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.

Fertility loss in senescing Arabidopsis ovules is controlled by the maternal sporophyte via a NAC transcription factor triad.

Flowers have a species-specific fertile period during which pollination and fertilization have to occur to initiate seed and fruit development. Unpollinated flowers remain receptive for mere hours in some species, and up to several weeks in others before flower senescence terminates fertility. As such, floral longevity is a key trait subject to both natural selection and plant breeding. Within the flower, the life span of the ovule containing the female gametophyte is decisive for fertilization and the initiation of seed development. Here, we show that unfertilized ovules in Arabidopsis thaliana undergo a senescence program that generates morphological and molecular hallmarks of canonical programmed cell death processes in the sporophytically derived ovule integuments. Transcriptome profiling of isolated aging ovules revealed substantial transcriptomic reprogramming during ovule senescence, and identified up-regulated transcription factors as candidate regulators of these processes. Combined mutation of three most-up-regulated NAC (NAM, ATAF1/2, and CUC2) transcription factors, NAP/ANAC029, SHYG/ANAC047, and ORE1/ANAC092, caused a substantial delay in ovule senescence and an extension of fertility in Arabidopsis ovules. These results suggest that timing of ovule senescence and duration of gametophyte receptivity are subject to genetic regulation controlled by the maternal sporophyte.

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.

The molecular chaperone mtHSC70-1 interacts with DjA30 to regulate female gametophyte development and fertility in Arabidopsis.

Arabidopsis mitochondria-targeted heat shock protein 70 (mtHSC70-1) plays important roles in the establishment of cytochrome c oxidase-dependent respiration and redox homeostasis during the vegetative growth of plants. Here, we report that knocking out the mtHSC70-1 gene led to a decrease in plant fertility; the fertility defect of the mutant was completely rescued by introducing the mtHSC70-1 gene. mtHSC70-1 mutants also showed defects in female gametophyte (FG) development, including delayed mitosis, abnormal nuclear position, and ectopic gene expression in the embryo sacs. In addition, we found that an Arabidopsis mitochondrial J-protein gene (DjA30) mutant, j30+/- , had defects in FG development and fertility similar to those of mtHSC70-1 mutant. mtHSC70-1 and DjA30 had similar expression patterns in FGs and interacted in vivo, suggesting that these two proteins might cooperate during female gametogenesis. Further, respiratory chain complex IV activity in mtHSC70-1 and DjA30 mutant embryo sacs was markedly downregulated; this led to the accumulation of mitochondrial reactive oxygen species (ROS). Scavenging excess ROS by introducing Mn-superoxide dismutase 1 or catalase 1 gene into the mtHSC70-1 mutant rescued FG development and fertility. Altogether, our results suggest that mtHSC70-1 and DjA30 are essential for the maintenance of ROS homeostasis in the embryo sacs and provide direct evidence for the roles of ROS homeostasis in embryo sac maturation and nuclear patterning, which might determine the fate of gametic and accessory cells.

Female gametophyte development is required for nucellar-tip degeneration during Arabidopsis ovule development.

MAIN CONCLUSION: Genetic ablation of the female gametophyte provides direct evidence for the existence of interregional communication during Arabidopsis ovule development and the importance of the female gametophyte in nucellar-tip degeneration. The angiosperm ovule consists of three regions: the female gametophyte, the nucellus, and the integuments, all of which develop synchronously and coordinately. Previously, interregional communication enabling cooperative ovule development had been proposed; however, the evidence for these communications mostly relies on the analysis of mutant phenotypes. To provide direct evidence, we specifically ablated the Arabidopsis female gametophyte by expressing the diphtheria toxin fragment A (DTA) under the female gametophyte-specific DD13 promoter and analyzed its effects on the development of the nucellus and the integuments. We found that the female gametophyte is not required for integument development or for the orientation and curvature of the ovule body, but is necessary for nucellar-tip degeneration. The results presented here provide direct evidence for communication from the female gametophyte to the nucellus and demonstrate that Arabidopsis ovules require interregional communication for cooperative development.

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.

Polyploidy and environmental stress response: a comparative study of fern gametophytes.

