ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization.

The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars.

Dual and opposing roles of EIN3 reveal a generation conflict during seed growth.

Seed size critically affects grain yield of crops and hence represents a key breeding target. The development of embryo-nourishing endosperm is a key driver of seed expansion. We here report unexpected dual roles of the transcription factor EIN3 in regulating seed size. These EIN3 functions have remained largely undiscovered because they oppose each other. Capitalizing on the analysis of multiple ethylene biosynthesis mutants, we demonstrate that EIN3 represses endosperm and seed development in a pathway regulated by ethylene. We, in addition, provide evidence that EIN3-mediated synergid nucleus disintegration promotes endosperm expansion. Interestingly, synergid nucleus disintegration is not affected in various ethylene biosynthesis mutants, suggesting that this promoting function of EIN3 is independent of ethylene. Whereas the growth-inhibitory ethylene-dependent EIN3 action appears to be encoded by sporophytic tissue, the growth-promoting role of EIN3 is induced by fertilization, revealing a generation conflict that converges toward the key signaling component EIN3.

Noninvasive Determination of CMV Serostatus From Dried Buccal Swab Samples: Assay Development, Validation, and Application to 1.2 Million Samples.

BACKGROUND: Buccal swab sampling constitutes an attractive noninvasive alternative to blood drawings for antibody serostatus assays. Here we describe a method to determine the cytomegalovirus immunoglobulin G (CMV IgG) serostatus from dried buccal swab samples. METHODS: Upon solubilization, CMV IgG is determined by an ELISA assay specifically adapted to cope with low IgG concentrations. The derived CMV titer is normalized against the total protein concentration to adjust for incorrectly or less efficiently sampled buccal swabs. Assay parameters were optimized on a set of 713 samples. RESULTS: Validation with 1784 samples revealed distinct results for > 80% of samples with 98.6% specificity and 99.1% sensitivity. Based on the analysis of 1.2 million samples we derived age- and sex-stratified CMV prevalence statistics for Germany, Poland, United Kingdom, and Chile. To confirm accuracy of the assay in routine operation, the CMV status of 6518 donors was reassessed by independent laboratories based on conventional blood samples revealing 96.9% specificity and 97.4% sensitivity. CONCLUSIONS: The assay accurately delivers the CMV IgG serostatus from dried buccal swab samples for > 80% of the participants. Thereby it provides a noninvasive alternative to plasma-based CMV monitoring for nondiagnostic purposes such as hematopoietic stem cell transplantation donor screening or population studies.

Reproductive Multitasking: The Female Gametophyte.

Fertilization of flowering plants requires the organization of complex tasks, many of which become integrated by the female gametophyte (FG). The FG is a few-celled haploid structure that orchestrates division of labor to coordinate successful interaction with the sperm cells and their transport vehicle, the pollen tube. As reproductive outcome is directly coupled to evolutionary success, the underlying mechanisms are under robust molecular control, including integrity check and repair mechanisms. Here, we review progress on understanding the development and function of the FG, starting with the functional megaspore, which represents the haploid founder cell of the FG. We highlight recent achievements that have greatly advanced our understanding of pollen tube attraction strategies and the mechanisms that regulate plant hybridization and gamete fusion. In addition, we discuss novel insights into plant polyploidization strategies that expand current concepts on the evolution of flowering plants.

Selective egg cell polyspermy bypasses the triploid block.

Polyploidization, the increase in genome copies, is considered a major driving force for speciation. We have recently provided the first direct in planta evidence for polyspermy induced polyploidization. Capitalizing on a novel sco1-based polyspermy assay, we here show that polyspermy can selectively polyploidize the egg cell, while rendering the genome size of the ploidy-sensitive central cell unaffected. This unprecedented result indicates that polyspermy can bypass the triploid block, which is an established postzygotic polyploidization barrier. In fact, we here show that most polyspermy-derived seeds are insensitive to the triploid block suppressor admetos. The robustness of polyspermy-derived plants is evidenced by the first transcript profiling of triparental plants and our observation that these idiosyncratic organisms segregate tetraploid offspring within a single generation. Polyspermy-derived triparental plants are thus comparable to triploids recovered from interploidy crosses. Our results expand current polyploidization concepts and have important implications for plant breeding.

