Central-cell-produced attractants control fertilization recovery.

Animal fertilization relies on hundreds of sperm racing toward the egg, whereas, in angiosperms, only two sperm cells are delivered by a pollen tube to the female gametes (egg cell and central cell) for double fertilization. However, unsuccessful fertilization under this one-pollen-tube design can be detrimental to seed production and plant survival. To mitigate this risk, unfertilized-gamete-controlled extra pollen tube entry has been evolved to bring more sperm cells and salvage fertilization. Despite its importance, the underlying molecular mechanism of this phenomenon remains unclear. In this study, we report that, in Arabidopsis, the central cell secretes peptides SALVAGER1 and SALVAGER2 in a directional manner to attract pollen tubes when the synergid-dependent attraction fails or is terminated by pollen tubes carrying infertile sperm cells. Moreover, loss of SALs impairs the fertilization recovery capacity of the ovules. Therefore, this research uncovers a female gamete-attraction system that salvages seed production for reproductive assurance.

Kin conflict in seed development: an interdependent but fractious collective.

Seeds are complex structures that unite diploid maternal tissues with filial tissues that may be haploid (gametophyte), diploid (embryo), or triploid (endosperm). Maternal tissues are predicted to favor smaller seeds than are favored by filial tissues, and filial genes of maternal origin are predicted to favor smaller seeds than are favored by filial genes of paternal origin. Consistent with these predictions, seed size is determined by an interplay between growth of maternal integuments, which limits seed size, and of filial endosperm, which promotes larger seeds. Within endosperm, genes of paternal origin favor delayed cellularization of endosperm and larger seeds, whereas genes of maternal origin favor early cellularization and smaller seeds. The ratio of maternal and paternal gene products in endosperm contributes to the failure of crosses between different ploidy levels of the same species and crosses between species. Maternally expressed small-interfering RNAs (siRNAs) are predicted to associate with growth-enhancing genes.

Spatiotemporal formation of the large vacuole regulated by the BIN2-VLG module is required for female gametophyte development in Arabidopsis.

In Arabidopsis thaliana, female gametophyte (FG) development is accompanied by the formation and expansion of the large vacuole in the FG; this is essential for FG expansion, nuclear polar localization, and cell fate determination. Arabidopsis VACUOLELESS GAMETOPHYTES (VLG) facilitates vesicular fusion to form large vacuole in the FG, but the regulation of VLG remains largely unknown. Here, we found that gain-of-function mutation of BRASSINOSTEROID INSENSITIVE2 (BIN2) (bin2-1) increases VLG abundance to induce the vacuole formation at stage FG1, and leads to abortion of FG. Loss-of-function mutation of BIN2 and its homologs (bin2-3 bil1 bil2) reduced VLG abundance and mimicked vlg/VLG phenotypes. Knocking down VLG in bin2-1 decreased the ratio of aberrant vacuole formation at stage FG1, whereas FG1-specific overexpression of VLG mimicked the bin2-1 phenotype. VLG partially rescued the bin2-3 bil1 bil2 phenotype, demonstrating that VLG acts downstream of BIN2. Mutation of VLG residues that are phosphorylated by BIN2 altered VLG stability and a phosphorylation mimic of VLG causes similar defects as did bin2-1. Therefore, BIN2 may function by interacting with and phosphorylating VLG in the FG to enhance its stability and abundance, thus facilitating vacuole formation. Our findings provide mechanistic insight into how the BIN2-VLG module regulates the spatiotemporal formation of the large vacuole in FG development.

Life cycle and reproduction dynamics of Bangiales in response to environmental stresses.

