A new "lock-and-key" system revealed for plant reproductive barriers.

Mate selection in flowering plants can occur very rapidly after male pollen contact on the female pistil, but the cellular regulators driving this process were poorly understood. In this issue of Cell, Lan et al. have discovered the components of a complex ligand-receptor system controlling pollen selection in Arabidopsis thaliana.

Antagonistic RALF peptides control an intergeneric hybridization barrier on Brassicaceae stigmas.

Pollen-pistil interactions establish interspecific/intergeneric pre-zygotic hybridization barriers in plants. The rejection of undesired pollen at the stigma is crucial to avoid outcrossing but can be overcome with the support of mentor pollen. The mechanisms underlying this hybridization barrier are largely unknown. Here, in Arabidopsis, we demonstrate that receptor-like kinases FERONIA/CURVY1/ANJEA/HERCULES RECEPTOR KINASE 1 and cell wall proteins LRX3/4/5 interact on papilla cell surfaces with autocrine stigmatic RALF1/22/23/33 peptide ligands (sRALFs) to establish a lock that blocks the penetration of undesired pollen tubes. Compatible pollen-derived RALF10/11/12/13/25/26/30 peptides (pRALFs) act as a key, outcompeting sRALFs and enabling pollen tube penetration. By treating Arabidopsis stigmas with synthetic pRALFs, we unlock the barrier, facilitating pollen tube penetration from distantly related Brassicaceae species and resulting in interspecific/intergeneric hybrid embryo formation. Therefore, we uncover a "lock-and-key" system governing the hybridization breadth of interspecific/intergeneric crosses in Brassicaceae. Manipulating this system holds promise for facilitating broad hybridization in crops.

Pollen Cell Wall Patterns Form from Modulated Phases.

The ornately geometric walls of pollen grains have inspired scientists for decades. We show that the evolved diversity of these patterns is entirely recapitulated by a biophysical model in which an initially uniform polysaccharide layer in the extracellular space, mechanically coupled to the cell membrane, phase separates to a spatially modulated state. Experiments reveal this process occurring in living cells. We observe that in ∼10% of extant species, this phase separation reaches equilibrium during development such that individual pollen grains are identical and perfectly reproducible. About 90% of species undergo an arrest of this process prior to equilibrium such that individual grains are similar but inexact copies. Equilibrium patterns have appeared multiple times during the evolution of seed plants, but selection does not favor these states. This framework for pattern development provides a route to rationalizing the surface textures of other secreted structures, such as cell walls and insect cuticle.

Induction of IL-10-producing type 2 innate lymphoid cells by allergen immunotherapy is associated with clinical response.

The role of innate immune cells in allergen immunotherapy that confers immune tolerance to the sensitizing allergen is unclear. Here, we report a role of interleukin-10-producing type 2 innate lymphoid cells (IL-10+ ILC2s) in modulating grass-pollen allergy. We demonstrate that KLRG1+ but not KLRG1- ILC2 produced IL-10 upon activation with IL-33 and retinoic acid. These cells attenuated Th responses and maintained epithelial cell integrity. IL-10+ KLRG1+ ILC2s were lower in patients with grass-pollen allergy when compared to healthy subjects. In a prospective, double-blind, placebo-controlled trial, we demonstrated that the competence of ILC2 to produce IL-10 was restored in patients who received grass-pollen sublingual immunotherapy. The underpinning mechanisms were associated with the modification of retinol metabolic pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathways in the ILCs. Altogether, our findings underscore the contribution of IL-10+ ILC2s in the disease-modifying effect by allergen immunotherapy.

Pesticide use negatively affects bumble bees across European landscapes.

Sustainable agriculture requires balancing crop yields with the effects of pesticides on non-target organisms, such as bees and other crop pollinators. Field studies demonstrated that agricultural use of neonicotinoid insecticides can negatively affect wild bee species1,2, leading to restrictions on these compounds3. However, besides neonicotinoids, field-based evidence of the effects of landscape pesticide exposure on wild bees is lacking. Bees encounter many pesticides in agricultural landscapes4-9 and the effects of this landscape exposure on colony growth and development of any bee species remains unknown. Here we show that the many pesticides found in bumble bee-collected pollen are associated with reduced colony performance during crop bloom, especially in simplified landscapes with intensive agricultural practices. Our results from 316 Bombus terrestris colonies at 106 agricultural sites across eight European countries confirm that the regulatory system fails to sufficiently prevent pesticide-related impacts on non-target organisms, even for a eusocial pollinator species in which colony size may buffer against such impacts10,11. These findings support the need for postapproval monitoring of both pesticide exposure and effects to confirm that the regulatory process is sufficiently protective in limiting the collateral environmental damage of agricultural pesticide use.

