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.

Chemical inhibition of Arabidopsis PIN-FORMED auxin transporters by the anti-inflammatory drug naproxen.

The phytohormone auxin plays central roles in many growth and developmental processes in plants. Development of chemical tools targeting the auxin pathway is useful for both plant biology and agriculture. Here we reveal that naproxen, a synthetic compound with anti-inflammatory activity in humans, acts as an auxin transport inhibitor targeting PIN-FORMED (PIN) transporters in plants. Physiological experiments indicate that exogenous naproxen treatment affects pleiotropic auxin-regulated developmental processes. Additional cellular and biochemical evidence indicates that naproxen suppresses auxin transport, specifically PIN-mediated auxin efflux. Moreover, biochemical and structural analyses confirm that naproxen binds directly to PIN1 protein via the same binding cavity as the indole-3-acetic acid substrate. Thus, by combining cellular, biochemical, and structural approaches, this study clearly establishes that naproxen is a PIN inhibitor and elucidates the underlying mechanisms. Further use of this compound may advance our understanding of the molecular mechanisms of PIN-mediated auxin transport and expand our toolkit in auxin biology and agriculture.

Structural insights into auxin recognition and efflux by Arabidopsis PIN1.

Polar auxin transport is unique to plants and coordinates their growth and development1,2. The PIN-FORMED (PIN) auxin transporters exhibit highly asymmetrical localizations at the plasma membrane and drive polar auxin transport3,4; however, their structures and transport mechanisms remain largely unknown. Here, we report three inward-facing conformation structures of Arabidopsis thaliana PIN1: the apo state, bound to the natural auxin indole-3-acetic acid (IAA), and in complex with the polar auxin transport inhibitor N-1-naphthylphthalamic acid (NPA). The transmembrane domain of PIN1 shares a conserved NhaA fold5. In the substrate-bound structure, IAA is coordinated by both hydrophobic stacking and hydrogen bonding. NPA competes with IAA for the same site at the intracellular pocket, but with a much higher affinity. These findings inform our understanding of the substrate recognition and transport mechanisms of PINs and set up a framework for future research on directional auxin transport, one of the most crucial processes underlying plant development.

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.

Functions and Mechanisms of SAC Phosphoinositide Phosphatases in Plants.

Phosphatidylinositol (PtdIns) is one type of phospholipid comprising an inositol head group and two fatty acid chains covalently linked to the diacylglycerol group. In addition to their roles as compositions of cell membranes, phosphorylated PtdIns derivatives, termed phosphoinositides, execute a wide range of regulatory functions. PtdIns can be phosphorylated by various lipid kinases at 3-, 4- and/or 5- hydroxyls of the inositol ring, and the phosphorylated forms, including PtdIns3P, PtdIns4P, PtdIns5P, PtdIns(3,5)P2, PtdIns(4,5)P2, can be reversibly dephosphorylated by distinct lipid phosphatases. Amongst many other types, the SUPPRESSOR OF ACTIN (SAC) family of phosphoinositide phosphatases recently emerged as important regulators in multiple growth and developmental processes in plants. Here, we review recent advances on the biological functions, cellular activities, and molecular mechanisms of SAC domain-containing phosphoinositide phosphatases in plants. With a focus on those studies in the model plant Arabidopsis thaliana together with progresses in other plants, we highlight the important roles of subcellular localizations and substrate preferences of various SAC isoforms in their functions.

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.

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.

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.