Red algae of the order Bangiales are notable for exhibiting flexible promotion of sexual and asexual reproductive processes by environmental stresses. This flexibility indicates that a trade-off between vegetative growth and reproduction occurs in response to environmental stresses that influence the timing of phase transition within the life cycle. Despite their high phylogenetic divergence, both filamentous and foliose red alga in the order Bangiales exhibit a haploid-diploid life cycle, with a haploid leafy or filamentous gametophyte (thallus) and a diploid filamentous sporophyte (conchocelis). Unlike haploid-diploid life cycles in other orders, the gametophyte in Bangiales is generated independently of meiosis; the regulation of this generation transition is not fully understood. Based on transcriptome and gene expression analyses, the originally proposed biphasic model for alternation of generations in Bangiales was recently updated to include a third stage. Along with the haploid gametophyte and diploid sporophyte, the triphasic framework recognizes a diploid conchosporophyte-a conchosporangium generated on the conchocelis-phase and previously considered to be part of the sporophyte. In addition to this sexual life cycle, some Bangiales species have an asexual life cycle in which vegetative cells of the thallus develop into haploid asexual spores, which are then released from the thallus to produce clonal thalli. Here, we summarize the current knowledge of the triphasic life cycle and life cycle trade-off in Neopyropia yezoensis and 'Bangia' sp. as model organisms for the Bangiales.

The single Marchantia polymorpha FERONIA homolog reveals an ancestral role in regulating cellular expansion and integrity.

Plant cells are surrounded by a cell wall, a rigid structure that is not only important for cell and organ shape, but is also crucial for intercellular communication and interactions with the environment. In the flowering plant Arabidopsis thaliana, the 17 members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family are involved in a multitude of physiological and developmental processes, making it difficult to assess their primary or ancestral function. To reduce genetic complexity, we characterized the single CrRLK1L gene of Marchantia polymorpha, MpFERONIA (MpFER). Plants with reduced MpFER levels show defects in vegetative development, i.e. rhizoid formation and cell expansion, and have reduced male fertility. In contrast, cell integrity and morphogenesis of the gametophyte are severely affected in Mpfer null mutants and MpFER overexpression lines. Thus, we conclude that the CrRLK1L gene family originated from a single gene with an ancestral function in cell expansion and the maintenance of cellular integrity. During land plant evolution, this ancestral gene diversified to fulfill a multitude of specialized physiological and developmental roles in the formation of both gametophytic and sporophytic structures essential to the life cycle of flowering plants.

The renaissance and enlightenment of Marchantia as a model system.

The liverwort Marchantia polymorpha has been utilized as a model for biological studies since the 18th century. In the past few decades, there has been a Renaissance in its utilization in genomic and genetic approaches to investigating physiological, developmental, and evolutionary aspects of land plant biology. The reasons for its adoption are similar to those of other genetic models, e.g. simple cultivation, ready access via its worldwide distribution, ease of crossing, facile genetics, and more recently, efficient transformation, genome editing, and genomic resources. The haploid gametophyte dominant life cycle of M. polymorpha is conducive to forward genetic approaches. The lack of ancient whole-genome duplications within liverworts facilitates reverse genetic approaches, and possibly related to this genomic stability, liverworts possess sex chromosomes that evolved in the ancestral liverwort. As a representative of one of the three bryophyte lineages, its phylogenetic position allows comparative approaches to provide insights into ancestral land plants. Given the karyotype and genome stability within liverworts, the resources developed for M. polymorpha have facilitated the development of related species as models for biological processes lacking in M. polymorpha.

Origin, loss, and regain of self-incompatibility in angiosperms.

The self-incompatibility (SI) system with the broadest taxonomic distribution in angiosperms is based on multiple S-locus F-box genes (SLFs) tightly linked to an S-RNase termed type-1. Multiple SLFs collaborate to detoxify nonself S-RNases while being unable to detoxify self S-RNases. However, it is unclear how such a system evolved, because in an ancestral system with a single SLF, many nonself S-RNases would not be detoxified, giving low cross-fertilization rates. In addition, how the system has been maintained in the face of whole-genome duplications (WGDs) or lost in other lineages remains unclear. Here we show that SLFs from a broad range of species can detoxify S-RNases from Petunia with a high detoxification probability, suggestive of an ancestral feature enabling cross-fertilization and subsequently modified as additional SLFs evolved. We further show, based on its genomic signatures, that type-1 was likely maintained in many lineages, despite WGD, through deletion of duplicate S-loci. In other lineages, SI was lost either through S-locus deletions or by retaining duplications. Two deletion lineages regained SI through type-2 (Brassicaceae) or type-4 (Primulaceae), and one duplication lineage through type-3 (Papaveraceae) mechanisms. Thus, our results reveal a highly dynamic process behind the origin, maintenance, loss, and regain of SI.