Elevated Southern Hemisphere moisture availability during glacial periods.

Late Pleistocene ice-age climates are routinely characterized as having imposed moisture stress on low- to mid-latitude ecosystems1-5. This idea is largely based on fossil pollen evidence for widespread, low-biomass glacial vegetation, interpreted as indicating climatic dryness6. However, woody plant growth is inhibited under low atmospheric CO2 (refs. 7,8), so understanding glacial environments requires the development of new palaeoclimate indicators that are independent of vegetation9. Here we show that, contrary to expectations, during the past 350 kyr, peaks in southern Australian climatic moisture availability were largely confined to glacial periods, including the Last Glacial Maximum, whereas warm interglacials were relatively dry. By measuring the timing of speleothem growth in the Southern Hemisphere subtropics, which today has a predominantly negative annual moisture balance, we developed a record of climatic moisture availability that is independent of vegetation and extends through multiple glacial-interglacial cycles. Our results demonstrate that a cool-moist response is consistent across the austral subtropics and, in part, may result from reduced evaporation under cool glacial temperatures. Insofar as cold glacial environments in the Southern Hemisphere subtropics have been portrayed as uniformly arid3,10,11, our findings suggest that their characterization as evolutionary or physiological obstacles to movement and expansion of animal, plant and, potentially, human populations10 should be reconsidered.

Osmosensor-mediated control of Ca2+ spiking in pollen germination.

Higher plants survive terrestrial water deficiency and fluctuation by arresting cellular activities (dehydration) and resuscitating processes (rehydration). However, how plants monitor water availability during rehydration is unknown. Although increases in hypo-osmolarity-induced cytosolic Ca2+ concentration (HOSCA) have long been postulated to be the mechanism for sensing hypo-osmolarity in rehydration1,2, the molecular basis remains unknown. Because osmolarity triggers membrane tension and the osmosensing specificity of osmosensing channels can only be determined in vivo3-5, these channels have been classified as a subtype of mechanosensors. Here we identify bona fide cell surface hypo-osmosensors in Arabidopsis and find that pollen Ca2+ spiking is controlled directly by water through these hypo-osmosensors-that is, Ca2+ spiking is the second messenger for water status. We developed a functional expression screen in Escherichia coli for hypo-osmosensitive channels and identified OSCA2.1, a member of the hyperosmolarity-gated calcium-permeable channel (OSCA) family of proteins6. We screened single and high-order OSCA mutants, and observed that the osca2.1/osca2.2 double-knockout mutant was impaired in pollen germination and HOSCA. OSCA2.1 and OSCA2.2 function as hypo-osmosensitive Ca2+-permeable channels in planta and in HEK293 cells. Decreasing osmolarity of the medium enhanced pollen Ca2+ oscillations, which were mediated by OSCA2.1 and OSCA2.2 and required for germination. OSCA2.1 and OSCA2.2 convert extracellular water status into Ca2+ spiking in pollen and may serve as essential hypo-osmosensors for tracking rehydration in plants.

100 ancient genomes show repeated population turnovers in Neolithic Denmark.

Major migration events in Holocene Eurasia have been characterized genetically at broad regional scales1-4. However, insights into the population dynamics in the contact zones are hampered by a lack of ancient genomic data sampled at high spatiotemporal resolution5-7. Here, to address this, we analysed shotgun-sequenced genomes from 100 skeletons spanning 7,300 years of the Mesolithic period, Neolithic period and Early Bronze Age in Denmark and integrated these with proxies for diet (13C and 15N content), mobility (87Sr/86Sr ratio) and vegetation cover (pollen). We observe that Danish Mesolithic individuals of the Maglemose, Kongemose and Ertebølle cultures form a distinct genetic cluster related to other Western European hunter-gatherers. Despite shifts in material culture they displayed genetic homogeneity from around 10,500 to 5,900 calibrated years before present, when Neolithic farmers with Anatolian-derived ancestry arrived. Although the Neolithic transition was delayed by more than a millennium relative to Central Europe, it was very abrupt and resulted in a population turnover with limited genetic contribution from local hunter-gatherers. The succeeding Neolithic population, associated with the Funnel Beaker culture, persisted for only about 1,000 years before immigrants with eastern Steppe-derived ancestry arrived. This second and equally rapid population replacement gave rise to the Single Grave culture with an ancestry profile more similar to present-day Danes. In our multiproxy dataset, these major demographic events are manifested as parallel shifts in genotype, phenotype, diet and land use.

