to gather and describe the most important information on the distribution of AGPs in gametophyte development. The present review provides a summary of the first reports about AGPs and the most recent knowledge about their functions during male and female gametophyte formation.

B-family subunits of protein phosphatase 2A are necessary for pollen development but not for female gametophyte development in Arabidopsis.

Protein phosphatase 2A (PP2A) is a heterotrimeric protein complex conserved among eukaryotes. The B subunit of PP2A determines the substrate specificity of the PP2A holoenzyme, and is classified into the B, B', B″ and B‴ families. Arabidopsis thaliana has two isoforms of the B-family subunit (ATBA and ATBB). A double knockout of their genes is lethal, but which developmental process is primarily impaired by the double knockout is unclear. Identifying such a process helps understand PP2A-mediated signaling more deeply. Here, genetic characterization of new knockout mutants for these genes shows that they are necessary for pollen development but not for female gametophyte development. Compared to wild-type pollen grains, the mutant pollen grains exhibited lower enzyme activities, germinated less frequently on stigmas, and exhibited the aberrant numbers of sperm cell nuclei, suggesting that ATBA and ATBB play pleiotropic roles in pollen development. The amino acids stabilizing the interaction between the human PP2A A and B-family subunits are conserved in an Arabidopsis A subunit (AtPP2AA2), ATBA and ATBB. His-tagged AtPP2AA2 co-immunoprecipitated with either Myc-tagged ATBA or Myc-tagged ATBB in vitro, confirming their interactions. Proteins that regulate pollen development and that undergo dephosphorylation are likely primary targets of ATBA and ATBB.

Development of the ovule and seed of Habanero chili pepper (Capsicum chinense Jacq.): Anatomical characterization and immunocytochemical patterns of pectin methyl-esterification.

Ovule and seed development in plants has long fascinated the scientific community given the complex cell coordination implicated in these processes. These cell events are highly conserved but are not necessarily representative of all plants. In this study, with the aim of obtaining information regarding the cellular patterns that follow the usual development of the ovule and the zygotic embryo, we carried out an integral anatomical study of the Capsicum chinense Jacq., floral buds and seeds at various days during maturation. This study allowed us to identify the main histo-morphological stages accompanying the transition of somatic cells into the macrospore, female gamete, and the zygotic embryogenesis. This knowledge is fundamental for future biotechnological research focused on solving the morphological recalcitrance observed during the in vitro induction of somatic or microspore embryogenesis in Capsicum. For the first time in C. chinense, we have described the hypostases, a putative source of plant growth regulators, and "the corrosion cavity", a space around the embryo. Additionally, the cell wall pectin-esterification status was investigated by immunohistology. At early stages of morphogenesis, the pectin is highly methyl-esterified; however, methyl-esterification decreases gradually throughout the process. A comparison of the results obtained here, together with the histo- and immunological changes occurring during the somatic and microspore embryogenesis, should help to elucidate the biochemical mechanisms that trigger the morphogenic events in Capsicum spp.

How many receptor-like kinases are required to operate a pollen tube.

Successful fertilization depends on active molecular dialogues that the male gametophyte can establish with the pistil and the female gametophyte. Pollen grains and stigmas must recognize each other; pollen tubes need to identify the pistil tissues they will penetrate, follow positional cues to exit the transmitting tract and finally, locate the ovules. These molecular dialogues directly affect pollen tube growth rate and orientation. Receptor-like kinases (RLKs) are natural candidates for the perception and decoding of extracellular signals and their transduction to downstream cytoplasmic interactors. Here, we update knowledge regarding how RLKs are involved in pollen tube growth, cell wall integrity and guidance. In addition, we use public data to build a pollen tube RLK interactome that might help direct experiments to elucidate the function of pollen RLKs and their associated proteins.

GID1 expression is associated with ovule development of sexual and apomictic plants.