A mitochondrial ADXR-ADX-P450 electron transport chain is essential for maternal gametophytic control of embryogenesis in Arabidopsis.

Mitochondrial adrenodoxins (ADXs) are small iron-sulfur proteins with electron transfer properties. In animals, ADXs transfer electrons between an adrenodoxin reductase (ADXR) and mitochondrial P450s, which is crucial for steroidogenesis. Here we show that a plant mitochondrial steroidogenic pathway, dependent on an ADXR-ADX-P450 shuttle, is essential for female gametogenesis and early embryogenesis through a maternal effect. The steroid profile of maternal and gametophytic tissues of wild-type (WT) and adxr ovules revealed that homocastasterone is the main steroid present in WT gametophytes and that its levels are reduced in the mutant ovules. The application of exogenous homocastasterone partially rescued adxr and P450 mutant phenotypes, indicating that gametophytic homocastasterone biosynthesis is affected in the mutants and that a deficiency of this hormone causes the phenotypic alterations observed. These findings also suggest not only a remarkable similarity between steroid biosynthetic pathways in plants and animals but also a common function during sexual reproduction.

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.

The structural organization of the outer tissues in the gametophytic stem of the umbrella moss Hypnodendron menziesii optimizes load bearing.

MAIN CONCLUSION: The ultrastructural design and biochemical organization of the significantly thickened outer tissues of the gametophytic stem of Hypnodendron menziesii optimizes load bearing of the stem. Hypnodendron menziesii is a bryoid umbrella moss growing in high humid conditions on the forest floors of New Zealand. The erect gametophyte bears up to eight whorls of branches in succession, spreading across the stem that bears the heavy weight of branches with highly hydrated leaves. Our investigation using a combination of light microscopy, transmission electron microscopy (TEM), scanning electron microscopy (SEM), and TEM-immunolabeling techniques provided novel information on the structural design and biochemical organization of greatly thickened cell walls of epidermal, hypodermal, and outermost cortical tissues, comparing underlying thin-walled cortical tissues in the gametophytic stem. Probing into the ultrastructure of the cell wall architecture of these target tissues by TEM and SEM revealed the cell walls to display a multilamellar organization, in addition to demonstrating the presence of an electron-dense substance in the cell wall, presumably flavonoids. The pattern of distribution and concentration of rhamnogalacturonan, homogalacturonan, and heteromannan, as determined by immunogold labeling, suggests that it is the combination of structural and molecular design of the cell wall that may optimize the mechanical function of the epidermal, hypodermal, and outer cortical tissues. Statistical relationships between the overall thickness of epidermal, hypodermal, and outer cortical cell walls, the lumen area of cells and the percentage area of cell wall occupied in these tissues at different heights of the stem, and thickness of secondary cell wall layers (L1-L4/5) were explored. The results of these analyses unequivocally support the contribution of outer tissues to the mechanical strength of the resilient stem.

Characterization of the fiber-like cortical cells in moss gametophytes.

MAIN CONCLUSION: Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-ß-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-ß-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Plasmodesmata dynamics in bryophyte model organisms: secondary formation and developmental modifications of structure and function.