Stigma receptors control intraspecies and interspecies barriers in Brassicaceae.

Flowering plants have evolved numerous intraspecific and interspecific prezygotic reproductive barriers to prevent production of unfavourable offspring1. Within a species, self-incompatibility (SI) is a widely utilized mechanism that rejects self-pollen2,3 to avoid inbreeding depression. Interspecific barriers restrain breeding between species and often follow the SI × self-compatible (SC) rule, that is, interspecific pollen is unilaterally incompatible (UI) on SI pistils but unilaterally compatible (UC) on SC pistils1,4-6. The molecular mechanisms underlying SI, UI, SC and UC and their interconnections in the Brassicaceae remain unclear. Here we demonstrate that the SI pollen determinant S-locus cysteine-rich protein/S-locus protein 11 (SCR/SP11)2,3 or a signal from UI pollen binds to the SI female determinant S-locus receptor kinase (SRK)2,3, recruits FERONIA (FER)7-9 and activates FER-mediated reactive oxygen species production in SI stigmas10,11 to reject incompatible pollen. For compatible responses, diverged pollen coat protein B-class12-14 from SC and UC pollen differentially trigger nitric oxide, nitrosate FER to suppress reactive oxygen species in SC stigmas to facilitate pollen growth in an intraspecies-preferential manner, maintaining species integrity. Our results show that SRK and FER integrate mechanisms underlying intraspecific and interspecific barriers and offer paths to achieve distant breeding in Brassicaceae crops.

Histone H2B.8 compacts flowering plant sperm through chromatin phase separation.

Sperm chromatin is typically transformed by protamines into a compact and transcriptionally inactive state1,2. Sperm cells of flowering plants lack protamines, yet they have small, transcriptionally active nuclei with chromatin condensed through an unknown mechanism3,4. Here we show that a histone variant, H2B.8, mediates sperm chromatin and nuclear condensation in Arabidopsis thaliana. Loss of H2B.8 causes enlarged sperm nuclei with dispersed chromatin, whereas ectopic expression in somatic cells produces smaller nuclei with aggregated chromatin. This result demonstrates that H2B.8 is sufficient for chromatin condensation. H2B.8 aggregates transcriptionally inactive AT-rich chromatin into phase-separated condensates, which facilitates nuclear compaction without reducing transcription. Reciprocal crosses show that mutation of h2b.8 reduces male transmission, which suggests that H2B.8-mediated sperm compaction is important for fertility. Altogether, our results reveal a new mechanism of nuclear compaction through global aggregation of unexpressed chromatin. We propose that H2B.8 is an evolutionary innovation of flowering plants that achieves nuclear condensation compatible with active transcription.

Competition for pollinators destabilizes plant coexistence.

Mounting concern over the global decline of pollinators has fuelled calls for investigating their role in maintaining plant diversity1,2. Theory predicts that competition for pollinators can stabilize interactions between plant species by providing opportunities for niche differentiation3, while at the same time can drive competitive imbalances that favour exclusion4. Here we empirically tested these contrasting effects by manipulating competition for pollinators in a way that predicts its long-term implications for plant coexistence. We subjected annual plant individuals situated across experimentally imposed gradients in neighbour density to either ambient insect pollination or a pollen supplementation treatment alleviating competition for pollinators. The vital rates of these individuals informed plant population dynamic models predicting the key theoretical metrics of species coexistence. Competition for pollinators generally destabilized the interactions between plant species, reducing the proportion of pairs expected to coexist. Interactions with pollinators also influenced the competitive imbalances between plant species, effects that are expected to strengthen with pollinator decline, potentially disrupting plant coexistence. Indeed, results from an experiment simulating pollinator decline showed that plant species experiencing greater reductions in floral visitation also suffered greater declines in population growth rate. Our results reveal that competition for pollinators may weaken plant coexistence by destabilizing interactions and contributing to competitive imbalances, information critical for interpreting the impacts of pollinator decline.