Ever since Darwin published his most famous book on the theory of evolution, scientists have sought to identify the mechanisms that drive the formation of new species. This is especially true for plant biologists who have long been fascinated by the extraordinary diversity of flowering plants.Many species of flowering plant first evolved after a dramatic increase in the DNA content of an individual plant, a process termed polyploidization. Most explanations for polyploidization involve a pollen grain making sperm that mistakenly contain two sets of chromosomes rather than one. Yet, it is difficult to reconcile this explanation with an important aspect of plant reproduction ­ the so-called "triploid block".Fertilization in flowering plants is more complicated than in animals. While one sperm fertilizes the egg cell to make the plant embryo, a second sperm from the same pollen grain must fertilize another cell to form the endosperm, the tissue that will nourish the embryo as it develops. This means that sperm with twice the normal number of chromosomes would affect the DNA content of both the embryo and the endosperm. Yet, an endosperm that receives extra paternal DNA typically halts the development of the seed via a process known as the triploid block, meaning it was not clear how often this process would actually result in a polyploid plant.In 2017, researchers reported that plants can, on rare occasions, generate polyploid offspring via a different route: the fertilization of one egg with two sperm rather than one. Now, Mao et al. ­ who include several researchers involved in the 2017 study ­ show that this process, termed "polyspermy", can introduce extra copies of DNA into just the egg cell, meaning it can bypass the triploid block of the endosperm.The experiments involved a model plant called Arabidopsis, and a screen of over 55,000 seeds identified about a dozen with embryos that had three parents, one mother and two fathers. Notably, most of these three-parent embryos developed in seeds that contained endosperm with the regular number of chromosomes and hence escaped the triploid block.These new results show that polyspermy provides plants with a means to essentially sneak extra copies of DNA 'behind the back' of the DNA-sensitive endosperm and into the next generation. They also give new insight in how polyploidization may have shaped the evolution of flowering plants and have important implications for agriculture where the breeding of new "hybrid" crops has often been limited by incompatibilities in the endosperm.

New advances and future directions in plant polyspermy.

Plants have evolved a battery of mechanisms that potentially act as polyspermy barriers. Supernumerary sperm fusion to one egg cell has consequently long remained a hypothetical concept. The recent discovery that polyspermy in flowering plants is not lethal but generates viable triploid plants is a game changer affecting the field of developmental biology, evolution, and plant breeding. The establishment of protocols to artificially induce polyspermy together with the development of a high-throughput assay to identify and trace polyspermic events in planta now provide powerful tools to unravel mechanisms of polyspermy regulation. These achievements are likely to open new avenues for animal polyspermy research as well, where forward genetic approaches are hampered by the fatal outcome of supernumerary sperm fusion.

Development and function of the flowering plant female gametophyte.

Flowering plants constitute an indispensable basis for the existence of most organisms, including humans. In a world characterized by rapid population growth and climate changes, understanding plant reproduction becomes increasingly important in order to respond to the resource shortage associated with this development. New technologies enabling powerful forward genetic approaches, comprehensive genome and transcriptome analyses, and sophisticated cell isolation and imaging have advanced our understanding of the molecular mechanisms underlying gamete formation and fertilization. In addition, these techniques have allowed us to explore the fascinating cellular crosstalk, which coordinates the intra- and interorganismic interactions that secure reproductive success. Here we review the basic principles underlying development of the germ cell-harboring female gametophyte in flowering plants. We start with the selection of the founder cells and end with the formation of a few-celled, highly specialized structure that operates on the basis of division of labor in order to generate the next generation.

Publisher Correction: Triparental plants provide direct evidence for polyspermy induced polyploidy.

This Article contained errors in Fig. 3 that were brought to our attention by the authors during the production process but, inadvertently, were not corrected before publication. The tick marks on the y-axis in panels b, f, and k, and the median line in the box-and-whisker plot for biparental diploid plants (BP) in panel i were shifted downwards by up to 2 mm. This has now been corrected in both the PDF and HTML versions of the Article.

Triparental plants provide direct evidence for polyspermy induced polyploidy.

It is considered an inviolable principle that sexually reproducing organisms have no more than two parents and fertilization of an egg by multiple sperm (polyspermy) is lethal in many eukaryotes. In flowering plants polyspermy has remained a hypothetical concept, due to the lack of tools to unambiguously identify and trace this event. We established a high-throughput polyspermy detection assay, which uncovered that supernumerary sperm fusion does occur in planta and can generate viable polyploid offspring. Moreover, polyspermy can give rise to seedlings with one mother and two fathers, challenging the bi-organismal concept of parentage. The polyspermy derived triploids are taller and produce bigger organs than plants resulting from a regular monospermic fertilization. In addition, we demonstrate the hybridization potential of polyspermy by instantly combining three different Arabidopsis accessions in one zygote. Our results provide direct evidence for polyspermy as a route towards polyploidy, which is considered a major plant speciation mechanism.

Molecular movement in the Arabidopsis thaliana female gametophyte.

KEY MESSAGE: Size limits on molecular movement among female gametes. Cellular decisions can be influenced by information communicated from neighboring cells. Communication can occur via signaling or through the direct transfer of molecules. Movement of RNAs and proteins has frequently been observed among symplastically connected plant cells. In flowering plants, the female gametes, the egg cell and central cell, are closely apposed within the female gametophyte. Here we investigated the ability of fluorescently labeled dyes and small RNAs to move from the Arabidopsis thaliana central cell to the egg apparatus following microinjection. These results define a size limit of at least 20 kDa for symplastic movement between the two gametes, somewhat larger than that previously observed in Torenia fournieri. Our results indicate that symplastic connectivity in Arabidopsis thaliana changes after fertilization and suggest that prior to fertilization mechanisms are in place to facilitate small RNA movement from the central cell to the egg cell and synergids.

Polyspermy barriers: a plant perspective.