MAIN CONCLUSION: Developing bryophytes differentially modify their plasmodesmata structure and function. Secondary plasmodesmata formation via twinning appears to be an ancestral trait. Plasmodesmata networks in hornwort sporophyte meristems resemble those of angiosperms. All land-plant taxa use plasmodesmata (PD) cell connections for symplasmic communication. In angiosperm development, PD networks undergo an extensive remodeling by structural and functional PD modifications, and by postcytokinetic formation of additional secondary PD (secPD). Since comparable information on PD dynamics is scarce for the embryophyte sister groups, we investigated maturating tissues of Anthoceros agrestis (hornwort), Physcomitrium patens (moss), and Marchantia polymorpha (liverwort). As in angiosperms, quantitative electron microscopy revealed secPD formation via twinning in gametophytes of all model bryophytes, which gives rise to laterally adjacent PD pairs or to complex branched PD. This finding suggests that PD twinning is an ancient evolutionary mechanism to adjust PD numbers during wall expansion. Moreover, all bryophyte gametophytes modify their existing PD via taxon-specific strategies resembling those of angiosperms. Development of type II-like PD morphotypes with enlarged diameters or formation of pit pairs might be required to maintain PD transport rates during wall thickening. Similar to angiosperm leaves, fluorescence redistribution after photobleaching revealed a considerable reduction of the PD permeability in maturating P. patens phyllids. In contrast to previous reports on monoplex meristems of bryophyte gametophytes with single initials, we observed targeted secPD formation in the multi-initial basal meristems of A. agrestis sporophytes. Their PD networks share typical features of multi-initial angiosperm meristems, which may hint at a putative homologous origin. We also discuss that monoplex and multi-initial meristems may require distinct types of PD networks, with or without secPD formation, to control maintenance of initial identity and positional signaling.

Ethylene controls three-dimensional growth involving reduced auxin levels in the moss Physcomitrium patens.

The conquest of land by plants was concomitant with, and possibly enabled by, the evolution of three-dimensional (3D) growth. The moss Physcomitrium patens provides a model system for elucidating molecular mechanisms in the initiation of 3D growth. Here, we investigate whether the phytohormone ethylene, which is believed to have been a signal before land plant emergence, plays a role in 3D growth regulation in P. patens. We report ethylene controls 3D gametophore formation, based on results from exogenously applied ethylene and genetic manipulation of PpEIN2, which is a central component in the ethylene signaling pathway. Overexpression (OE) of PpEIN2 activates ethylene responses and leads to earlier formation of gametophores with fewer gametophores produced thereafter, phenocopying ethylene-treated wild-type. Conversely, Ppein2 knockout mutants, which are ethylene insensitive, show initially delayed gametophore formation with more gametophores produced later. Furthermore, pharmacological and biochemical analyses reveal auxin levels are decreased in the OE lines but increased in the knockout mutants. Our results suggest that evolutionarily, ethylene and auxin molecular networks were recruited to build the plant body plan in ancestral land plants. This might have played a role in enabling ancient plants to acclimate to the continental surfaces of the planet.

A phytochrome/phototropin chimeric photoreceptor promotes growth of fern gametophytes under limited light conditions.

Many ferns thrive even in low-light niches such as under an angiosperm forest canopy. However, the shade adaptation strategy of ferns is not well understood. Phytochrome 3/neochrome (phy3/neo) is an unconventional photoreceptor, found in the fern Adiantum capillus-veneris, that controls both red and blue light-dependent phototropism and chloroplast photorelocation, which are considered to improve photosynthetic efficiency in ferns. Here we show that phy3/neo localizes not only at the plasma membrane but also in the nucleus. Since both phototropism and chloroplast photorelocation are mediated by membrane-associated phototropin photoreceptors, we speculated that nucleus-localized phy3/neo possesses a previously undescribed biological function. We reveal that phy3/neo directly interacts with Adiantum cryptochrome 3 (cry3) in the nucleus. Plant cryptochromes are blue light receptors that transcriptionally regulate photomorphogenesis; therefore, phy3/neo may function via cry3 to synchronize light-mediated development with phototropism and chloroplast photorelocation to promote fern growth under low-light conditions. Furthermore, we demonstrate that phy3/neo regulates the expression of the Cyclin-like gene AcCyc1 and promotes prothallium expansion growth. These findings provide insight into the shade adaptation strategy of ferns and suggest that phy3/neo plays a substantial role in the survival and growth of ferns during the tiny gametophytic stage under low-light conditions, such as those on the forest floor.