A receptor-channel trio conducts Ca2+ signalling for pollen tube reception.

Precise signalling between pollen tubes and synergid cells in the ovule initiates fertilization in flowering plants1. Contact of the pollen tube with the ovule triggers calcium spiking in the synergids2,3 that induces pollen tube rupture and sperm release. This process, termed pollen tube reception, entails the action of three synergid-expressed proteins in Arabidopsis: FERONIA (FER), a receptor-like kinase; LORELEI (LRE), a glycosylphosphatidylinositol-anchored protein; and NORTIA (NTA), a transmembrane protein of unknown function4-6. Genetic analyses have placed these three proteins in the same pathway; however, it remains unknown how they work together to enable synergid-pollen tube communication. Here we identify two pollen-tube-derived small peptides7 that belong to the rapid alkalinization factor (RALF) family8 as ligands for the FER-LRE co-receptor, which in turn recruits NTA to the plasma membrane. NTA functions as a calmodulin-gated calcium channel required for calcium spiking in the synergid. We also reconstitute the biochemical pathway in which FER-LRE perceives pollen-tube-derived peptides to activate the NTA calcium channel and initiate calcium spiking, a second messenger for pollen tube reception. The FER-LRE-NTA trio therefore forms a previously unanticipated receptor-channel complex in the female cell to recognize male signals and trigger the fertilization process.

Standard Deviations: The Biological Bases of Transmission Ratio Distortion.

The rule of Mendelian inheritance is remarkably robust, but deviations from the equal transmission of alternative alleles at a locus [a.k.a. transmission ratio distortion (TRD)] are also commonly observed in genetic mapping populations. Such TRD reveals locus-specific selection acting at some point between the diploid heterozygous parents and progeny genotyping and therefore can provide novel insight into otherwise-hidden genetic and evolutionary processes. Most of the classic selfish genetic elements were discovered through their biasing of transmission, but many unselfish evolutionary and developmental processes can also generate TRD. In this review, we describe methodologies for detecting TRD in mapping populations, detail the arenas and genetic interactions that shape TRD during plant and animal reproduction, and summarize patterns of TRD from across the genetic mapping literature. Finally, we point to new experimental approaches that can accelerate both detection of TRD and characterization of the underlying genetic mechanisms.

The adaptor protein ECAP, the corepressor LEUNIG, and the transcription factor BEH3 interact and regulate microsporocyte generation in Arabidopsis.

Histospecification and morphogenesis of anthers during development in Arabidopsis (Arabidopsis thaliana) are well understood. However, the regulatory mechanism of microsporocyte generation at the pre-meiotic stage remains unclear, especially how archesporial cells are specified and differentiate into 2 cell lineages with distinct developmental fates. SPOROCYTELESS (SPL) is a key reproductive gene that is activated during early anther development and remains active. In this study, we demonstrated that the EAR motif-containing adaptor protein (ECAP) interacts with the Gro/Tup1 family corepressor LEUNIG (LUG) and the BES1/BZR1 HOMOLOG3 (BEH3) transcription factor to form a transcription activator complex, epigenetically regulating SPL transcription. SPL participates in microsporocyte generation by modulating the specification of archesporial cells and the archesporial cell-derived differentiation of somatic and reproductive cell layers. This study illustrates the regulation of SPL expression by the ECAP-LUG-BEH3 complex, which is essential for the generation of microsporocytes. Moreover, our findings identified ECAP as a key transcription regulator that can combine with different partners to regulate gene expression in distinct ways, thereby facilitating diverse processes in various aspects of plant development.

Arabidopsis class A S-acyl transferases modify the pollen receptors LIP1 and PRK1 to regulate pollen tube guidance.