A common denominator of sexual reproduction in many eukaryotic species is the exposure of an egg to excess sperm to maximize the chances of reproductive success. To avoid potential harmful or deleterious consequences of supernumerary sperm fusion to a single female gamete (polyspermy), many eukaryotes, including plants, have evolved barriers preventing polyspermy. Typically, these checkpoints are implemented at different stages in the reproduction process. The virtual absence of unambiguous reports of naturally occurring egg cell polyspermy in flowering plants is likely reflecting the success of this multiphasic strategy and highlights the difficulty to trace this presumably rare event. We here focus on potential polyspermy avoidance mechanisms in plants and discuss them in light of analogous processes in animals.

Live and let die: a REM complex promotes fertilization through synergid cell death in Arabidopsis.

Fertilization in flowering plants requires a complex series of coordinated events involving interaction between the male and female gametophyte. We report here molecular data on one of the key events underpinning this process - the death of the receptive synergid cell and the coincident bursting of the pollen tube inside the ovule to release the sperm. We show that two REM transcription factors, VALKYRIE (VAL) and VERDANDI (VDD), both targets of the ovule identity MADS-box complex SEEDSTICK-SEPALLATA3, interact to control the death of the receptive synergid cell. In vdd-1/+ mutants and VAL_RNAi lines, we find that GAMETOPHYTIC FACTOR 2 (GFA2), which is required for synergid degeneration, is downregulated, whereas expression of FERONIA (FER) and MYB98, which are necessary for pollen tube attraction and perception, remain unaffected. We also demonstrate that the vdd-1/+ phenotype can be rescued by expressing VDD or GFA2 in the synergid cells. Taken together, our findings reveal that the death of the receptive synergid cell is essential for maintenance of the following generations, and that a complex comprising VDD and VAL regulates this event.

Rapid Elimination of the Persistent Synergid through a Cell Fusion Mechanism.

In flowering plants, fertilization-dependent degeneration of the persistent synergid cell ensures one-on-one pairings of male and female gametes. Here, we report that the fusion of the persistent synergid cell and the endosperm selectively inactivates the persistent synergid cell in Arabidopsis thaliana. The synergid-endosperm fusion causes rapid dilution of pre-secreted pollen tube attractant in the persistent synergid cell and selective disorganization of the synergid nucleus during the endosperm proliferation, preventing attractions of excess number of pollen tubes (polytubey). The synergid-endosperm fusion is induced by fertilization of the central cell, while the egg cell fertilization predominantly activates ethylene signaling, an inducer of the synergid nuclear disorganization. Therefore, two female gametes (the egg and the central cell) control independent pathways yet coordinately accomplish the elimination of the persistent synergid cell by double fertilization.

Love is a battlefield: programmed cell death during fertilization.

Plant development and growth is sustained by the constant generation of tremendous amounts of cells, which become integrated into various types of tissues and organs. What is all too often overlooked is that this thriving life also requires the targeted degeneration of selected cells, which undergo cell death according to genetically encoded programmes or environmental stimuli. The side-by-side existence of generation and demise is particularly evident in the haploid phase of the flowering plants cycle. Here, the lifespan of terminally differentiated accessory cells contrasts with that of germ cells, which by definition live on to form the next generation. In fact, with research in recent years it is becoming increasingly clear that the gametophytes of flowering plants constitute an attractive and powerful system for investigating the molecular mechanisms underlying selective cell death.

Ethylene signaling is required for synergid degeneration and the establishment of a pollen tube block.

In flowering plants, sperm cells are delivered by pollen tubes, which are attracted by two egg-cell-adjoining synergids. Successful fertilization terminates pollen tube attraction; however, the underlying mechanisms are not understood. Here, we show that the process of fertilization activates an EIN3- and EIN2-dependent ethylene-response cascade necessary for synergid cell death and the concomitant establishment of a pollen tube block. Microinjection of the ethylene precursor ACC into the female gametophyte or constitutive ethylene response results in premature synergid disintegration. This indicates that the requirement of fertilization for synergid degeneration and associated establishment of a pollen tube block can be bypassed by mimicking a postfertilization ethylene burst. Surprisingly, the persistent synergid in ethylene-hyposensitive plants adopts the molecular profile and cell-cycle regime of the biparental embryo-nourishing tissue, suggesting that ethylene signaling prevents the formation of an asexual maternal endosperm fraction.

When Double is not Twice as Much.

Gene and genome duplications provide a playground for various selective pressures and contribute significantly to genome complexity. It is assumed that the genomes of all major eukaryotic lineages possess duplicated regions that result from gene and genome duplication. There is evidence that the model plant Arabidopsis has been subjected to at least three whole-genome duplication events over the last 150-200 million years. As a result, many cellular processes are governed by redundantly acting gene families. Plants pass through two distinct life phases with a haploid gametophytic alternating with a diploid sporophytic generation. This ontogenetic difference in gene copy number has important implications for the outcome of deleterious mutations, which are masked by the second gene copy in diploid systems but expressed in a dominant fashion in haploid organisms. As a consequence, maintaining the activity of duplicated genes might be particularly advantageous during the haploid gametophytic generation. Here, we describe the distinctive features associated with the alteration of generations and discuss how activity profiles of duplicated genes might get modulated in a life phase dependent fashion.