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.

Projecting kelp (Ecklonia radiata) gametophyte thermal adaptation and persistence under climate change.

BACKGROUND AND AIMS: Kelp forests underpin temperate marine ecosystems but are declining due to ocean warming, causing loss of associated ecosystem services. Projections suggest significant future decline but often only consider the persistence of adult sporophytes. Kelps have a biphasic life cycle, and the haploid gametophyte can be more thermally tolerant than the sporophyte. Therefore, projections may be altered when considering the thermal tolerance of gametophytes. METHODS: We undertook thermal tolerance experiments to quantify the effect of temperature on gametophyte survival, relative growth rate (RGR) and sex ratio for three genetically distinct populations of Ecklonia radiata gametophytes from comparatively high, mid- and low latitudes (43°, 33° and 30°S). We then used these data to project the likely consequences of climate-induced thermal change on gametophyte persistence and performance across its eastern Australian range, using generalized additive and linear models. KEY RESULTS: All populations were adapted to local temperatures and their thermal maximum was 2-3 °C above current maximum in situ temperatures. The lowest latitude population was most thermally tolerant (~70 % survival up to 27 °C), while survival and RGR decreased beyond 25.5 and 20.5 °C for the mid- and low-latitude populations, respectively. Sex ratios were skewed towards females with increased temperature in the low- and high-latitude populations. Spatially explicit model projections under future ocean warming (2050-centred) revealed a minimal decline in survival (0-30 %) across populations, relative to present-day predictions. RGRs were also projected to decline minimally (0-2 % d-1). CONCLUSIONS: Our results contrast with projections for the sporophyte stage of E. radiata, which suggest a 257-km range contraction concurrent with loss of the low-latitude population by 2100. Thermal adaptation in E. radiata gametophytes suggests this life stage is likely resilient to future ocean warming and is unlikely to be a bottleneck for the future persistence of kelp.

Thermal performance curves identify seasonal and site-specific variation in the development of Ecklonia radiata (Phaeophyceae) gametophytes and sporophytes.

Rapid ocean warming is affecting kelp forests globally. While the sporophyte life stage has been well studied for many species, the microscopic life stages of laminarian kelps have been understudied, particularly regarding spatial and temporal variations in thermal tolerance and their interaction. We investigated the thermal tolerance of growth, survival, development, and fertilization of Ecklonia radiata gametophytes, derived from zoospores sampled from two sites in Tasmania, Australia, throughout a year, over a temperature gradient (3-30°C). For growth we found a relatively stable thermal optimum at ~20.5°C and stable thermal maxima (25.3-27.7°C). The magnitude of growth was highly variable and depended on season and site, with no consistent spatial pattern for growth and gametophyte size. Survival also had a relatively stable thermal optimum of ~17°C, 3°C below the optimum for growth. Gametophytes grew to single cells between 5 and 25°C, but sporophytes were only observed between 10 and 20°C, indicating reproductive failure outside this range. The results reveal complex effects of source population and season of collection on gametophyte performance in E. radiata, with implications when comparing results from material collected at different localities and times. In Tasmania, gametophytes grow considerably below the estimated thermal maxima and thermal optima that are currently only reached during summer heatwaves, whereas optima for survival (~17°C) are frequently reached and surpassed during heatwaves, which may affect the persistence and recruitment of E. radiata in a warmer climate.

Identification of reproductive sex-biased gene expression in Asparagopsis taxiformis (lineage 6) gametophytes.