KEY MESSAGE: BbrizGID1 is expressed in the nucellus of apomictic Brachiaria brizantha, previous to aposporous initial differentiation. AtGID1a overexpression triggers differentiation of Arabidopsis thaliana MMC-like cells, suggesting its involvement in ovule development. GIBBERELLIN-INSENSITIVE DWARF1 (GID1) is a gibberellin receptor previously identified in plants and associated with reproductive development, including ovule formation. In this work, we characterized the Brachiaria brizantha GID1 gene (BbrizGID1). BbrizGID1 showed up to 92% similarity to GID1-like gibberellin receptors of other plants of the Poaceae family and around 58% to GID1-like gibberellin receptors of Arabidopsis thaliana. BbrizGID1 was more expressed in ovaries at megasporogenesis than in ovaries at megagametogenesis of both sexual and apomictic plants. In ovules, BbrizGID1 transcripts were detected in the megaspore mother cell (MMC) of sexual and apomictic B. brizantha. Only in the apomictic plants, expression was also observed in the surrounding nucellar cells, a region in which aposporous initial cells differentiate to form the aposporic embryo sac. AtGID1a ectopic expression in Arabidopsis determines the formation of MMC-like cells in the nucellus, close to the MMC, that did not own MMC identity. Our results suggest that GID1 might be involved in the proper differentiation of a single MMC during ovule development and provide valuable information on the role of GID1 in sexual and apomictic reproduction.

Developmental landmarks during floral ontogeny of jalapeño chili pepper (Capsicum annuum L.) and the effect of gibberellin on ovary growth.

Pepper (Capsicum annuum L.) is an important horticultural crop in many regions of the world. The final shape and size of the fruit are known to be determined at a very early step of flower development. During flower development hormonal treatments using gibberellins seem to promote growth resulting in higher yield and fruit quality. However, the morphological changes that occur in the pepper flowers after these treatments are largely unknown. In the present study, we provide a description of floral development landmarks of jalapeño chili pepper (cultivar Huichol), divided in nine representative stages from its initiation until the opening of the bud. We established a correlation among external flower development and the time and pattern of reproductive organogenesis. Male and female gametogenesis progression was used to define specific landmarks during flower maturation. The pattern of expression of key genes involved in gibberellin metabolism and response was also evaluated in the nine flower stages. The proposed development framework was used to analyze the effect of gibberellin treatments in the development of the flower. We observed both an effect of the treatment in the histology of the ovary tissue and an increase in the level of expression of CaGA2ox1 and CaGID1b genes. The developmental stages we defined for this species are very useful to analyze the molecular and morphological changes after hormonal treatments.

Arabidopsis LORELEI, a Maternally Expressed Imprinted Gene, Promotes Early Seed Development.

In flowering plants, the female gametophyte controls pollen tube reception immediately before fertilization and regulates seed development immediately after fertilization, although the controlling mechanisms remain poorly understood. Previously, we showed that LORELEI (LRE), which encodes a putative glycosylphosphatidylinositol-anchored membrane protein, is critical for pollen tube reception by the female gametophyte before fertilization and the initiation of seed development after fertilization. Here, we show that LRE is expressed in the synergid, egg, and central cells of the female gametophyte and in the zygote and proliferating endosperm of the Arabidopsis (Arabidopsis thaliana) seed. Interestingly, LRE expression in the developing seeds was primarily from the matrigenic LRE allele, indicating that LRE expression is imprinted. However, LRE was biallelically expressed in 8-d-old seedlings, indicating that the patrigenic allele does not remain silenced throughout the sporophytic generation. Regulation of imprinted LRE expression is likely novel, as LRE was not expressed in pollen or pollen tubes of mutants defective for MET1, DDM1, RNA-dependent DNA methylation, or MSI-dependent histone methylation. Additionally, the patrigenic LRE allele inherited from these mutants was not expressed in seeds. Surprisingly, and contrary to the predictions of the parental conflict hypothesis, LRE promotes growth in seeds, as loss of the matrigenic but not the patrigenic LRE allele caused delayed initiation of seed development. Our results showed that LRE is a rare imprinted gene that functions immediately after double fertilization and supported the model that a passage through the female gametophyte establishes monoalleleic expression of LRE in seeds and controls early seed development.

Gametophyte Development Needs Mitochondrial Coproporphyrinogen III Oxidase Function.