Climate change is rapidly altering natural habitats and generating complex patterns of environmental stress. Ferns are major components of many forest understories and, given their independent gametophyte generation, may experience unique pressures in emerging temperature and drought regimes. Polyploidy is widespread in ferns and may provide a selective advantage in these rapidly changing environments. This work aimed to understand whether the gametophytes of allopolyploid ferns respond differently to climate-related physiological stress than their diploid parents. The experimental approach involved a multifactorial design with 27 treatment combinations including exposure to multiple levels of drought and temperature over three treatment durations, with recovery measured at multiple timepoints. We measured Chl fluorescence from over 2000 gametophytes to evaluate stress avoidance and tolerance in diploid and polyploid species. Polyploids generally showed a greater ability to avoid and/or tolerate a range of stress conditions compared with their diploid counterparts, suggesting that polyploidy may confer enhanced flexibility and resilience under climate stress. Overall, these results suggest that polyploidy may provide some resilience to climate change in mixed ploidy populations. However, all species remain susceptible to the impacts of extreme drought and heat stress.

A word of caution: T-DNA-associated mutagenesis in plant reproduction research.

T-DNA transformation is prevalent in Arabidopsis research and has expanded to a broad range of crops and model plants. While major progress has been made in optimizing the Agrobacterium-mediated transformation process for various species, a variety of pitfalls associated with the T-DNA insertion may lead to the misinterpretation of T-DNA mutant analysis. Indeed, secondary mutagenesis either on the integration site or elsewhere in the genome, together with epigenetic interactions between T-DNA inserts or frequent genomic rearrangements, can be tricky to differentiate from the effect of the knockout of the gene of interest. These are mainly the case for genomic rearrangements that become balanced in filial generations without consequential phenotypical defects, which may be confusing particularly for studies that aim to investigate fertility and gametogenesis. As a cautionary note to the plant research community studying gametogenesis, we here report an overview of the consequences of T-DNA-induced secondary mutagenesis with emphasis on the genomic imbalance on gametogenesis. Additionally, we present a simple guideline to evaluate the T-DNA-mutagenized transgenic lines to decrease the risk of faulty analysis with minimal experimental effort.

Development of pollinated and unpollinated ovules in Ginkgo biloba: unravelling the role of pollen in ovule tissue maturation.

In gymnosperms such as Ginkgo biloba, the arrival of pollen plays a key role in ovule development, before fertilization occurs. Accordingly, G. biloba female plants geographically isolated from male plants abort all their ovules after the pollination drop emission, which is the event that allows the ovule to capture pollen grains. To decipher the mechanism induced by pollination required to avoid ovule senescence and then abortion, we compared the transcriptomes of pollinated and unpollinated ovules at three time points after the end of the emission of pollination drop. Transcriptomic and in situ expression analyses revealed that several key genes involved in programmed cell death such as senescence and apoptosis, DNA replication, and cell cycle regulation were differentially expressed in unpollinated ovules compared to pollinated ovules. We provide evidence that the pollen captured by the pollination drop affects auxin local accumulation and might cause deregulation of key genes required for the ovule's programmed cell death, activating both the cell cycle regulation and DNA replication genes.

Wheat ESCRT-III protein TaSAL1 regulates male gametophyte transmission and controls tillering and heading date.

Charged multivesicular protein 1 (CHMP1) is a member of the endosomal sorting complex required for transport-III (ESCRT-III) complex that targets membrane localized signaling receptors to intralumenal vesicles in the multivesicular body of the endosome and eventually to the lysosome for degradation. Although CHMP1 plays roles in various plant growth and development processes, little is known about its function in wheat. In this study, we systematically analysed the members of the ESCRT-III complex in wheat (Triticum aestivum) and found that their orthologs were highly conserved in eukaryotic evolution. We identified CHMP1 homologous genes, TaSAL1s, and found that they were constitutively expressed in wheat tissues and essential for plant reproduction. Subcellular localization assays showed these proteins aggregated with and closely associated with the endoplasmic reticulum when ectopically expressed in tobacco leaves. We also found these proteins were toxic and caused leaf death. A genetic and reciprocal cross analysis revealed that TaSAL1 leads to defects in male gametophyte biogenesis. Moreover, phenotypic and metabolomic analysis showed that TaSAL1 may regulate tillering and heading date through phytohormone pathways. Overall, our results highlight the role of CHMP1 in wheat, particularly in male gametophyte biogenesis, with implications for improving plant growth and developing new strategies for plant breeding and genetic engineering.