The sub-tropical red seaweed Asparagopsis taxiformis is of significant interest due to its ability to store halogenated compounds, including bromoform, which can mitigate methane production in ruminants. Significant scale-up of aquaculture production of this seaweed is required; however, relatively little is known about the molecular mechanisms that control fundamental physiological processes, including the regulatory factors that determine sexual dimorphism in gametophytes. In this study, we used comparative RNA-sequencing analysis between different morphological parts of mature male and female A. taxiformis (lineage 6) gametophytes that resulted in greater number of sex-biased gene expression in tips (containing the reproductive structures for both sexes), compared with the somatic main axis and rhizomes. Further comparative RNA-seq against immature tips was used to identify 62 reproductive sex-biased genes (59 male-biased, 3 female-biased). Of the reproductive male-biased genes, 46% had an unknown function, while others were predicted to be regulatory factors and enzymes involved in signaling. We found that bromoform content obtained from female samples (8.5 ± 1.0 mg·g-1 dry weight) was ~10% higher on average than that of male samples (6.5 ± 1.0 mg·g-1 dry weight), although no significant difference was observed (p > 0.05). There was also no significant difference in the marine bromoform biosynthesis locus gene expression. In summary, our comparative RNA-sequencing analysis provides a first insight into the potential molecular factors relevant to gametogenesis and sexual differentiation in A. taxiformis, with potential benefits for identification of sex-specific markers.

Gametophytic epigenetic regulators, MEDEA and DEMETER, synergistically suppress ectopic shoot formation in Arabidopsis.

KEY MESSAGE: The gametophytic epigenetic regulators, MEA and DME, extend their synergistic role to the sporophytic development by regulating the meristematic activity via restricting the gene expression in the shoot apex. The gametophyte-to-sporophyte transition facilitates the alternation of generations in a plant life cycle. The epigenetic regulators DEMETER (DME) and MEDEA (MEA) synergistically control central cell proliferation and differentiation, ensuring proper gametophyte-to-sporophyte transition in Arabidopsis. Mutant alleles of DME and MEA are female gametophyte lethal, eluding the recovery of recessive homozygotes to examine their role in the sporophyte. Here, we exploited the paternal transmission of these mutant alleles coupled with CENH3-haploid inducer to generate mea-1;dme-2 sporophytes. Strikingly, the simultaneous loss of function of MEA and DME leads to the emergence of ectopic shoot meristems at the apical pole of the plant body axis. DME and MEA are expressed in the developing shoot apex and regulate the expression of various shoot-promoting factors. Chromatin immunoprecipitation (ChIP), DNA methylation, and gene expression analysis revealed several shoot regulators as potential targets of MEA and DME. RNA interference-mediated transcriptional downregulation of shoot-promoting factors STM, CUC2, and PLT5 rescued the twin-plant phenotype to WT in 9-23% of mea-1-/-;dme-2-/- plants. Our findings reveal a previously unrecognized synergistic role of MEA and DME in restricting the meristematic activity at the shoot apex during sporophytic development.

Unreduced spore formation in a spontaneous chimeric pinnule in an artificially produced haploid Anisocampium niponicum (Athyriaceae, Polypodiales).

Haploid sporophytes of Anisocampium niponicum with 2n = 40, were produced artificially by induced apogamy in vitro. They were subsequently transplanted into pots and two of them have been cultivated for the investigation of sporogenesis and/or production of chimera for more than 20 years. Haploid A. niponicum is sterile, but an abnormal chimeric pinnule that developed spontaneously in a single frond produced sporangia with spores. Each sporangium bore approximately 32 spores that were almost uniform in size. Sowing of these spores resulted in 50 gametophytes. Of 20 gametophytes cultured individually, five produced sporophytes apogamously after eight months. Both the gametophytes and subsequent apogamous sporophytes showed a chromosome number of 2n = 40. Our study demonstrates that a haploid sporophyte offspring can be produced from a haploid mother sporophyte via haploid spores. Since asexual reproduction is a prominent evolutionary process in ferns, the reproduction of a haploid A. niponicum sporophyte by unreduced spore formation might help to elucidate how apogamous ferns occur and evolve.