Tetrapyrrole biosynthesis is one of the most essential metabolic pathways in almost all organisms. Coproporphyrinogen III oxidase (CPO) catalyzes the conversion of coproporphyrinogen III into protoporphyrinogen IX in this pathway. Here, we report that mutation in the Arabidopsis (Arabidopsis thaliana) CPO-coding gene At5g63290 (AtHEMN1) adversely affects silique length, ovule number, and seed set. Athemn1 mutant alleles were transmitted via both male and female gametes, but homozygous mutants were never recovered. Plants carrying Athemn1 mutant alleles showed defects in gametophyte development, including nonviable pollen and embryo sacs with unfused polar nuclei. Improper differentiation of the central cell led to defects in endosperm development. Consequently, embryo development was arrested at the globular stage. The mutant phenotype was completely rescued by transgenic expression of AtHEMN1 Promoter and transcript analyses indicated that AtHEMN1 is expressed mainly in floral tissues and developing seeds. AtHEMN1-green fluorescent protein fusion protein was found targeted to mitochondria. Loss of AtHEMN1 function increased coproporphyrinogen III level and reduced protoporphyrinogen IX level, suggesting the impairment of tetrapyrrole biosynthesis. Blockage of tetrapyrrole biosynthesis in the AtHEMN1 mutant led to increased reactive oxygen species (ROS) accumulation in anthers and embryo sacs, as evidenced by nitroblue tetrazolium staining. Our results suggest that the accumulated ROS disrupts mitochondrial function by altering their membrane polarity in floral tissues. This study highlights the role of mitochondrial ROS homeostasis in gametophyte and seed development and sheds new light on tetrapyrrole/heme biosynthesis in plant mitochondria.

Loss of pollen-specific phospholipase NOT LIKE DAD triggers gynogenesis in maize.

Gynogenesis is an asexual mode of reproduction common to animals and plants, in which stimuli from the sperm cell trigger the development of the unfertilized egg cell into a haploid embryo. Fine mapping restricted a major maize QTL (quantitative trait locus) responsible for the aptitude of inducer lines to trigger gynogenesis to a zone containing a single gene NOT LIKE DAD (NLD) coding for a patatin-like phospholipase A. In all surveyed inducer lines, NLD carries a 4-bp insertion leading to a predicted truncated protein. This frameshift mutation is responsible for haploid induction because complementation with wild-type NLD abolishes the haploid induction capacity. Activity of the NLD promoter is restricted to mature pollen and pollen tube. The translational NLD::citrine fusion protein likely localizes to the sperm cell plasma membrane. In Arabidopsis roots, the truncated protein is no longer localized to the plasma membrane, contrary to the wild-type NLD protein. In conclusion, an intact pollen-specific phospholipase is required for successful sexual reproduction and its targeted disruption may allow establishing powerful haploid breeding tools in numerous crops.

The DC1-domain protein VACUOLELESS GAMETOPHYTES is essential for development of female and male gametophytes in Arabidopsis.

In this work we identified VACUOLELESS GAMETOPHYTES (VLG) as a DC1 domain-containing protein present in the endomembrane system and essential for development of both female and male gametophytes. VLG was originally annotated as a gene coding for a protein of unknown function containing DC1 domains. DC1 domains are cysteine- and histidine-rich zinc finger domains found exclusively in the plant kingdom that have been named on the basis of similarity with the C1 domain present in protein kinase C (PKC). In Arabidopsis, both male and female gametophytes are characterized by the formation of a large vacuole early in development; this is absent in vlg mutant plants. As a consequence, development is arrested in embryo sacs and pollen grains at the first mitotic division. VLG is specifically located in multivesicular bodies or pre-vacuolar compartments, and our results suggest that vesicular fusion is affected in the mutants, disrupting vacuole formation. Supporting this idea, AtPVA12 - a member of the SNARE vesicle-associated protein family and previously related to a sterol-binding protein, was identified as a VLG interactor. A role for VLG is proposed mediating vesicular fusion in plants as part of the sterol trafficking machinery required for vacuole biogenesis in plants.