OsRH52A, a DEAD-box protein, regulates functional megaspore specification and is required for embryo sac development in rice.

The development of the embryo sac is an important factor that affects seed setting in rice. Numerous genes associated with embryo sac (ES) development have been identified in plants; however, the function of the DEAD-box RNA helicase family genes is poorly known in rice. Here, we characterized a rice DEAD-box protein, RH52A, which is localized in the nucleus and cytoplasm and highly expressed in the floral organs. The knockout mutant rh52a displayed partial ES sterility, including degeneration of the ES (21%) and the presence of a double-female-gametophyte (DFG) structure (11.8%). The DFG developed from two functional megaspores near the chalazal end in one ovule, and 3.4% of DFGs were able to fertilize via the sac near the micropylar pole in rh52a. RH52A was found to interact with MFS1 and ZIP4, both of which play a role in homologous recombination in rice meiosis. RNA-sequencing identified 234 down-regulated differentially expressed genes associated with reproductive development, including two, MSP1 and HSA1b, required for female germline cell specification. Taken together, our study demonstrates that RH52A is essential for the development of the rice embryo sac and provides cytological details regarding the formation of DFGs.

Polycomb proteins RING1A/B promote H2A monoubiquitination to regulate female gametophyte development in Arabidopsis.

A functional female gametophyte is the basis of successful sexual reproduction in flowering plants. During female gametophyte development, the megaspore mother cell (MMC), which differentiates from a single subepidermal somatic cell in the nucellus, undergoes meiosis to produce four megaspores; only the one at the chalazal end, referred to as the functional megaspore (FM), then undergoes three rounds of mitosis and develops into a mature embryo sac. Here, we report that RING1A and RING1B (RING1A/B), two functionally redundant Polycomb proteins in Arabidopsis, are critical for female gametophyte development. Mutations of RING1A/B resulted in defects in the specification of the MMC and the FM, and in the subsequent mitosis of the FM, thereby leading to aborted ovules. Detailed analysis revealed that several genes essential for female gametophyte development were ectopically expressed in the ring1a ring1b mutant, including Argonaute (AGO) family genes and critical transcription factors. Furthermore, RING1A/B bound to some of these genes to promote H2A monoubiquitination (H2Aub). Taken together, our study shows that RING1A/B promote H2Aub modification at key genes for female gametophyte development, suppressing their expression to ensure that the development progresses correctly.

An adventurous journey toward and away from fern apomixis: Insights from genome size and spore abortion patterns.

PREMISE: Apomixis in ferns is relatively common and obligatory. Sterile hybrids may restore fertility via apomixis at a cost of long-term genetic stagnation. In this study, we outlined apomixis as a possible temporary phase leading to sexuality and analyzed factors relating to transitioning to and away from apomixis, such as unreduced and reduced spore formation in apomict and apo-sex hybrid ferns. METHODS: We analyzed the genome size of 15 fern species or hybrids ("taxa") via flow cytometry. The number of reduced and unreduced gametophytes was established as a proxy for viable spore formation of either type. We also calculated the spore abortion ratio (sign of reduced spores) in several taxa, including the apo-sex hybrid Dryopteris × critica and its 16 apomictically formed offspring. RESULTS: Four of 15 sampled taxa yielded offspring variable in genome size. Specifically, each variable taxon formed one viable reduced plant among 12-451 sampled gametophytes per taxon. Thus, haploid spore formation in the studied apomicts was very rare but possible. Spore abortion analyses indicated gradually decreasing abortion (haploid spore formation) over time. In Dryopteris × critica, abortion decreased from 93.8% to mean 89.5% in one generation. CONCLUSIONS: Our results support apomixis as a transitionary phase toward sexuality. Newly formed apomicts hybridize with sexual relatives and continue to form haploid spores early on. Thus, they may get the genomic content necessary for regular meiosis and restore sexuality. If the missing relative goes extinct, the lineage gets locked into apomixis as may be the case with the Dryopteris affinis complex.