Protein S-acylation catalyzed by protein S-acyl transferases (PATs) is a reversible lipid modification regulating protein targeting, stability, and interaction profiles. PATs are encoded by large gene families in plants, and many proteins including receptor-like cytoplasmic kinases (RLCKs) and receptor-like kinases (RLKs) are subject to S-acylation. However, few PATs have been assigned substrates, and few S-acylated proteins have known upstream enzymes. We report that Arabidopsis (Arabidopsis thaliana) class A PATs redundantly mediate pollen tube guidance and participate in the S-acylation of POLLEN RECEPTOR KINASE1 (PRK1) and LOST IN POLLEN TUBE GUIDANCE1 (LIP1), a critical RLK or RLCK for pollen tube guidance, respectively. PAT1, PAT2, PAT3, PAT4, and PAT8, collectively named PENTAPAT for simplicity, are enriched in pollen and show similar subcellular distribution. Functional loss of PENTAPAT reduces seed set due to male gametophytic defects. Specifically, pentapat pollen tubes are compromised in directional growth. We determine that PRK1 and LIP1 interact with PENTAPAT, and their S-acylation is reduced in pentapat pollen. The plasma membrane (PM) association of LIP1 is reduced in pentapat pollen, whereas point mutations reducing PRK1 S-acylation affect its affinity with its interacting proteins. Our results suggest a key role of S-acylation in pollen tube guidance through modulating PM receptor complexes.

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Evolution of the thermostability of actin-depolymerizing factors enhances the adaptation of pollen germination to high temperature.

Double fertilization in many flowering plants (angiosperms) often occurs during the hot summer season, but the mechanisms that enable angiosperms to adapt specifically to high temperatures are largely unknown. The actin cytoskeleton is essential for pollen germination and the polarized growth of pollen tubes, yet how this process responds to high temperatures remains unclear. Here, we reveal that the high thermal stability of 11 Arabidopsis (Arabidopsis thaliana) actin-depolymerizing factors (ADFs) is significantly different: ADFs that specifically accumulate in tip-growing cells (pollen and root hairs) exhibit high thermal stability. Through ancestral protein reconstruction, we found that subclass II ADFs (expressed specifically in pollen) have undergone a dynamic wave-like evolution of the retention, loss, and regeneration of thermostable sites. Additionally, the sites of AtADF7 with high thermal stability are conserved in ADFs specific to angiosperm pollen. Moreover, the high thermal stability of ADFs is required to regulate actin dynamics and turnover at high temperatures to promote pollen germination. Collectively, these findings suggest strategies for the adaptation of sexual reproduction to high temperature in angiosperms at the cell biology level.

Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA.

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Fertilized egg cells secrete endopeptidases to avoid polytubey.

Upon gamete fusion, animal egg cells secrete proteases from cortical granules to establish a fertilization envelope as a block to polyspermy1-4. Fertilization in flowering plants is more complex and involves the delivery of two non-motile sperm cells by pollen tubes5,6. Simultaneous penetration of ovules by multiple pollen tubes (polytubey) is usually avoided, thus indirectly preventing polyspermy7,8. How plant egg cells regulate the rejection of extra tubes after successful fertilization is not known. Here we report that the aspartic endopeptidases ECS1 and ECS2 are secreted to the extracellular space from a cortical network located at the apical domain of the Arabidopsis egg cell. This reaction is triggered only after successful fertilization. ECS1 and ECS2 are exclusively expressed in the egg cell and transcripts are degraded immediately after gamete fusion. ECS1 and ESC2 specifically cleave the pollen tube attractor LURE1. As a consequence, polytubey is frequent in ecs1 ecs2 double mutants. Ectopic secretion of these endopeptidases from synergid cells led to a decrease in the levels of LURE1 and reduced the rate of pollen tube attraction. Together, these findings demonstrate that plant egg cells sense successful fertilization and elucidate a mechanism as to how a relatively fast post-fertilization block to polytubey is established by fertilization-induced degradation of attraction factors.

Pollinators contribute to the maintenance of flowering plant diversity.

Mechanisms that favour rare species are key to the maintenance of diverse communities1-3. One of the most critical tasks for conservation of flowering plant biodiversity is to understand how plant-pollinator interactions contribute to the maintenance of rare species4-7. Here we show that niche partitioning in pollinator use and asymmetric facilitation confer fitness advantage of rarer species in a biodiversity hotspot using phylogenetic structural equation modelling that integrates plant-pollinator and interspecific pollen transfer networks with floral functional traits. Co-flowering species filtered pollinators via floral traits, and rarer species showed greater pollinator specialization leading to higher pollination-mediated male and female fitness than more abundant species. When plants shared pollinator resources, asymmetric facilitation via pollen transport dynamics benefitted the rarer species at the cost of more abundant species, serving as an alternative diversity-promoting mechanism. Our results emphasize the importance of community-wide plant-pollinator interactions that affect reproduction for biodiversity maintenance.