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.

Morphological and functional evolution of gametophytes in epilithic Hymenasplenium murakami-hatanakae (Aspleniaceae): The fifth family capable of producing the independent gametophytes.

The fern independent gametophytes that can maintain populations by vegetative reproduction without conspecific sporophytes have been considered an unusual phenomenon found in some epiphytic or epilithic species of Hymenophyllaceae, Pteridaceae, Lomariopsidaceae, and Polypodiaceae. By chance, the discovery of mysterious strap-like gametophytes on Izu-Oshima Island, Japan, has led to the hypothesis that Hymenasplenium murakami-hatanakae, a fern species belonging to Aspleniaceae, can also form independent gametophytes. Our investigation revealed gametophyte populations of H. murakami-hatanakae on three islands in the Izu Islands. Based on chloroplast DNA analysis of the gametophyte and sporophyte populations, the gametophytes were found to be maintained by vegetative reproduction without a new supply of spores from sporophytes. A comparison of the surrounding vegetation at the collection sites showed that environmental factors such as light and humidity may influence the maintenance of gametophyte populations. These results clearly show that H. murakami-hatanakae is one of the ferns capable of forming independent gametophytes. This is the first report of independent gametophytes from the suborder Aspleniineae (eupolypod II). The discovery of the independent gametophyte within a phylogenetic lineage previously thought not to form independent gametophytes will provide important insights into the morphological and functional evolution of gametophytes in ferns.

Terrestrial mosses as a substrate and potential host for cyanobacteria, green algae and diatoms.

Most studies of terrestrial bryophytes as natural substrates for photosynthetic microorganisms have been performed in the polar regions, where bryophytes are an important part of the ecosystem. As they remain green throughout the year, bryophytes may also be an ideal substrate for epiphytic organisms in temperate regions. The present study investigated the colonization potential and diversity of microalgae on selected plant species in riparian forest and spruce monoculture in a temperate region. It examines whether the presence of algae is related to substrate humidity, the micromorphology of gametophyte or the seasonal availability of substrate. The taxonomic diversity of algae was studied. Cyanobacteria and green algae were cultured on BG-11 agar medium, while diatoms were identified in permanent diatomaceous slides. The alpha- and beta-diversity indices were calculated, and the communities were compared using Bray-Curtis distances and multidimensional correspondence analyses. Our findings indicate that the largest number of alga species were diatoms; however, their presence was only observed in riparian forest and was associated with high humidity. Both aerophilic and freshwater taxa were noted, the latter carried by water from nearby aquatic ecosystem. Green algae were present in both phytocoenoses and humidity appears to have no substantial effect on the degree of colonization; their diversity was low and the group consisted of terrestrial taxa. In two bryophytes growing at the highest humidity, cyanobacteria were only identified in culture. The key factor influencing the degree of microalgae colonization was the humidity of the substrate, which was related to the distance from water.

Transcription Factors and Their Regulatory Roles in the Male Gametophyte Development of Flowering Plants.

Male gametophyte development in plants relies on the functions of numerous genes, whose expression is regulated by transcription factors (TFs), non-coding RNAs, hormones, and diverse environmental stresses. Several excellent reviews are available that address the genes and enzymes associated with male gametophyte development, especially pollen wall formation. Growing evidence from genetic studies, transcriptome analysis, and gene-by-gene studies suggests that TFs coordinate with epigenetic machinery to regulate the expression of these genes and enzymes for the sequential male gametophyte development. However, very little summarization has been performed to comprehensively review their intricate regulatory roles and discuss their downstream targets and upstream regulators in this unique process. In the present review, we highlight the research progress on the regulatory roles of TF families in the male gametophyte development of flowering plants. The transcriptional regulation, epigenetic control, and other regulators of TFs involved in male gametophyte development are also addressed.

Genomic Signatures of Sexual Selection on Pollen-Expressed Genes in Arabis alpina.

Fertilization in angiosperms involves the germination of pollen on the stigma, followed by the extrusion of a pollen tube that elongates through the style and delivers two sperm cells to the embryo sac. Sexual selection could occur throughout this process when male gametophytes compete for fertilization. The strength of sexual selection during pollen competition should be affected by the number of genotypes deposited on the stigma. As increased self-fertilization reduces the number of mating partners, and the genetic diversity and heterozygosity of populations, it should thereby reduce the intensity of sexual selection during pollen competition. Despite the prevalence of mating system shifts, few studies have directly compared the molecular signatures of sexual selection during pollen competition in populations with different mating systems. Here we analyzed whole-genome sequences from natural populations of Arabis alpina, a species showing mating system variation across its distribution, to test whether shifts from cross- to self-fertilization result in molecular signatures consistent with sexual selection on genes involved in pollen competition. We found evidence for efficient purifying selection on genes expressed in vegetative pollen, and overall weaker selection on sperm-expressed genes. This pattern was robust when controlling for gene expression level and specificity. In agreement with the expectation that sexual selection intensifies under cross-fertilization, we found that the efficacy of purifying selection on male gametophyte-expressed genes was significantly stronger in genetically more diverse and outbred populations. Our results show that intra-sexual competition shapes the evolution of pollen-expressed genes, and that its strength fades with increasing self-fertilization rates.

Three-Dimensional Morphological Analysis Revealed the Cell Patterning Bases for the Sexual Dimorphism Development in the Liverwort Marchantia polymorpha.

In land plants, sexual dimorphism can develop in both diploid sporophytes and haploid gametophytes. While developmental processes of sexual dimorphism have been extensively studied in the sporophytic reproductive organs of model flowering plants such as stamens and carpels of Arabidopsis thaliana, those occurring in gametophyte generation are less well characterized due to the lack of amenable model systems. In this study, we performed three-dimensional morphological analyses of gametophytic sexual branch differentiation in the liverwort Marchantia polymorpha, using high-depth confocal imaging and a computational cell segmentation technique. Our analysis revealed that the specification of germline precursors initiates in a very early stage of sexual branch development, where incipient branch primordia are barely recognizable in the apical notch region. Moreover, spatial distribution patterns of germline precursors differ between males and females from the initial stage of primordium development in a manner dependent on the master sexual differentiation regulator MpFGMYB. At later stages, distribution patterns of germline precursors predict the sex-specific gametangia arrangement and receptacle morphologies seen in mature sexual branches. Taken together, our data suggest a tightly coupled progression of germline segregation and sexual dimorphism development in M. polymorpha.

Sporogenesis, gametophyte development and embryogenesis in Glehnia littoralis.

BACKGROUND: Glehnia littoralis is an economic herb with both medicinal and edible uses. It also has important ecological value and special phylogenetic status as it is a monotypic genus species distributing around beach. Little information on its reproductive biology has been reported so far, which has hindered conservation and application of this species. In this study, we observed morphological changes from buds emergence to seeds formation and internal changes during sporogenesis, gametophyte development and embryo and endosperm development of G. littoralis using paraffin-embedded-sectioning and stereo microscope. RESULTS: The results showed that the stages of internal development events of G. littoralis corresponded to obvious external morphological changes, most of developmental features were consistent with other Apiaceae species. The development of male and female gametophytes was not synchronized in the same flower, however, exhibited temporal overlap. From mid-late April to mid-May, the anther primordial and ovule primordial developed into the trinucleate pollen grain and eight-nuclear embryo sac, respectively. From late-May to mid-July, the zygote developed into mature embryo. In addition, some defects in gynoecium or ovule development and abnormal embryo and endosperm development were found. We induced that the possible causes of abortion in G. littoralis were as follows: nutrient limitation, poor pollination and fertilization, and bad weather. CONCLUSIONS: This study revealed the whole process and morphological characteristics of the development of reproductive organ in G. littoralis, which not only provided important data for the study of systematic and conservation biology, but also provided a theoretical basis for cross breeding.